Phytohaemagglutinin-stimulated Peripheral Blood Mononuclear Cells Research Articles

New Values of Teucrium species: in Vitro Study of Cytotoxic Activities of Secondary Metabolites

The cytotoxicity of seven Teucrium species, a long time ago used as a food spices, for beverages and teas preparing, as well as therapeutics for digestive and respiratory diseases, were examined against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human chronic myelogenous leukemia K562 and human breast adenocarcinoma MDA-MB-361 cells. MTT assay was used for determination of target cell survival. The most prominent cytotoxic effect was observed against K562 cells, especially by T. scordioides, T. montanum and T. botrys. All Teucrium extracts showed good cytotoxic activity on HeLa cells, but very low cytotoxic effect on MDA-MB-361 cells. In addition, the cytotoxic activities of T. scordioides and T. montanum extract were tested on healthy resting and phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PHA-stimulated PBMC). T. scordioides and T. montanum extracts at concentration of 200 µg/ml reduced the resting PBMC and PHA-stimulated PBMC survival up to 10% and 20%, while the reduction of K562 cell survival at the same concentration of extracts was 94% and 97%, respectively. These results point to selectivity in their antitumor actions. Teucrium species can be regarded as promising candidates for natural plant sources of effective biological compounds as a supplements in the food industry, as well as for therapeutic use.

Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.

Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. In vitro experiment. Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control. The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.

Expression or secretion of IL-17 in the peripheral blood mononuclear cells from patients with dilated cardiomyopathy

Objective — The objective of the study was to investigate the relation between interleukin-17 (IL-17) level in the plasma and in peripheral blood mononuclear cells (PBMCs), and dilated cardiomyopathy (DCM) disease severity.Methods and results — 30 patients with DCM and 20 normal adults as control were studied. IL-17 protein level in plasma, PBMC culture supernatants, and phytohaemagglutinin-stimulated PBMC culture supernatants were measured with ELISA. IL-17 mRNA expression in PBMCs was detected by reverse transcription-polymerase chain reaction. Results showed that the IL-17 protein level in PHA-stimulated PBMC culture supernatants or its mRNA level in the PHA-stimulated PBMC, but not in plasma or in PBMC culture supernatants, was significantly elevated in DCM patients compared with normal control subjects.The IL-17 protein level in cultured supernatants and the IL-17 mRNA level in the PBMCs were strongly correlated with the heart function of DCM patients.Conclusions — The ability to express IL-17 protein or mRNA in PBMC is abnormal and the change strongly correlates with the heart function of DCM patients.

Detection of circulating FcɛR2/CD23+ monocytes in patients with rheumatic diseases

Recently, in vitro studies have demonstrated that expression of Fc epsilon R2/CD23 on normal monocytes can be specifically induced by IL-4. In order to investigate the interaction of IL-4 and monocytes in rheumatic diseases, flow cytometry studies were performed. Elevated numbers of circulating Fc epsilon R2/CD23+ monocytes were detected in patients with progressive systemic sclerosis (PSS) as compared with controls. In addition, supernatants derived from phytohaemagglutinin-stimulated peripheral blood mononuclear cells of PSS patients contained high activity to induce Fc epsilon R2/CD23 on CD14+ monocytes. An increased frequency of Fc epsilon R2/CD23+ monocytes was also observed in rheumatoid arthritis, and sequential studies in patients with systemic lupus erythematosus showed a close relationship between Fc epsilon R2/CD23+ monocytes and disease activity. It is suggested that IL-4 has an important role in the pathogenesis of PSS by activating monocytes, and might also contribute to monocyte activation in other rheumatic diseases.

3′-Azido-2′,3′-Dideoxythymidine (Zidovudine) Uptake Mechanisms in T Lymphocytes

The nucleoside reverse transcriptase inhibitors (NRTIs) make up a family of antiretroviral drugs widely used in the treatment of HIV-1 infection. Human concentrative nucleoside transporters and equilibrative nucleoside transporters, encoded by SLC28 and SLC29 gene families, respectively, are known to be involved in the uptake of a variety of nucleoside analogues used in anticancer treatment. We therefore examined whether SLC28- and SLC29-encoded proteins contribute to the entry of these NRTls into the human leukaemic T-cell line Molt-4. Cis-inhibition experiments demonstrated that nucleoside transporters have a negligible role in antiviral drug uptake. Moreover, the previously identified 3'-azido-2',3'-dideoxythymidine (zidovudine; AZT) carriers, organic anion transporters (organic anion transporter [hOATs], members of the SLC22 gene family) have not been detected in T cells, either functionally or at the mRNA level, thus ruling out a role for hOATs in antiviral drug uptake in these cells. Nevertheless, the data provided here argue against the hypothesis of simple diffusion across the plasma membrane as the unique mechanism of AZT uptake. Actually, this pyrimidine derivative seems to have a temperature-sensitive route of entrance, a finding that, along with the evidence that, AZT inhibits its own uptake and its transport into phytohaemagglutinin-stimulated peripheral blood mononuclear cells is upregulated, strongly support the idea that AZT uptake into T-cells is associated with a mediated and regulated, transport mechanism.

Measles virus induces apoptosis in uninfected bystander T cells and leads to granzyme B and caspase activation in peripheral blood mononuclear cell cultures.

Measles causes lymphopenia and depresses cell-mediated immunity, but the mechanisms of immunosuppression and cell loss are poorly known. We have used an in vitro model of measles virus (MV)-infected peripheral blood mononuclear cells (PBMCs) and phytohaemagglutinin-stimulated PBMCs in order to assess MV-leucocyte interactions. Cell population undergoing apoptosis was measured by flow cytometry and Annexin-V-fluos staining. The expression of Fas, FasL, TNRF1, and Bcl-2 was analyzed by flow cytometry and Western blotting, and activation of caspase cascade was measured using a colourimetric caspase substrate set. The effects of caspase inhibitors were detected by flow cytometry. Measles virus was able to infect monocytes, but interestingly induced apoptosis in uninfected T cells, indicating that induction of apoptosis in T cells is mediated by MV-infected adherent cells. Only 1% of T cells contained MV antigen day 3 p.i. Interestingly the percentage of early apoptotic T cells at the same time was 35%, showing that apoptosis was not the result of MV infection in T cells. Measles virus-induced Fas but not FasL or TNFR1 expression on PMBC, as well as activation of granzyme B and caspase cascade. Simultaneously, overexpression of Bcl-2 protein was detected. Caspase inhibitor decreased the amount of apoptotic T cells. Measles virus-infected monocytes induce apoptosis in uninfected T cells, suggesting that infected monocytes probably interact via cell-surface molecules with uninfected T cells and induce apoptosis by indirect mechanisms. Apoptosis of the lymphocytes may contribute to the pathogenesis of MV-induced immunosuppression and cell loss.

CD1d-restricted natural killer T cells are potent targets for human immunodeficiency virus infection.

Invariant human natural killer T cells (NKT) express a restricted T-cell receptor (TCR) Valpha24Vbeta11 repertoire. These cells share both phenotypic and functional similarities between NK and T cells. Given the emerging role of NKT cells as critical cells in bridging the gap between innate and adaptive immunity, we examined their susceptibility to productive human immunodeficiency virus (HIV) infection by T-tropic, M-tropic, and primary isolates of HIV. We generated three human NKT cell clones (CA5, CA29, and CA31). Phenotypic characterization of these Valpha24+ Vbeta11+ clones indicated that they were predominately positive for CD4, CD161, HLA-DR, CD38, CD45RO, and CD95 expression. The NKT cell clones expressed significantly more surface CCR5 molecules/cell and lower CXCR4 molecules/cell than phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMC). Consistent with the surface expression of CCR5 and CXCR4, the NKT clones were also selectively susceptible to HIV M-tropic, T-tropic, and primary isolate infection, as evaluated by both HIV p24 enzyme-linked immunosorbent assay and intracellular staining of HIV proteins. The amount of p24 production was dependent on the NKT clone studied and the HIV strain used. Clones CA29 and CA31 were also susceptible to HIV IIIB infection. The virions produced by these clones were able to productively infect PHA-stimulated PBMCs with the same kinetics as for primary infection of CD4+ blast. Collectively, this data demonstrates that NKT cells can be a target for productive HIV infection but with a lag in the time to peak p24 production.

Inhibition of cytokine production by the traditional oriental medicine, ‘Gamcho-Sasim-Tang’ in mitogen-stimulated peripheral blood mononuclear cells from Adamantiades-Behçet's patients

Gamcho-Sasim-Tang (GS-Tang) is a traditional Chinese medication, which has been successfully used in Korea for the treatment of Adamantiades-Behçet's disease (ABD). We investigated the modulation effects of GS-Tang on cytokine production from phytohaemagglutinin-stimulated peripheral blood mononuclear cells of Behçet's patients. ABD is a systemic inflammatory disorder and might involve immune dysfunction. Cytokines involved in the regulation of inflammatory reactions and immune responses may play a role in the pathogenesis of ABD. GS-Tang (1 mg/ml) significantly inhibited the production of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), compared to absence of GS-Tang (by 42.0±6.6% inhibition for TNF-α and 95.9±5.7% for IL-1β, P<0.05). GS-Tang also inhibited the production of IFN-γ, immunoregulatory T helper cell type 1 cytokine, by 80.2±5.3% ( P=0.001). The inhibitory effects of GS-Tang on cytokine production showed dose-dependent manner. Our results suggest that GS-Tang might have anti-inflammatory and immuno-regulatory effects through the cytokine modulation.

Cytokine production by blood mononuclear cells from in-patients with anorexia nervosa.

Although protein-energy malnutrition is a common cause of immunodeficiency, the immune function in underweight anorexia nervosa (AN) patients usually seems to be better preserved than would be expected. However, a deranged cytokine production and its consequences are currently being investigated in these patients. This study was aimed at measuring, over time, the capacity of peripheral blood mononuclear cells (PBMC) from AN in-patients to produce several cytokines involved in the regulation of immune responses. The in vitro production of interferon (IFN)-gamma, interleukin (IL)-2, tumour necrosis factor (TNF)-alpha, IL-6 and IL-1 beta by phytohaemagglutinin-stimulated PBMC were assessed on forty female adolescents with AN. These measures were carried out twice, upon hospital admission and at discharge, which occurred on average after 1 month. Thirty-five control subjects were also studied. Cytokines were measured by ELISA kits. The production of TNF-alpha and IL-6 was lower and production of IL-1 beta higher in AN patients than in the control group at both time points of assessment. Refeeding for 1 month was not enough time to reverse these differences and patients still had a low body weight at discharge. IFN-gamma production was lower in the patients than in control subjects only at discharge and no differences were found in IL-2 production between both groups. The results suggest that a mechanism involving modifications in the secretion pattern of proinflammatory cytokines could explain some immune function findings in underweight AN patients.

Cytokine production by blood mononuclear cells from in-patients with anorexia nervosa

Although protein–energy malnutrition is a common cause of immunodeficiency, the immune function in underweight anorexia nervosa (AN) patients usually seems to be better preserved than would be expected. However, a deranged cytokine production and its consequences are currently being investigated in these patients. This study was aimed at measuring, over time, the capacity of peripheral blood mononuclear cells (PBMC) from AN in-patients to produce several cytokines involved in the regulation of immune responses. Thein vitroproduction of interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α, IL-6 and IL-1β by phytohaemagglutinin-stimulated PBMC were assessed on forty female adolescents with AN. These measures were carried out twice, upon hospital admission and at discharge, which occurred on average after 1 month. Thirty-five control subjects were also studied. Cytokines were measured by ELISA kits. The production of TNF-α and IL-6 was lower and production of IL-1β higher in AN patients than in the control group at both time points of assessment. Refeeding for 1 month was not enough time to reverse these differences and patients still had a low body weight at discharge. IFN-γ production was lower in the patients than in control subjects only at discharge and no differences were found in IL-2 production between both groups. The results suggest that a mechanism involving modifications in the secretion pattern of proinflammatory cytokines could explain some immune function findings in underweight AN patients.

Cytokine profile of patients with mycosis fungoides and the immunomodulatory effect of AS101.

Cytokines are known to play a major role in the pathogenesis of mycosis fungoides, a cutaneous malignant neoplasm of CD 4 T cells. In the present study, we investigated the effect of AS101, a tellurium-based compound with immunomodulating properties, on the pattern of lymphokine production by peripheral blood mononuclear cells (PBMCs) from patients with mycosis fungoides. PBMCs were isolated from 35 patients with mycosis fungoides stage IA and IB before initiation of treatment and from 20 healthy sex and age-matched controls. Unstimulated and phytohaemagglutinin-stimulated PBMCs were tested with and without the addition of AS101. The production of interferon-gamma, interleukin 2 (IL-2), IL-2 receptor (IL-2R), interleukin 5 (IL-5) and interleukin 10 (IL-10) was determined by enzyme-linked immunosorbent assays. The effects of AS-101 on mycosis fungoides PBMCs were compared to those of healthy donor PBMCs. Significantly higher levels of IL-2R, IL-5 and IL-10 and significantly lower levels of interferon-gamma were found in the patients compared to the controls. There was no significant difference between the groups in the production of IL-2. AS101 inhibited the production of IL-2R, IL-5 and IL-10 and induced a significant increase in IL-2 levels in the mycosis fungoides PBMCs. These findings may have important clinical implications for the possible therapeutic benefit of AS101 in mycosis fungoides.

Alteration in the responsiveness to tumour necrosis factor-alpha is crucial for maximal expression of monocyte chemoattractant protein-1 in human neutrophils.

We previously reported delayed expression of monocyte chemoattractant protein-1 (MCP-1) in human neutrophils cultured with a cytokine-rich crude supernatant of phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PHA-sup). Tumour necrosis factor-alpha (TNF-alpha) contained in the PHA-sup played a key role in this event, but there appeared to be another factor(s) in the same supernatant that co-operated with TNF-alpha for maximal MCP-1 expression. In the present study, we reduced TNF-alpha concentrations in the PHA-sup to minimal levels using anti-TNF-alpha affinity columns (TNF-depleted-sup) and investigated the co-operation between TNF-alpha and TNF-depleted-sup. Nine hours of preincubation with TNF-depleted-sup altered the responsiveness of neutrophils to TNF-alpha and enabled TNF-alpha to increase the level of MCP-1 expression to a maximal level within 4 hr. The priming effect was not due to the increased expression of cell-surface TNF receptors. However, the activation of primed cells by TNF-alpha was clearly through TNF receptor-p55. Finally, the activity in the TNF-depleted-sup that co-operated with TNF-alpha was eluted at 60 000 MW on high-performance liquid chromatography-gel filtration. Thus, delayed neutrophil expression of MCP-1 is regulated by a cytokine-dependent mechanism that induces neutrophils to enter a 'mature' stage.

Infectious human herpesvirus 8 in a healthy North American blood donor

Molecular studies have provided strong evidence for the association of human herpesvirus 8 (HHV-8) with Kaposi's sarcoma. These data have been supported by serological studies, which have also suggested that HHV-8 can be found in the healthy population. We report the presence of infectious HHV-8 in a healthy donor to a North American blood bank. We examined the peripheral blood mononuclear cells or CD19 cells of blood donors by PCR for evidence of HHV-8 infection. The CD19 cells were separated from peripheral blood mononuclear cells by immunomagnetic-bead selection. To enhance detection of HHV-8, the CD19 cells from eleven unsystematically selected blood donors were activated with phorbol ester and recombinant interleukin-6; the culture fluid was filtered and inoculated onto HHV-8-negative target CD19 cells that had been prepared from phytohaemagglutinin-stimulated peripheral blood mononuclear cells. These inoculated target cells were cultured for 3 days and then analysed for HHV-8 sequences by PCR. Serum samples were tested for antibodies to HHV-8 by an indirect immunofluorescence assay. One blood donor was consistently found to be infected with HHV-8 by PCR after the cell-culture activation procedure. He was seropositive for the virus. The HHV-8 recovered was infectious, as shown by a reverse-transcription-PCR technique that detected HHV-8 RNA in the inoculated target cells. These data provide the first indication that HHV-8 can be recovered from the blood of a healthy individual, a blood donor, and that the virus is infectious. This observation suggests that HHV-8 could be transmitted by blood transfusion, a possibility that merits further study.

A potent human interleukin-4 antagonist stimulates the proliferation of murine cells expressing the human interleukin-4 binding chain.

A single-amino-acid substitution mutant form of human interleukin-4 (hIL-4), Y124D.hIL-4, has been described previously as an antagonist of the effects of hIL-4 on various human cells. The murine T-cell leukemic cell line CT.h4S, which expresses the human IL-4 receptor, proliferates in response to both hIL-4 and murine IL-4. Although Y124D.hIL-4 antagonizes the proliferative effects of hIL-4 on human phytohaemagglutinin-stimulated peripheral blood mononuclear cells, Y124D.hIL-4 is a potent stimulator for CT.h4S cells. Molecular modelling studies were performed to investigate the stability of different conformations of residue 124 as well as the efficiency of different molecular mechanics force fields in homology modelling. We suggest that the aspartate substitution alters the C-terminal end of the D-helix in such way that the analogue still binds to the human IL-4 receptor alpha-chain and signals through the murine gamma c-chain. In contrast, the Y124D.hIL-4/IL-4 receptor complex cannot signal through the human gamma c-chain.

Decreased production of soluble interleukin 2 receptor by phytohaemagglutinin-stimulated peripheral blood mononuclear cells in patients with breast cancer after adjuvant therapy

Decreased production of soluble interleukin 2 receptor by phytohaemagglutinin-stimulated peripheral blood mononuclear cells in patients with breast cancer after adjuvant therapy

Immune induction of major histocompatibility complex antigens on a human thyroid cell line (SGHTL-34)

Expression of major histocompatibility complex antigens by epithelial cells may play a role in the aetiology of autoimmune disorders. We have studied the effect of gamma-interferon on SGHTL-34, a human thyroid cell line which constitutively expresses class I but not class II antigens. gamma-Interferon induced the expression of class II and increased the expression of class I molecules (assessed by flow cytofluorimetry) in a dose-dependent manner. Thyrotrophin or phytohaemagglutinin had no effect on either class I or class II expression. However, a supernatant from phytohaemagglutinin-stimulated peripheral blood mononuclear cells, containing 6400 U gamma-interferon/ml, was an effective inducer of both class I and class II antigens. These data clarify earlier studies using primary thyroid cultures, which are contaminated with cells of the immune system.

Increased detection of human immunodeficiency virus antigen carriers by simultaneous assay of plasma and extracts from resting and phytohaemagglutinin-stimulated peripheral blood mononuclear cells

Antigenaemia due to human immunodeficiency virus (HIV) is thought to be significant either before the appearance of a specific antibody response, or after its decline during terminal stages. In order to increase the rate of detection of HIV antigen carriers, regardless of the stage or despite the presence of specific serum antibodies, we assayed, simultaneously, plasma samples and extracts from resting and phytohaemagglutinin-stimulated mononuclear cells from 25 infected, antibody-positive individuals and 10 healthy, antibody-negative female volunteer blood donors. We detected the presence of HIV antigen in at least one of the three types of specimens obtained from all the 25 infected subjects but in none of the 10 healthy blood donors. This approach might prove most useful for the study of patients with controversial or equivocal antibody test results.

Lymphocyte activation by HIV-1 envelope glycoprotein.

Cell activation by phytohaemagglutinin, phorbol ester and by the supernatant of phytohaemagglutinin-stimulated peripheral blood mononuclear cells induces the expression and cytopathic effects of latent human immunodeficiency virus type-1 (HIV-1) in vitro. The lymphocyte surface protein CD4 has been identified as a receptor for HIV-1 and binds the viral envelope glycoprotein (gp120). In the light of evidence indicating that one natural function of CD4 is as a growth factor receptor, we examined the ability of native gp120 to activate resting CD4-bearing lymphocytes. Our results indicate that gp120 has innate biological activity as a result of a specific interaction with CD4, inducing increases in intracellular levels of inositol trisphosphate and of calcium, and in interleukin-2 receptor expression and cell motility.