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Immunology Jun 1980Human peripheral blood mononuclear cells were stimulated with pokeweed mitogen (PWM) to study the role of mononuclear phagocytic cells (MNP) in lymphocyte proliferation....
Human peripheral blood mononuclear cells were stimulated with pokeweed mitogen (PWM) to study the role of mononuclear phagocytic cells (MNP) in lymphocyte proliferation. MNP were identified by cytoplasmic alpha-naphthyl acetate esterase, by the capacity of phagocytosis and by lysozyme synthesis. It appeared that after 3 days of stimulation with PWM all MNP disappeared from the cultures and remained absent during prolonged culture in mitogen-free medium. In non-stimulated cultures MNP remained. The disappearance of MNP from cell cultures was caused by a lymphocyte-derived factor, which was transferable by cell-free supernatants of stimulated mononuclear cells. From experiments in which cultures were treated with different concentations of PWM and from pre-culture experiments, it could be shown that in vitro lymphocyte proliferation required both non-stimulated lymphocytes and freshly prepared MNP. In addition, the decreasing concentration of PWM as stimulating agent, resulted in a decreasing proliferation of lymphocytes, which was inversely proportional to the presence of MNP.
Topics: Cell Division; Cells, Cultured; Humans; Lymphocyte Activation; Muramidase; Naphthol AS D Esterase; Phagocytes; Phagocytosis; Pokeweed Mitogens; Time Factors
PubMed: 7409856
DOI: No ID Found -
Clinical and Experimental Immunology Jan 1977Lymphocytes from twenty-five patients with atopic dermatitis were investigated for their in vitro reactivity to stimulation with tuberculin (PPD), lipopolysaccharide...
Lymphocytes from twenty-five patients with atopic dermatitis were investigated for their in vitro reactivity to stimulation with tuberculin (PPD), lipopolysaccharide (LPS), phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). The response to a low dose of Con A was increased, and the reactivity in unstimulated cultures tended to be lower than similar cultures from the control group. Addition of inactivated autologous plasma to the cultures had an inhibitory effect, when the plasma came from patients with high levels of IgE. The patients' in vitro reactivity to PPD in a leucocyte migration test was equal to that found in normal persons and no effect was observed after addition of autologous serum. The mean percentage of E rosette forming cells was significantly reduced in patients with high levels of IgE. The number of EAC rosette forming cells was within normal range. It is hypothesized that the observations could reflect the existence of suppressor mechanisms in patients where the immune system is strongly stimulated.
Topics: Adolescent; Adult; Chemotaxis, Leukocyte; Child; Dermatitis, Atopic; Humans; Immunoglobulin E; In Vitro Techniques; Leukocyte Count; Lymphocyte Activation; Lymphocytes; Middle Aged; Mitogens; Tuberculin
PubMed: 849645
DOI: No ID Found -
Kidney International Mar 1986Spontaneous in vitro IgA synthesis by peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy was elevated; 419 +/- 71 ng/10(6) cells (Mean +/- SEM)...
Spontaneous in vitro IgA synthesis by peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy was elevated; 419 +/- 71 ng/10(6) cells (Mean +/- SEM) compared with controls; 217 +/- 35 (P less than 0.02). Pokeweed mitogen (PWM) stimulated IgA synthesis was also elevated in patients; 4326 +/- 1140 ng/10(6) cells (Mean +/- SEM) versus 1458 +/- 406 (P less than 0.02) but the PWM stimulation index for patients did not differ significantly from that of the controls. Concanavalin A (Con A) suppression of PWM stimulated IgA synthesis resulted in the generation of similar quantities of IgA by PBMC from both patients and controls but the percentage suppression was significantly elevated in patients; 87 +/- 5 (Mean +/- SEM) versus 58 +/- 10 (P less than 0.05). Synthesis of IgG and IgM followed the same pattern as that described for IgA. T and B cells from patients and controls were cultured alone and in various co-culture permutations. Enriched B cells of patients demonstrated a selectively increased capacity for IgA production; 266 +/- 106 ng/5 X 10(5) cells (Mean +/- SEM) compared with controls; 42 +/- 9 (P less than 0.01) and this parameter correlated significantly with serum IgA concentrations (R = 0.77, P less than 0.05). Overall analysis of co-culture data showed no significant difference between the influences of autologous, control or patient T cells on immunoglobulin synthesis by normal B cells. Autolymphocytotoxic antibodies were not detected and, compared with controls, patient sera had no differential effect on numbers of IgA producing cells generated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Adolescent; Adult; Aged; B-Lymphocytes; Cells, Cultured; Concanavalin A; Culture Media; Enzyme-Linked Immunosorbent Assay; Female; Glomerulonephritis, IGA; Humans; Immunoglobulin A; Lymphocyte Activation; Male; Middle Aged; Monocytes; Pokeweed Mitogens; T-Lymphocytes
PubMed: 3486314
DOI: 10.1038/ki.1986.57 -
Clinical and Experimental Immunology Dec 1984Peripheral blood B lymphocytes, depleted of adherent cells, from 10 patients with multiple myeloma were cultured in the presence of PWM with autologous or donor T...
Peripheral blood B lymphocytes, depleted of adherent cells, from 10 patients with multiple myeloma were cultured in the presence of PWM with autologous or donor T lymphocytes. The results show that: (1) co-cultures with allogeneic T lymphocytes produced more plasma cells than those with autologous ones; (2) the kappa/lambda ratio overlapped the values obtained in normal controls, irrespective of the light chain produced by the neoplastic plasma cells and (3) the immunological phenotype of plasma cells obtained from PWM stimulated peripheral B cells (RFA2+, RFA3+, A10+) was clearly different from that one of myelomatous plasma cells (RFA2-, RFA3-, A10+). These data confirm the T cell imbalance already seen in myeloma patients; moreover they show that PWM responsive B cell are functionally normal and phenotypically different from bone marrow myeloma cells. These results support the view that most of the peripheral B lymphocytes, previously identified as monoclonal are in fact normal cells bearing adherent monoclonal Ig molecules.
Topics: Antibodies, Monoclonal; B-Lymphocytes; Bone Marrow; Cells, Cultured; Fluorescent Antibody Technique; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Lymphocyte Activation; Multiple Myeloma; Phenotype; Plasma Cells; Pokeweed Mitogens; Receptors, Antigen, B-Cell
PubMed: 6439450
DOI: No ID Found -
Clinical and Experimental Immunology Oct 1981T lymphocyte subpopulations were studied in 40 patients with scleroderma (PSS), 26 of whom were studied simultaneously for lymphoproliferative responses to...
T lymphocyte subpopulations were studied in 40 patients with scleroderma (PSS), 26 of whom were studied simultaneously for lymphoproliferative responses to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PSS patients exhibited a reduction relative to 42 age- and sex-matched controls in the absolute number and percentage of early E rosettes, late E rosettes and E rosettes formed with aminoethylisothiouronium bromide (AET) treated sheep red blood cells. There was no difference between patients and controls in the proportions of B lymphocytes. PSS patients exhibited normal lymphocyte transformation responses to PHA and ConA and an augmented response to PWM. The mitogen responses did not correlate with the absolute number or percentage of lymphocytes or T and B lymphocyte subpopulations. No correlation was observed between any immunological variable studied and the extent of skin or organ involvement, disease duration or therapy.
Topics: Adult; Aged; B-Lymphocytes; Concanavalin A; Female; Humans; Immunoglobulin G; In Vitro Techniques; Lymphocyte Activation; Male; Middle Aged; Mitogens; Phytohemagglutinins; Pokeweed Mitogens; Rosette Formation; Scleroderma, Systemic; T-Lymphocytes
PubMed: 6978216
DOI: No ID Found -
Acta Poloniae Pharmaceutica 2009Several new derivatives of 5-hydrazino-3-methyl-4-isothiazolecarboxylic ethyl esters were synthesized. Using 4-aminoacetophenone, the hydrazine group was transformed in...
Several new derivatives of 5-hydrazino-3-methyl-4-isothiazolecarboxylic ethyl esters were synthesized. Using 4-aminoacetophenone, the hydrazine group was transformed in position 5 in the hydrazone which reacted with the isocyanates, aldehydes and sugars. Thirteen newly synthesized compounds were tested for their ability to affects the immunological response in vitro in several rodent models. The immunoregulatory properties of the compounds were differential and dose-dependent. The strongest activity was exhibited by 5-{N'-[1-4{-4-[3-(-methoxyphenyl)-ureidol]-phenylethylidene]-hydrazino}-3-methyl-4-isothiazolecarboxylic acid ethyl ester (compound 3a). The compound strongly inhibited the secondary, humoral immune response to sheep erythrocytes and the proliferative response of mouse splenocytes to concanavalin A and pokeweed mitogen. The immunotropic activities of the new isothiazole derivatives and potential application of the compounds in therapy are discussed.
Topics: Animals; B-Lymphocytes; Carboxylic Acids; Concanavalin A; Dose-Response Relationship, Drug; Erythrocytes; Immunologic Factors; Mice; Mice, Inbred CBA; Pokeweed Mitogens; Rats; Rats, Wistar; Sheep; Spleen; Structure-Activity Relationship; T-Lymphocytes; Thiazoles
PubMed: 19894646
DOI: No ID Found -
BMC Cancer Sep 2004Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly...
BACKGROUND
Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA).
METHODS
Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant.
RESULTS
The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed.
CONCLUSION
Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC.
Topics: Biopsy; Carcinoma; Herpesvirus 4, Human; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Immunophenotyping; In Vitro Techniques; Interleukin-10; Interleukin-2; Interleukin-6; Interleukins; Lymphocytes; Lymphocytes, Tumor-Infiltrating; Nasopharyngeal Neoplasms; Pokeweed Mitogens; Reference Values
PubMed: 15450122
DOI: 10.1186/1471-2407-4-68 -
The Journal of Clinical Investigation Mar 1985The cell surface marker profile and functional analysis of peripheral blood lymphocytes from 11 Japanese adult T cell leukemia patients were studied. The phenotypic...
The cell surface marker profile and functional analysis of peripheral blood lymphocytes from 11 Japanese adult T cell leukemia patients were studied. The phenotypic analysis of Japanese adult T cell leukemia (ATL) cells by a series of 13 monoclonal antibodies showed that all ATL cells are anti-T4 reactive but some differ in their expression of T3, T11, and T12 antigens. Thus, considerable phenotypic heterogeneity exists in these populations of leukemia cells. When analyzed in functional assays, ATL cells were suppressive when added to a pokeweed mitogen- (PWM) driven Ig synthesis system. However, the suppression mechanism seemed to be more complex than originally conceived. ATL cells examined in this study seem to function mainly as an inducer of suppressor cells, and as such, activate normal T8 precursors of suppressor cells rather than function as suppressor effector cells. In addition, no evidence was obtained to suggest that suppression of PWM-stimulated IgG synthesis was mediated by natural killer (NK) activity of ATL cells. Rather, ATL cells seem to be markedly deficient in NK activity. These studies suggest that the majority of ATL cells tested are representative of and seem to be the leukemic counterparts of the T4+ suppressor inducer subset.
Topics: Adult; Aged; Antibodies, Monoclonal; Antigens, Surface; B-Lymphocytes; Deltaretrovirus; Female; Humans; Immunoglobulin G; Killer Cells, Natural; Leukemia; Lymphocyte Activation; Male; Middle Aged; Phenotype; Pokeweed Mitogens; Retroviridae Infections; T-Lymphocytes; T-Lymphocytes, Regulatory
PubMed: 2858496
DOI: 10.1172/JCI111780 -
The Journal of Experimental Medicine Nov 1983Glucocorticosteroids (GCS) added to otherwise unstimulated cultures of human peripheral blood mononuclear cells (PBMC) induce the synthesis and secretion of all classes...
Glucocorticosteroids (GCS) added to otherwise unstimulated cultures of human peripheral blood mononuclear cells (PBMC) induce the synthesis and secretion of all classes of immunoglobulin. The magnitude of this response is similar to that seen with other polyclonal B cell activators such as pokeweed mitogen (PWM), and like that of PWM, the steroid effect is dependent on both T cells and monocytes. To determine the cellular target for GCS in these cultures, separated populations of T cells and non-T cells were preincubated with steroids and then recombined. No immunoglobulin was produced in any of these preincubation experiments. As a different approach to this question, supernatants were collected from various cell populations following stimulation with PWM, concanavalin A (Con A), phytohemagglutinin (PHA), alloantigens, or GCS. These supernatants were tested for their effects on GCS-induced Ig production by B cells. Supernatants from 3-d cultures of unstimulated, as well as GCS-treated, PBMC contained a T cell-replacing factor that permitted T-depleted PBMC to produce Ig upon steroid stimulation. This supernatant factor (TRF-S) could be produced in the absence of steroid stimulation, but both the factor and GCS were necessary for the induction of Ig synthesis. Production of the TRF-S required the presence of both T cells and adherent cells in culture and was found in the highest concentrations at 3-4 d of culture. Supernatants from cultures stimulated with PWM, PHA, Con A, and alloantigens did not contain detectable TRF-S activity, and TRF-S was unable to replace helper T cells for PWM-induced Ig production. TRF-S required the presence of adherent cells in the T cell-depleted responder population for its action. Further, it was effective in inducing Ig production along with GCS in the presence of a sufficient concentration of cyclosporin A to block all T cell helper activity for primary responses of PBMC to PWM or GCS. TRF-S was inactivated by trypsin treatment, heating to 56 degrees C, freezing, lyophilization, and storage at 4 degrees C for greater than 3 wk. Its molecular weight is probably 10,000 daltons or more, since TRF-S activity is not rapidly dialyzable. These experiments indicate that GCS-induced Ig production by human B cells does not require the presence of intact T cells in the cultures and therefore the steroids are not exerting their influence directly on T suppressor or T helper cells. Furthermore, they demonstrate a previously unrecognized cytokine that induces the differentiation of human B cells to Ig production in the presence of GCS.
Topics: B-Lymphocytes; Chemical Phenomena; Chemistry, Physical; Concanavalin A; Dexamethasone; Humans; Immunoglobulins; Interleukin-5; Lymphokines; Monocytes; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocytes
PubMed: 6605406
DOI: 10.1084/jem.158.5.1473 -
The Journal of Clinical Investigation May 1980The effect of d-penicillamine (Pen) and mixtures of Pen and copper sulfate on the capacity of normal human peripheral blood mononuclear cells (PBM) to generate...
The effect of d-penicillamine (Pen) and mixtures of Pen and copper sulfate on the capacity of normal human peripheral blood mononuclear cells (PBM) to generate immunoglobulin-secreting cells (ISC) in response to the T-cell-dependent polyclonal B-cell activators pokeweed mitogen (PWM) and staphylococcal protein A (SPA) was examined. PBM obtained from normal individuals were incubated for 1-2 h at 37 degrees C with medium alone, Pen, CuSO(4), or a mixture of Pen and CuSO(4). After washing, the cells were incubated for 6-7 d with PWM or SPA and then, with a reverse hemolytic plaque assay, assayed for the number of ISC generated. Preincubation of PBM with either Pen (100 mug/ml) or CuSO(4) (2 mug/ml) did not alter the subsequent capacity of the cells to generate ISC in response to PWM or SPA. In contrast, responsiveness to both mitogens was nearly abolished when PBM were similarly preincubated with a mixture of Pen and CuSO(4). Inhibition of responsiveness could not be ascribed to cell death, carry-over of the inhibitors, or an alteration in the concentration of PWM or the length of incubation yielding maximum responses. Co-culture experiments demonstrated that Pen and CuSO(4) preincubation had not caused augmented suppressor cell function. Experiments in which PBM were separated into adherent and nonadherent populations indicated that Pen and CuSO(4) preincubation inhibited the responsiveness of the nonadherent cells but did not alter the accessory cell function of monocytes. To determine whether Pen and CuSO(4) preincubation effected T- or B-cell function, PBM were separated into B- and T-cell-enriched populations, individually preincubated with Pen and CuSO(4), and then co-cultured with PWM. The results indicated that Pen and CuSO(4) markedly inhibited helper T-cell function and had little effect on the capacity of B cells to generate ISC. The observation that in the presence of CuSO(4) Pen inhibits helper T-cell activity may, in part, explain the therapeutic efficacy of Pen in rheumatoid arthritis and especially the capacity of Pen therapy to decrease antiglobulin titers in treated patients.
Topics: Adult; B-Lymphocytes; Copper; Humans; Immunoglobulins; In Vitro Techniques; Penicillamine; Pokeweed Mitogens; Sulfates; T-Lymphocytes
PubMed: 6965944
DOI: 10.1172/JCI109759