Absence Of Phytohemagglutinin Research Articles

Potential of Lactobacillus acidophilus to modulate cytokine production by peripheral blood monocytes in patients with endometriosis.

Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. Peripheral blood monocytes cells (PBMCs) may have altered function to some extent in women with endometriosis. Lactobacillus acidophilus is a probiotic bacterium within the human body with the ability of alleviating many inflammatory diseases. Here, we examined the effect of L. acidophilus on PBMCs of endometriosis patients. In this study, peripheral blood samples were obtained from endometriosis patients (n=11) and non-endometriosis individuals (n=11). After isolation of peripheral blood mononuclear cells with Ficoll, cells were cultured in the presence and absence of phytohemagglutinin. Also, these cells were co-cultured with 1×106 CFU/ml of L. acidophilus. IL-6 and IL-1 cytokines were measured by ELISA method and the two groups were evaluated and compared. The results showed that in endometriosis patients, the production of pro-inflammatory cytokines, including IL-1 and IL-6, by PBMC was increased compared to non-endometriosis subjects, and stimuli such as PHA intensified this elevation. Also, L. acidophilus increased the levels of pro-inflammatory cytokines including IL-1 and IL-6. However, the production of these cytokines decreased due to the modulatory properties of bacterial cells after 48 h. According to the results of the current study, IL-1 and IL-6 production was significantly increased in PMBCs of endometriosis patients compared to that of the healthy controls. Also, Lactobacillus acidophilus was considered as an antigenic compound and induced IL-1 and IL-6 production. According to these results, probiotics can be further used for the treatment of endometriosis patients and more investigations are needed to confirm these results.

A streamlined proliferation assay using mixed lymphocytes for evaluation of human mesenchymal stem cell immunomodulation activity

BackgroundMesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival. MethodsPBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 107/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50–60,000 cells/well and cultured in 96 well plates for 4–48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0–5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader. ResultsPBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased <20% at the highest PHA concentrations. Co-culture with MSCs suppressed PBMC proliferation dependent upon MSC passage number, source, and prior growth conditions. Total time to complete the ATP assay was under an hour including incubations. With minimal manipulations in the assay, intra- and inter- assay variations averaged 11.1 and 15.7% respectively. ConclusionsDirect co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.

Cytokine Regulation from Human Peripheral Blood Leukocytes Cultured In Vitro with Silver Doped Bioactive Glasses Microparticles.

Bioactive glasses (BG) applications include tissue engineering for bone regeneration, coating for implants, and scaffolds for wound healing. BG can be conjugated to ions like silver, which might add some antimicrobial properties to this biomaterial. The immunomodulatory activity of ion-doped bioactive glasses particles was not investigated before. The aim of this work was to evaluate the cytotoxic and immunomodulatory effect of BG and silver-doped bioactive glass (BGAg) in human peripheral blood cells. BG and BGAg samples belonging to the system 58SiO2•(36-x)CaO·6P2O5·xAg2O, where x = 0 and 1 mol%, respectively, were synthesized via sol–gel method and characterized. Cytotoxicity, modulation of cytokine production (TNF-α, IL-1β, IL-6, IL-4, and IL-10), and oxidative stress response were investigated in human polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) cultures. Cell viability in the presence of BG or BGAg was concentration-dependent. In addition, BGAg presented higher PBMCs toxicity (LC50 = 0.005%) when compared to BG (LC50 = 0.106%). Interestingly, interleukin4 was produced by PBMCs in response to BG and BGAg in absence of phytohemagglutinin (PHA) and did not modulate PHA-induced cytokine levels. Subtoxic concentrations (0.031% for BG and 0.0008% for BGAg) did not change other cytokines in PBMCs nor reactive oxygen species (ROS) production by PMN. However, BG and BGAg particles decreased zymosan-induced ROS levels in PMN. Although ion incorporation increased BG cytotoxicity, the bioactive glass particles demonstrated a in vitro anti-inflammatory potencial. Future studies are needed to clarify the scavenger potential of the BG/BGAg particles/scaffolds as well as elucidate the effect of the anti-inflammatory potential in modulating tissue growth in vivo.

Evaluation of the anti-oxidant and anti-inflammatory effects of the methanolic extract of Ferula szowitsiana root on PHA-induced inflammation in human lymphocytes

Inflammation is defined as a defensive response of the body against either the endogenous or exogenous triggers, while this process becomes chronic leading to various disorders such as asthma, cancers, and multiple sclerosis. Recently, pharmacological properties of different constituents of F. szowitsiana have been reported. In the present study, we aimed to evaluate the anti-oxidative and anti-inflammatory effects of the methanolic extract of F. szowitsiana root on human isolated lymphocytes. The effects of either F. szowitsiana (10, 40 and 160 μg/ml) or dexamethasone (0.1 mM) were evaluated on the levels of cell proliferation, reactive oxygen species (ROS), nitric oxide (NO) production, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities, and total glutathione content (GSH) as well as the secretion of inflammatory cytokines [interleukin 6 (IL-6) and tumor necrosis factor (TNF)-α] in the presence or absence of phytohemagglutinin (PHA) stimulation (n = 8 for each group). PHA stimulation notably elevated ROS, NO, MDA, IL-6, and TNF-α levels as well as diminished GSH, CAT and SOD levels. In PHA-stimulated, the results also revealed that F. szowitsiana (10–160 µg/ml) significantly decreased MDA, ROS, NO, IL-6 and TNF-α levels as well as increased CAT, SOD and GSH levels. Collectively, F. szowitsiana is able to attenuate the overproduction of inflammatory and oxidative stress markers in the presence of PHA-stimulated T lymphocytes, while to propagate the anti-oxidative defense. Contextually, the plant has promising healing effects in the different inflammatory disorders associated with the interference of the acquired immune system such as multiple sclerosis and asthma.

Correlation between IL-17A/F, IL-23, IL-35 and IL-12/-23 (p40) levels in peripheral blood lymphocyte cultures and disease activity in Behcet's patients.

Behcet's disease is a chronic multisystemic disease with remissions and relapses. Several studies have shown that immune mechanisms play an important role in the development of the disease. In order to assess the association of disease activity with IL-17A/F, IL-23, IL-12/23 (p40) and IL-35 expression, we aimed to investigate production of these cytokines in peripheral blood mononuclear cells (PBMCs) from Behcet's patients and normal controls. Furthermore, we included Systemic Lupus Erythematosus (SLE) as disease control to evaluate the specificity of our data for immunopathogenesis of BD. Totally 15 active, 15 inactive Behcet's patients, 12 active and 12 inactive SLE patients and 12 healthy volunteers were enrolled in the study. Peripheral blood mononuclear cells were separated, lymphocyte cultures were performed and IL-17A/F, IL-12/23 p(40), IL-23, IL-35 cytokine levels were measured by ELISA in culture supernatants in the presence or absence of phytohemagglutinin (PHA) on time-dependent manner. IL-17 A/F levels increased parallel to IL-23 levels in Behcet's and SLE patients. Compared to healthy controls, IL-17 A/F levels were higher in active Behcet's and SLE patients; on the contrary, levels of IL-35 were lower. IL-17A/F, IL-12/23 (p40) and IL-23 levels were detectable most frequently in active Behcet's patients followed by active SLE patients. Our results indicate that IL-17 A/F, IL-23 and IL-12/23 (p40) may play role in the immunopathogenesis of BD so as Th17 and Th1 cell responses. Since IL-35 levels were lower in active Behcet's patients compared to inactive patients and healthy controls, there may be a plasticity between Th17 and Treg cells according to the state of disease activity.

Medicinal plant extracts modulate respiratory burst and proliferation activity of rainbow trout (Oncorhynchus mykiss) leukocytes.

The present study was designed to investigate the immunomodulatory effects of Aloe vera, Curcuma longa, Echinacea purpurea, Lavandula officinalis, Origanum vulgare, Panax ginseng, and Rheum officinale extracts on leukocytes purified from rainbow trout (Oncorhynchus mykiss) head kidney. The cells were cultured in a medium containing increasing doses of extracts; afterwards, they were tested for reactive oxygen species production after stimulation with phorbol myristate acetate (PMA) and proliferation in the presence or absence of phytohemagglutinin from Phaseolus vulgaris (PHA-P). After a 2-h exposure, the extracts of L. officinalis, O. vulgare, and R. officinale strongly reduced the oxidative burst activity of PMA-stimulated leukocytes, in a dose-dependent manner (P≤0.05). A. vera, C. longa, E. purpurea, and P. ginseng extracts reduced this response with lower efficacy and especially at lower concentrations. On the contrary, the highest concentration of ginseng extract stimulated the respiratory burst of leukocytes compared to untreated control cells. After a 72-h exposure, the extracts of L. officinalis, R. officinale, C. longa, E. purpurea, and P. ginseng had a clear dose-dependent stimulatory effect on leukocyte proliferation (P≤0.05). The results suggest that these medicinal plants can be considered as reliable sources of new antioxidants or immunostimulants to be used in aquaculture.

The effect of dimethyl fumarate on gene expression and the level of cytokines related to different T helper cell subsets in peripheral blood mononuclear cells of patients with psoriasis.

Fumaric acid esters such as dimethyl fumarate (DMF) have proven to be effective in the treatment of psoriasis. In view of the role of Th17 in the pathogenesis of psoriasis, the present study was conducted to investigate the effects of DMF on Th1, Th2, and Th17 responses in patients. Peripheral blood mononuclear cells (PBMCs) were isolated from psoriasis patients and healthy individuals and were cultured in the presence or absence of phytohemagglutinin and DMF. The cell supernatants were removed to measure cytokine secretion, and the lymphocytes were used for real-time polymerase chain reaction to establish gene expression. An increase in gene expression of interferon-γ (IFN-γ), as a marker for Th1 activity, and interleukin-17 (IL-17), granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-22 representing the Th17 subset in the PBMCs of patients in comparison with those of control subjects was observed. Culture of PBMCs from psoriasis patients and controls in the presence of DMF decreased IFN-γ and increased IL-4 gene expression in both groups. Treatment with DMF could significantly decrease IL-17, GM-CSF, and IL-22 mRNA levels in the PBMCs of patients. Decreased release of IFN-γ and GM-CSF cytokine secretion after DMF treatment was also observed in PBMC cultures of patients and controls. These data show the effectiveness of DMF in modulating Th17 cells in addition to Th1/Th2 cells and reflect one of the underlying mechanisms of action of DMF in psoriasis. These findings may also support the possible benefits of using fumarate in the treatment of other autoimmune diseases in the pathogeneses of which Th1 and Th17 cells play major roles.

Chemical and Biological Activity of Leaf Extracts of Chromolaena Leivensis

The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90% viability at a concentration of 100 microg/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 microg/mL showed toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was produced by 3,5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells treated with 50 microg/mL of 3,5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 microg/mL, at time intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of 25 microg/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 microg/mL slightly increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids at concentrations of 50 and 100 microg/mL slightly increased the proliferation of fibroblasts.

Differential but beneficial effect of phytohemagglutinin on efficiency of in vitro porcine embryo production by somatic cell nuclear transfer or in vitro fertilization

In vitro production of porcine embryos has become routine in most laboratories but the yield and quality of the resultant blastocysts remains sub-optimal. Phytohemagglutinin (PHA) is an N-acetylgalactosamine/galactose sugar-specific lectin with a wide variety of biological activities including mitogenesis, mediation of cell recognition and agglutination of cells. This study was therefore, designed to investigate the effect of PHA on the preimplantation development and quality of parthenogenetic, somatic cell nuclear transferred (SCNT) and in vitro fertilized (IVF) porcine embryos cultured in the absence or presence of PHA. Analysis showed that, supplementation of PHA significantly improved the blastocyst rate of parthenogenetic (70.6 +/- 0.2 vs. 51.4 +/- 0.6%) and SCNT (27.7 +/- 1.7 vs. 12.5 +/- 0.3%) embryos but not IVF embryos (25.0 +/- 14.3 vs. 20.1 +/- 12.7%). Nonetheless, PHA-treated blastocysts had higher hatching ability and contained higher cell number than control blastocysts in all the groups (P < 0.05). TUNEL labeling revealed that blastocysts cultured in the presence of PHA were less predisposed to biochemical apoptosis and showed lower indices of TUNEL, fragmentation and total apoptosis than those cultured in the absence of PHA (P < 0.05). Real time qRT-PCR analysis of parthenogenetic blastocysts revealed that PHA decreased the expression ratio of BAX:BCL-xL transcripts. Therefore, our study suggests that PHA improves the blastocyst yield and quality by enhancing blastocyst expansion, hatching and total cell number and decreasing the apoptosis. However, PHA has a differential effect on development rate of IVF derived embryos. These results may represent an approach towards achieving better preimplantation development of porcine embryos in vitro.

Chicken lines differ in production of interferon-like activity by peripheral white blood cells stimulated with phytohemagglutinin

Interferon (INF) activity was evaluated in the supernatants from peripheral white blood cells (WBC) of chickens from six lines. The WBC were cultured in flasks or 24-well plates with medium or medium and phytohemagglutinin (PHA). After 2 to 5 d, duplicate supernatant samples were tested for INF activity, i.e., the log2 titer inhibiting 50% destruction of the cytopathic effect of vesicular stomatitis virus on primary chick embryo fibroblasts. Also triplicate WBC samples were tested for proliferation by [H3]-thymidine labeling and scintillation counting. In the absence of PHA, INF was significant for only two lines, i.e., 7(2) (two trials) and C (one trial). With PHA the level of INF produced was similar if flasks were sampled daily or on successive days. The INF levels were highest using 10 or 20 microg/mL PHA, but line differences were best distinguished using 5 or 10 microg/mL. In three trials there was a low correlation between PHA-stimulated WBC proliferation and INF titer (r > or = 0.30; P < 0.05). It was concluded that supernatants from chicken WBC stimulated with 10 microg/mL PHA contain INF, and inbred Lines 72 and C repeatedly produce more INF than inbred Lines 63 and 15I(5). This is the first evidence for line differences in INF production in chickens, and these lines may be useful for characterization of the relevant genes and their importance in immune response(s) and disease resistance.

Interleukin-6 production does not increase with age.

Investigators have reported an increase, decrease, or no effect of age on interleukin-6 (IL-6) production. Differences in experimental conditions and the health status of subjects may explain these contradicting results. Because the subjects used in most of the previous studies were not carefully screened for health, we investigated the effect of age on IL-6 production in healthy young and elderly subjects. Twenty young (aged 20-30 years) and 26 elderly (>65 years) men completed the study. Each subject was screened for good health, undergoing physical examinations and laboratory tests. Circulating IL-6 levels were not significantly different between young and elderly subjects. A subgroup of subjects representing both young and elderly volunteers had high (>1000 pg/ml) circulating levels of IL-6. However, circulating IL-6 levels were low (<100 pg/ml) in the majority of subjects in both age groups. Peripheral blood mononuclear cells (PBMC) were cultured for IL-6 production in the presence or absence of phytohemagglutinin (PHA) or concanavalin (Con)A for 48 hours. Unstimulated secretion of IL-6 by PBMC cultured in autologous plasma (AP) or fetal bovine serum (FBS) was detectable in the majority of cultures. Age did not influence this spontaneous secretion of IL-6. PBMC stimulation with PHA or ConA significantly increased IL-6 production, but age did not affect the ability of PBMC to secrete IL-6 after stimulation when cultured in FBS. IL-6 production by PBMC cultured in AP and stimulated with PHA was not affected by age. However, when stimulated with ConA, PBMC from the elderly subjects produced less IL-6 than PBMC from the young subjects. Because IL-6 has been suggested to contribute to the age-related increase in prostaglandin (PG)E2 and nitric oxide (NO) production, we investigated the effect of age on the production of IL-6 by murine peritoneal macrophages (Mphi) as well as the effect of IL-6 on the production of other Mphi inflammatory products. Similar to the findings in humans, mouse age did not influence the level of IL-6 produced by Mphi. These data suggest that in healthy subjects, increased production of IL-6 is not a normal consequence of aging. Previously reported higher IL-6 levels in elderly subjects might reflect an underlying, undiagnosed disease state. PGE2 and NO production were not affected by the addition of IL-6 to Mphi from young mice or anti-IL-6 antibody to Mphi from old mice. Thus, IL-6 does not appear to influence the Mphi production of selected inflammatory molecules.

The effect of cocoa procyanidins on the transcription and secretion of interleukin 1β in peripheral blood mononuclear cells

Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1β. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1β gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1β expression of PHA-stimulated cells by 1–15%, while the larger oligomers (pentamer-decamer) increased expression by 4–52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

The Appearance of the CD4CD8 Phenotype on Activated T Cells:: Possible Role of Antigen Transfer

Stimulation of peripheral blood lymphocytes (PBL) with phytohemagglutinin (PHA) strikingly increased the proportion of CD4+CD8+ cells. Highly purified CD4+ and CD8+ lymphocyte populations cultured in the presence of PHA consistently failed to coexpress the CD8 and CD4 markers. Similarly, exposure of highly purified CD4+ cells to PHA and recombinant interleukin-2 resulted in augmented expression of CD25 but failed to induce the expression of CD8. When purified preparations of either CD4+ or CD8+ cells were activated separately for 3 days and incubated together for an additional 5 h, a considerable proportion of CD4+CD8+ cells was found in the mixture. Cycloheximide treatment did not prevent the appearance of the CD8 marker on CD4 cells. CD4+CD8+ cells isolated from PBL exposed for 3 days to PHA lost their CD8 antigenicity within 24–48 h in the absence of PHA. Increased levels of soluble CD4 and CD8 antigens were found in supernatant fluids of PHA-stimulated cells. T cells failed, however, to bind soluble markers even after prolonged incubation in the presence of supernatant fluids. Our studies show that activation of CD4+ cells per se does not elicit the CD4+CD8+ phenotype and that soluble T cell markers do not bind to T cells. Rather, it seems that direct cell–cell contact is required for the transfer of CD8 molecules from CD8+ cells to the membrane of CD4+ cells.

Cytokine production by adherent and non-adherent mononuclear cells in chronic fatigue syndrome

It has been suggested that cytokines play a role in certain clinical manifestations of chronic fatigue syndrome (CFS). In this study adherent (monocytes) and non-adherent (lymphocytes) mononuclear cells were stimulated in the presence or absence of phytohemagglutinin (PHA) or lipopolysaccharide (LPS), respectively, and supernatants were assayed for IL-6, TNF-α, and IL-10 by ELISA. IL-6 was also measured at the mRNA level by polymerase chain reaction. The levels of spontaneously (unstimulated) produced TNF-α by non-adherent lymphocytes and spontaneously produced IL-6 by both adherent monocytes and non-adherent lymphocytes were significantly increased as compared to simultaneously studied matched controls. The abnormality of IL-6 was also observed at mRNA level. In contrast, spontaneously produced IL-10 by both adherent and non-adherent cells and by PHA-activated non-adherent cells were decreased. This preliminary study suggests that an aberrant production of cytokines in CFS may play a role in the pathogenesis and in some of the clinical manifestations of CFS.

Characterization of rat lymphocyte primary culture for the development of an in-vitro mutagenesis assay: effect of interleukin-2 and 2-mercaptoethanol on the activities of intermediary metabolism enzymes and cell proliferation.

Efficient energy utilization is essential for cell growth; in an attempt to improve the growth conditions of the rat T-lymphocyte culture model for potential use in studying the mutagenic activity of carcinogens in vitro, we have investigated the effects of phytohemagglutinin (PHA), interleukin-2 (IL-2) and 2-mercaptoethanol (2-ME) on the activities of intermediary metabolism enzymes and cell proliferation. Isolated lymphocytes were cultured in the presence and absence of PHA, IL-2, or 2-ME. The intermediary metabolism enzymes investigated were glutamate dehydrogenase, glutamate-pyruvate transaminase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, pyruvate kinase, and fatty acid synthetase (FAS). Measurable activity of all enzymes investigated, except for FAS, was detected in PHA-stimulated cells cultured with IL-2 or 2-ME. The unstimulated lymphocytes had significantly lower enzyme activity than stimulated cells. The combination of all three agents showed increased enzyme activity. This increase in activity brought about by the combination of the three agents was not reproduced by either agent acting alone. In general, the increase in enzyme activity correlated with cell proliferation as measured by [3H]thymidine uptake in PHA-stimulated cultures containing IL-2 and/or 2-ME. The results suggest that the addition of exogenous IL-2 and 2-ME enhances metabolic function and may be beneficial in in vitro culture of rat lymphocytes.

Soluble class I antigen secretion by peripheral blood lymphocytes in multiple sclerosis.

We have quantified the soluble class I antigen (sHLA) secretion by peripheral blood lymphocytes of 26 multiple sclerosis (MS) patients. Thirteen of them were in a stable phase of the disease, 6 on relapse and 7 suffered from a progressive MS. sHLA secretion was reduced in the presence or absence of phytohemagglutinin in patients with either active or stable MS, being normal after stimulation with a monoclonal antibody anti-CD3. In MS patients, lymphocyte proliferation and immunoglobulin secretion were found to be similar to those of 29 healthy blood donors who comprised the group of controls. These results reflect systemic anomalies in the cell activation process in MS, which seem to be independent of the disease activity. Whether these alterations are specific to MS or are common to other inflammatory CNS diseases remains to be elucidated.

Evolution of a CD3+CD4+ alpha/beta T-cell receptor+ mature T-cell clone from CD3-CD7+ sorted human bone marrow cells.

In order to study extrathymic differentiation in vitro, CD7+CD3- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCRδ1 (TCRγ/δ-specific) or WT31 (TCR2, α/β-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3+, CD4+, CD8-, TCR2+, TCR1-, and was further characterized as CD2+, CD45RO+, CD16-, and CD56-. The presence of mRNA for TCR α and γ but not ,and γ chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted “natural killer (NK)-like” lysis of K562 target cells, with no autocytotoxicity detected. Tle NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor α and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-δ, and GM-CSF in these cells after stimulation with PHA and B-LCL. These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.

Immunostimulatory factor(s) in the porcine uterine fluids

The effects of porcine uterine fluid filtrate (PUF) on lymphocyte activity were examined. PUF was added to porcine lymphocyte cultures in the presence or absence of phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). Lymphocyte response was determined by 3H-thymidine incorporation. Results indicated that PUF enhanced PHA-, Con A-, and PWM-induced lymphocyte mitogenesis by 222%, 207%, and 261%, respectively, as compared to control. The stimulatory effect of PUF was unlikely to be due to the presence of interleukin-2 in PUF, by evidence that PUF alone did not support cytotoxic T-lymphocyte proliferation, nor was the stimulatory effect due to microbial contamination, because treatment of PUF through PyroBind columns to remove endotoxin did not affect its activity. Incubation of PUF at 56C for 40 min did not alter the enhancement effect. Treatment of PUF with trypsin or pronase for up to 24 hr did not change its activity. In mixed lymphocytes reaction, PUF augmented 3H-thymidine uptake by 242%. The molecular (mol) mass of the immunostimulatory activity in PUF was examined by size-exclusion HPLC, Sephadex G-50, and G-15 gel filtration, and it was found to be 0.8 kDa and < 0.5 kDa. Extracts of porcine endometrium also enhanced PHA-induced lymphocyte mitogenesis, suggesting that the factor(s) may be of uterine origin. In conclusion, we have identified an factor(s) in PUF capable of stimulating lymphocyte mitogenesis and mixed lymphocyte reaction.

Inorganic arsenic effects on human lymphocyte stimulation and proliferation

Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10 −7, 10 −8 and 10 −9 M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10 −9 M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and poliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.

ＡＴＬ，ＨＴＬＶ‐Ｉキャリアにおける末梢血単核球培養上清中ＩＬ‐２濃度およびＩＬ‐２・レセプター発現

It has been reported that human T-cell leukemia virus type I (HTLV-I) is closely associated with the development of adult T-cell leukemia (ATL). Furthermore, it has become to be known that the interleukin-2 (IL-2)/interleukin-2 receptor (IL-2R) system in ATL plays an important role in HTLV-I-related leukemogenesis.In the study, IL-2 production and IL-2R expression of mononuclear cells (MNCs) in the blood of patients with HTLV-I-related disease were examined by cultivating the MNCs in the presence or absence of phytohemagglutinin (PHA). The sandwich radioimmunoassay method was used for quantitation of IL-2 in the supernatant of the medium, and the direct immunofluorescence method was employed for the detection of IL-2R.Spontaneous IL-2 production from MNCs was seen in 3 of 8 patients with acute and lymphoma type of ATL, in 6 of 9 with chronic type of ATL, in 6 of 10 with smouldering type of ATL, and in 20 of 32 HTLV-I carriers. However, IL-2 production was not induced by PHA stimulation in 7 of 8 patients with acute and lymphoma type of ATL, in 7 of 9 with chronic type of ATL, in 3 of 10 with smouldering type of ATL, and in 10 of 32 HTLV-I carriers.IL-2R expression on MNCs from these patients with HTLV-I-related disease increased by the cultivation. However, the rate of increase of the IL-2R expression in the PHA-stimulated group (IL-2R expression after culture/that of fresh cells) decreased in association with the progression of HTLV-I-related disease.These data revealed that MNCs from not only patients with ATL but also from some HTLV-I carriers showed abnormal IL-2 production and IL-2R expression. In other words, the IL-2/IL-2R system abonormality present in ATL also exists in some HTLV-I carriers. Further study of HTLV-I carriers with this IL-2/IL-2R abnormality is necessary to predict whether ATL develops in the patients.