Cultures Of Human Mononuclear Cells Research Articles

Ribonucleic acid isolation from human mononuclear cell culture with magnetic beads pre-enrichment for molecular analysis

Ribonucleic acid isolation from human mononuclear cell culture with magnetic beads pre-enrichment for molecular analysis

Novel Strategy in Searching for Natural Compounds with Anti-Aging and Rejuvenating Potential.

We suggest a novel approach for searching natural compounds with anti-aging and rejuvenation potential using cell cultures, with a high potential for the further in vivo applications. The present paper discusses ways of defining age for cell populations with large numbers of cells and suggests a method of assessing how young or old a cell population is based on a cell age profile approach. This approach uses experimental distributions of the cells over the cell cycle stages, acquired using flow cytometry. This paper discusses how such a profile should evolve under homeostatic maintenance of cell numbers in the proliferation niches. We describe promising results from experiments on a commercial substance claiming rejuvenating and anti-aging activity acting upon the cultures of human mononuclear cells and dermal fibroblasts. The chosen substance promotes a shift towards larger proportion of cells in synthesis and proliferation stages, and increases cell culture longevity. Further, we describe promising in vivo testing results of a selected food supplement. Based on the described concept of cell age profile and available test results, a strategy to search for natural compounds with regenerative, anti-aging and rejuvenation potential is suggested and proposed for wider and thorough testing. Proposed methodology of age assessment is rather generic and can be used for quantitative assessment of the anti-aging and rejuvenation potential of different interventions. Further research aimed at the tests of the suggested strategy using more substances and different interventions, and the thorough studies of molecular mechanisms related to the action of the substance used for testing the suggested search methodology, are needed.

2-3% fetal calf serum in cell media is sufficient for the development and growth of human mononuclear cell cultures

Aim: It is a common practice that 10-20% fetal calf serum (FCS) is being used in the cell culture media for the development and maintenance of human mononuclear cells as well as other cell cultures. FCS is produced through a painful process involving animals and is one of the major cost factors. The reduction in the volume of FCS can make it less unethical and reduce the costs strongly. Materials and Methods: Therefore 2-3 % FCS and 10% FCS were used in the development of different mononuclear cell cultures for making comparisons and different parameters like morphology, production of antibodies, isolation of DNA/RNA with mini-column and magnetic beads cell isolations along with the presence of human internal controls with real-time PCR were tested. Results: It was found that there was no difference between 2-3 % FCS and 10 % FCS containing media in the above-said parameters. More than a hundred such cell cultures were developed and many of them were maintained for more than 3 years. Conclusion: 2-3 % FCS is sufficient for the development and maintenance of human mononuclear cell cultures over a long period of time.

ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation.

Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways.

Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4(+) and CD8(+) lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform-specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4(+) and CD8(+) counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4(+) and CD8(+) lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity.

Roles of myeloperoxidase and GAPDH in interferon-gamma production of GM-CSF-dependent macrophages.

Interferon (IFN)-gamma is highly expressed in atherosclerotic lesions and may have an important role in atherogenesis. Myeloperoxidase (MPO), the most abundant protein in neutrophils, is a marker of plaque vulnerability and a possible bridge between inflammation and cardiovascular disease. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has also been implicated in the pathogenesis of atherosclerosis. The present study investigated the role of neutrophil activation in atherosclerosis. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IFN-gamma protein by GM-CSF-dependent-macrophages was investigated by enzyme-linked immunosorbent assay after stimulation with MPO. GM-CSF enhanced macrophage expression of the mannose receptor (CD206), which is involved in MPO uptake. MPO increased IFN-gamma production by GM-CSF-dependent macrophages in a concentration-dependent manner. Pretreatment of macrophages with small interfering RNA (siRNA) for CD206 or extracellular signal-regulated kinase (ERK)-2 attenuated IFN-gamma production, while siRNA for ERK-1 did not. GAPDH is known to bind to adenylate/uridylate (AU)-rich elements of RNA and may influence IFN-gamma protein expression by binding to the AU-rich element of IFN-gamma mRNA. Interestingly, pretreatment with siRNA for GAPDH significantly reduced IFN-gamma production by macrophages, while it did not affect TF protein expression. In conclusion, MPO upregulates IFN-gamma production by GM-CSF-dependent-macrophages via the CD206/ERK-2 signaling pathway, while silencing GAPDH reduces IFN-gamma production.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation.

Contrasting Mechanisms of Interferon‐α Inhibition by Intravenous Immunoglobulin After Induction by Immune Complexes Versus Toll‐like Receptor Agonists

Plasmacytoid dendritic cells (PDCs) produce high concentrations of interferon-α (IFNα) following exposure to immune complexes containing nucleic acids. We previously reported that serum from healthy donors inhibits IFNα production by PDCs in response to systemic lupus erythematosus (SLE) immune complexes, and that inhibition is mediated, in part, by IgG. IgG is the major component of intravenous immunoglobulin and is well known to exert antiinflammatory properties. Although suppression of inflammation by the sialylated subfraction of IgG has been implicated in some models, the mechanism of IFNα inhibition by IgG and the importance of sialylation have not been studied. SLE immune complexes or synthetic Toll-like receptor (TLR) agonists were used to stimulate total or individual cell-depleted human mononuclear cell cultures in the presence or absence of IgG, Fc fragments, F(ab')2 fragments, and their sialylated or unsialylated subfractions. Cytokines were quantified by enzyme-linked immunosorbent assay. We identified 2 distinct mechanisms by which IgG inhibits IFNα production. First, IgG Fc fragments inhibited SLE immune complex-stimulated IFNα production via a sialic acid-independent mechanism, by inhibiting immune complex binding to Fcγ receptor IIa on PDCs. In contrast, the F(ab')2 fragment of the sialylation-enriched fraction of IgG inhibited TLR-7 or TLR-9 agonist-induced IFNα production but did not require the sialic acid residue itself. The inhibitory activity of IgG on TLR agonist-induced IFNα required monocyte production of prostaglandin E2, a potent suppressor of IFNα production by PDCs. IgG attenuates IFNα production by PDCs by both cell surface receptor and intracellular pathways, depending on the nature of the inducing stimulus.

Biochemical and spatial control of human pluripotent stem cell mesoderm formation

Purpose: This study aims to develop the surface architecture enabling selective in-situ capturing of endothelial progenitor cells (EPCs) and subsequent rapid endothelialization on blood-contacting surfaces under arterial flow. First, based on ex vivo cellular potentials on various surface-bound candidate ligands, the best-suited pair of cell receptor exclusively expressed on endothelial lineage cells and surface-bound ligand is defined. Second, prolonged activation of intracellular signaling transduction pathways of EPCs adhered on surface-bound ligand is examined. Lastly, in-situ capture of EPCs and endothelialization in a porcein model is verified. Methods: Methods included (1) covalent bonding of molecules [vascular endothelial growth factor (VEGF) and two VEGF receptor antibodies and Tie-1 and -2 antibodies] on vinyl alcoholcopolymer; (2) culture of human mononuclear cells(MCs) on these proteinbound substrates and histocytochemical analyses; and (3) implantation of stent in coronaries. Results: Proteins were covalently bound to the copolymer surface via activation of hydroxyl group. Among molecules examined, only VEGF exhibited high adhesion and proliferation characteristics and a quite high differentiation potential of MCs with culture time. Day-order continuous activation of intracellular signaling transduction pathways (phosphorylation of VEGF receptor, FAK, ERK and Akt) was observed for ECs adhered on VEGF-bound substrate. Implantation study using VEGF-bound stents showed that cells expressing VEGF receptor adhered on stent surface followed by endothelialization was observed within several days. Conclusions: This study concludes that surface-bound VEGF enables selective capture of EPCs and complete endothelialization in vivo, and simple surface architecture promise to exert non-thrombogenic potential to cardiovascular devices.

In situ capture technology of endothelial progenitor cells

Purpose: This study aims to develop the surface architecture enabling selective in-situ capturing of endothelial progenitor cells (EPCs) and subsequent rapid endothelialization on blood-contacting surfaces under arterial flow. First, based on ex vivo cellular potentials on various surface-bound candidate ligands, the best-suited pair of cell receptor exclusively expressed on endothelial lineage cells and surface-bound ligand is defined. Second, prolonged activation of intracellular signaling transduction pathways of EPCs adhered on surface-bound ligand is examined. Lastly, in-situ capture of EPCs and endothelialization in a porcein model is verified. Methods: Methods included (1) covalent bonding of molecules [vascular endothelial growth factor (VEGF) and two VEGF receptor antibodies and Tie-1 and -2 antibodies] on vinyl alcoholcopolymer; (2) culture of human mononuclear cells(MCs) on these proteinbound substrates and histocytochemical analyses; and (3) implantation of stent in coronaries. Results: Proteins were covalently bound to the copolymer surface via activation of hydroxyl group. Among molecules examined, only VEGF exhibited high adhesion and proliferation characteristics and a quite high differentiation potential of MCs with culture time. Day-order continuous activation of intracellular signaling transduction pathways (phosphorylation of VEGF receptor, FAK, ERK and Akt) was observed for ECs adhered on VEGF-bound substrate. Implantation study using VEGF-bound stents showed that cells expressing VEGF receptor adhered on stent surface followed by endothelialization was observed within several days. Conclusions: This study concludes that surface-bound VEGF enables selective capture of EPCs and complete endothelialization in vivo, and simple surface architecture promise to exert non-thrombogenic potential to cardiovascular devices.

Triterpene Acids from Rose Hip Powder Inhibit Self‐antigen‐ and LPS‐induced Cytokine Production and CD4+ T‐cell Proliferation in Human Mononuclear Cell Cultures

A triterpene acid mixture consisting of oleanolic, ursolic and betulinic acid isolated from a standardized rose hip powder (Rosa canina L.) has been shown to inhibit interleukin (IL)-6 release from Mono Mac 6 cells. The present study examined the effects of the triterpene acid mixture on the cytokine production and proliferation of CD4⁺ T cells and CD19⁺ B cells induced by a self-antigen, human thyroglobulin and by lipopolysaccharide in cultures of normal mononuclear cells. The triterpene acid mixture inhibited the production of tumor necrosis factor-α and IL-6 with estimated IC₅₀ values in the range 35-56 µg/mL, the Th1 cytokines interferon-γ and IL-2 (IC₅₀ values 10-20 µg/mL) and the antiinflammatory cytokine IL-10 (IC₅₀ values 18-21 µg/mL). Moreover, the mixture also inhibited CD4⁺ T-cell and CD19⁺ B-cell proliferation (IC₅₀ value 22 and 12 µg/mL, respectively). Together, these data demonstrate that oleanolic, ursolic and betulinic acid are active immunomodulatory constituents of the standardized rose hip powder. However, since the estimated IC₅₀ values are in the µg/mL range, it is questionable whether the content of the triterpene acids in the standardized rose hip powder, alone, can explain the reported clinical effects.

The Envelope Protein of a Human Endogenous Retrovirus-W Family Activates Innate Immunity through CD14/TLR4 and Promotes Th1-Like Responses

Multiple sclerosis-associated retroviral element (MSRV) is a retroviral element, the sequence of which served to define the W family of human endogenous retroviruses. MSRV viral particles display proinflammatory activities both in vitro in human mononuclear cell cultures and in vivo in a humanized SCID mice model. To understand the molecular basis of such properties, we have investigated the inflammatory potential of the surface unit of the MSRV envelope protein (ENV-SU), the fraction that is poised to naturally interact with host cells. We report in this study that MSRV ENV-SU induces, in a specific manner, human monocytes to produce major proinflammatory cytokines through engagement of CD14 and TLR4, which are pattern recognition receptors of primary importance in innate immunity. ENV-SU could also trigger a maturation process in human dendritic cells. Finally, ENV-SU endowed dendritic cells with the capacity to support a Th1-like type of Th cell differentiation. The data are discussed in the context of immune responses and chronic proinflammatory disorders.

TGF‐β and TNF‐α producing effects of losartan and amlodipine on human mononuclear cell culture

The modulation of cytokine release, which affects adhesion of leucocytes to endothelial cells, and proliferation of peripheral blood mononuclear cells with antihypertensive drugs was explored. In the present study, mononuclear cells were incubated with losartan and amlodipine at concentrations of 10(-6), 10(-5) and 10(-4) mol/L for 6 h. Transforming growth factor (TGF)-beta and tumour necrosis factor (TNF)-alpha levels were measured. Proliferation of mononuclear cells were assessed at the same concentrations of amlodipine and losartan with the methylthiazoletetrazolium (MTT) test. Amlodipine was found to induce TGF-beta synthesis from mononuclear cells with increasing concentrations, while it was found to inhibit TNF-alpha secretion with increasing concentrations. In contrast, losartan was found to induce TGF-beta and TNF-alpha secretion with increasing concentrations. Anti-atherosclerotic effects of amlodipine and losartan might be through increased secretion of TGF-beta from mononuclear cells. Different results at different concentrations might be due to the pharmocokinetic differences of these drugs.

Optimisation of the CT.h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1×10 4 CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5×10 4 responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1×10 6 responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (⩾day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.

Interaction of Mycobacterium tuberculosis-induced transforming growth factor beta1 and interleukin-10.

Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-beta1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-beta1 independently suppressed the production of PPD-induced gamma interferon (IFN-gamma) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-gamma in cultures containing both rTGF-beta1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-beta1 but not with rIL-10. Suppression of PPD-induced IFN-gamma in PBMC containing both rTGF-beta1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P < 0.05) than cultures containing TGF-beta1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-beta1 and IL-10 together enhanced PPD-induced IFN-gamma in PBMC in a synergistic manner. Thus, TGF-beta1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-gamma, and TGF-beta1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.

Enhancement of TNF-alpha production by ganglioside GM2 in human mononuclear cell culture.

Some gangliosides have been regarded as autoantigens of immune-mediated neurological disorders such as Guillain-Barré syndrome (GBS), Miller Fisher syndrome and multifocal motor neuropathy. On the other hand, proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), may be important in the pathogenesis of some neuroimmunological disorders. To clarify the interactions between immune cells and gangliosides, we investigated the effects of gangliosides on the production of proinflammatory cytokines in peripheral blood mononuclear cell (PBMC) cultures. We found that ganglioside GM2 markedly enhances the production of TNF-alpha and that TNF-alpha induction by coated GM2 is still more marked. These findings suggest that immune cells, especially monocytes/macrophages, cause inflammation upon encountering GM2.

Influence of mild hypothermia on cytokine gene expression in the culture of human mononuclear cells

Clinical studies demonstrated that moderate hypothermia may improve neurological outcome after severe head injury. On the other hand hypothermia is associated with increased incidence of infection complications. Recent studies suggest that mild hypothermia directly impaires natural host defenses — leukocyte mobility, phagocytosis and reactive oxygen species production and antibody production.

Immunomodulatory Activity of β-Casein Permeate Medium Fermented by Lactic Acid Bacteria

During fermentation, lactic acid bacteria may be able to release components that possess immunomodulatory activity. This activity was investigated in several culture supernatants arising from lactic acid bacteria cultured in a medium composed primarily of UF permeate of bovine milk; β-CN was added as the sole protein source. Only a Lactobacillus helveticus supernatant allowed the modulation (both suppression and enhancement) of lymphocyte proliferation in vitro on human peripheral blood lymphocytes, but L. helveticus did not modulate the cytotoxic activity of natural killer cells or of lymphokine-activated killer cells. The addition of different quantities of culture supernatant to cultures of human mononuclear cells, stimulated by the mitogen concanavalin A, significantly increased the production of interferon-γ and decreased the production of interleukin-2 and the expression of the α-chain of the interleukin-2 receptor (p55), all of which appear to be correlated with the decrease in lymphocyte proliferation. Our results suggest that the culture supernatant activity might be related to interaction with monocyte-macrophage and T helper cells, especially Th1-like cells.

Human T Cells Are Highly Sensitive to Suppression of Mitogenesis by Polycyclic Aromatic Hydrocarbons and This Effect Is Differentially Reversed by α-Naphthoflavone

The immunosuppressive effects of polycyclic aromatic hydrocarbons (PAHs) on immune responses in rodents, bothin vivoandin vitro,have been widely documented. However, few studies have addressed the immunotoxicity of PAHs in the human system. In this report, we examined the toxic effects of nine different PAHs on human peripheral blood T cell mitogenesis. We found that benzo(a)pyrene (BaP), 3-methylcholanthrene (3-MC), and 7,12-dimethylbenz(a)anthracene (DMBA) were highly immunotoxic in the human system, while dibenz(a,c)anthracene (DAC) and dibenz(a,h)anthracene (DAH) were of intermediate toxicity, 9,10-di- methylanthracene (DMA), benzo(e)pyrene (BeP), and ben- z(a)anthracene (BA) were mildly immunotoxic, and anthracene (ANTH) had no measureable toxicity at the concentrations tested. Our results using human lymphocytes differed from previous studies in rodents, in that BaP and 3-MC were the most immunotoxic PAHs in the human mitogenesis assay, while DMBA has long been regarded as the PAH that is most potently toxic to rodent T cell responses. We also showed that α-naphthoflavone (ANF), which functions as both anAhreceptor antagonist and an inhibitor of cytochrome P450 activity, was able to block the suppressive effects of both BaP and DMBA, but not 3-MC. This suggests that the immunotoxicity of 3-MC may be mediated through a different mechanism than BaP or DMBA. Addition of four different BaP metabolites directly to cultures of human mononuclear cells showed that the 7,8-dihydrodiol metabolite was the most toxic, and that this toxicity could be completely blocked by equimolar and 10-fold greater concentrations of ANF. The 7,8-dihydrodiol metabolite was probably further metabolized to the 7,8-diol epoxide, the toxicity of which could not be effectively reversed by ANF. The 4,5-epoxide metabolite was apparently cytotoxic at high concentrations (10 μm), while the 7-hydroxy metabolite had no overtly negative effects on proliferation.