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Bioscience, Biotechnology, and... Jul 2004We investigated native structures and mitogenic properties of pokeweed lectin-D isoforms (PL-D1 and -D2) on human peripheral blood lymphocytes along with other...
We investigated native structures and mitogenic properties of pokeweed lectin-D isoforms (PL-D1 and -D2) on human peripheral blood lymphocytes along with other isolectins (PL-A to -C). Both native PL-D isoforms appeared to behave as monomers. PL-D2 proliferated the lymphocytes like PL-C, whereas PL-D1 had no mitogenicity. PL-D1 acquired mitogenic activity after trimming of the C-terminal dipeptide.
Topics: Cell Proliferation; Dose-Response Relationship, Drug; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; Mitosis; Pokeweed Mitogens; Protein Isoforms; Ribosome Inactivating Proteins; Tetrazolium Salts
PubMed: 15277769
DOI: 10.1271/bbb.68.1591 -
The Journal of Clinical Investigation Dec 1985The neoplastic T cells of a series of seven patients with chronic T-cell neoplasia were tested for helper activity on pokeweed mitogen (PWM)-induced and interleukin 2...
The neoplastic T cells of a series of seven patients with chronic T-cell neoplasia were tested for helper activity on pokeweed mitogen (PWM)-induced and interleukin 2 (IL-2)-induced Ig synthesis. The neoplastic T cells of all patients had a T3+4+8-11+I1- phenotype but differed in expression of the 3A1 antigen. The neoplastic T cells of three patients had helper activity on both PWM- and IL-2-driven Ig synthesis, and in addition produced IL-2 in response to PWM stimulation. Two of these patients had hypergammaglobulinemia. In contrast, the neoplastic T cells in the remaining four patients did not produce IL-2 and did not support PWM-driven Ig synthesis. The T4+ cells of these four patients, however, provided excellent helper activity on IL-2-driven Ig synthesis. These findings emphasize the role of IL-2 in T cell-dependent Ig synthesis and clearly show that IL-2 production is required for helper activity in the PWM-driven system. It is concluded that the combined use of PWM- and IL-2-driven Ig synthesis systems allows separate analysis of IL-2 production and T-helper activity in health and disease.
Topics: Aged; Antibodies, Monoclonal; Antibody Formation; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Cells, Cultured; Female; Humans; Hypergammaglobulinemia; Interleukin-2; Leukemia; Male; Middle Aged; Pokeweed Mitogens; Sezary Syndrome; T-Lymphocytes; T-Lymphocytes, Helper-Inducer
PubMed: 2934407
DOI: 10.1172/JCI112219 -
Cellular Immunology Sep 1996As shown previously, native or recombinant (r) human platelet factor 4 (PF4) alleviates the suppression induced by Con A or dimaprit, a histamine type 2 receptor (H2-R)... (Comparative Study)
Comparative Study
Induction of inhibitory activity for B cell differentiation in human CD8 T cells with pokeweed mitogen, dimaprit, and cAMP upregulating agents: countersuppressive effect of platelet factor 4.
As shown previously, native or recombinant (r) human platelet factor 4 (PF4) alleviates the suppression induced by Con A or dimaprit, a histamine type 2 receptor (H2-R) agonist, in a murine system. The effect of rPF4 on human peripheral blood cells has now been studied, using as a model pokeweed mitogen (PWM)-induced, T-cell-mediated suppression of Ig-secreting cell (ISC) formation by Staphylococcus aureus and rIL-2 activated B cells. PWM, but not phytohemagglutinin (PHA), induced inhibitory activity in mitomycin-treated CD8+ T cells, but not unfractionated or CD4+ T cells, for both ISC formation and B cell proliferation. rPF4 and its C-terminal tridecapeptide alleviated the suppressive effect of PWM-activated CD8+ T cells on ISC production but not on proliferation. Heparin did not prevent this immunoregulatory activity of PF4. Neutralizing antibody to TGF-beta, but not to IFN-gamma or TNF-alpha, alleviated the suppression of ISC formation in some of the experiments. The H2-R appeared to play a part in inducing suppression, because the H2-R antagonist, cimetidine, prevented the PWM-induced suppression of ISC production. Furthermore, dimaprit induced suppression of ISC formation when added instead of PWM at the start of culture. Incubation of CD8+ T cells with dimaprit for only 3 hr prior to coculture with S. aureus + IL-2 activated B cells decreased the ISC response. This suppression was also alleviated by addition of rPF4 to the coculture. Similar to dimaprit, known cAMP upregulating agents, such as forskolin, dibutyryl cAMP, and cAMP analog, all induced this immunoregulatory activity in T cells. Moreover, the effect of dimaprit was prevented by the specific protein kinase A inhibitor, HA1004, suggesting strongly that upregulation of cAMP played a role in the H2-R-mediated effect. Cell contact appeared to be necessary, since supernatants from dimaprit or PWM activated T cells failed to suppress ISC production. We suggest that the known ability of PF4 to prevent TGF-beta-mediated effects on endothelial and other target cells may be involved in the alleviating effect of PF4 on the cell-contact-dependent CD8+ T-cell-mediated B cell suppression.
Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibody-Producing Cells; B-Lymphocytes; Binding, Competitive; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Division; Cell-Free System; Cells, Cultured; Cimetidine; Cyclic AMP; Dimaprit; Heparin; Humans; Immunosuppressive Agents; Interferon-gamma; Interleukin-2; Lymphocyte Activation; Peptide Fragments; Platelet Factor 4; Pokeweed Mitogens; Staphylococcus aureus; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation
PubMed: 8964082
DOI: 10.1006/cimm.1996.0234 -
European Journal of Immunology Jun 2003To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM....
To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.
Topics: Animals; CD4-Positive T-Lymphocytes; Concanavalin A; Dipeptidyl Peptidase 4; Female; Immunization; Immunoglobulin Class Switching; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Interferon-gamma; Interleukin-2; Interleukin-4; Interleukins; Killer Cells, Natural; Lymphocyte Activation; Lymphokines; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pokeweed Mitogens; Specific Pathogen-Free Organisms; Spleen
PubMed: 12778469
DOI: 10.1002/eji.200323469 -
American Journal of Veterinary Research Aug 2000To compare the in vitro immunosuppressive effects of cyclosporine and 4 novel immunosuppressive drugs on lymphocytes in whole blood collected from healthy cats. (Comparative Study)
Comparative Study
OBJECTIVE
To compare the in vitro immunosuppressive effects of cyclosporine and 4 novel immunosuppressive drugs on lymphocytes in whole blood collected from healthy cats.
SAMPLE POPULATION
Whole blood samples collected from 10 healthy adult domestic shorthair cats.
PROCEDURE
Mitogen-stimulated lymphocyte proliferation in whole blood incubated with and without various concentrations of cyclosporine, tacrolimus, sirolimus, mycophenolic acid (MPA), or A771726 was measured by use of [3H]thymidine incorporation. Drug concentrations that resulted in a 50% inhibition of mitogen-induced proliferation (IC50) were calculated. Lymphocyte viability was determined by use of the trypan blue dye exclusion method.
RESULTS
An obvious dose-response relationship for the antiproliferative effects of each drug was detected. Mean IC50 determined with concanavalin A was 46 nM for cyclosporine, 9 nM for tacrolimus, 12 nM for sirolimus, 16 nM for MPA, and 30 mM for A771726, whereas with pokeweed mitogen, mean IC50 was 33 nM for cyclosporine, 5 nM for tacrolimus, 15 nM for sirolimus, 14 nM for mycophenolic acid, and 25 mM for A771726. Mitogen-stimulated and nonstimulated lymphocytes remained viable, regardless of drug evaluated.
CONCLUSIONS AND CLINICAL RELEVANCE
Tacrolimus, sirolimus, MPA, and A771726 inhibited in vitro mitogen-stimulated proliferation of feline lymphocytes in a dose-dependent manner. These novel immunosuppressive drugs may be useful for management of immune-mediated inflammatory diseases and prevention and treatment of rejection in cats that undergo organ transplantation.
Topics: Animals; Cats; Cell Division; Coloring Agents; Concanavalin A; Cyclosporine; Dose-Response Relationship, Drug; Immunosuppressive Agents; Inhibitory Concentration 50; Isoxazoles; Leflunomide; Lymphocyte Activation; Mycophenolic Acid; Pokeweed Mitogens; Scintillation Counting; Sirolimus; Tacrolimus; Trypan Blue
PubMed: 10951980
DOI: 10.2460/ajvr.2000.61.906 -
Clinical and Vaccine Immunology : CVI Aug 2009The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance...
The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4(+) and CD8(+) T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at -70 degrees C for < or =3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at -70 degrees C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
Topics: Blood; Candida; Cell Proliferation; Cell Survival; Flow Cytometry; Freezing; Human Experimentation; Humans; L-Selectin; Leukocyte Common Antigens; Lymphocyte Count; Lymphocyte Subsets; Pokeweed Mitogens; Specimen Handling; T-Lymphocytes; Tetanus
PubMed: 19515870
DOI: 10.1128/CVI.00342-08 -
Japanese Journal of Cancer Research :... Jun 1992In order to obtain more potent lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy, pokeweed mitogen (PWM) was added to the culture medium for the...
In order to obtain more potent lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy, pokeweed mitogen (PWM) was added to the culture medium for the initial 24-48 h of culturing. The proliferation rate of PWM-stimulated LAK cells reached about 1000-fold after 3-week culture. This rate was nearly the same as that of LAK cells stimulated by 10 ng/ml of OKT3, the mouse anti-CD3 monoclonal antibody. However, the cytotoxicity of PWM-stimulated LAK cells was significantly more potent than that of OKT3-stimulated LAK cells. Phenotypic analysis revealed that PWM-stimulated LAK cells were CD3+CD56(+)-dominant while OKT3-stimulated LAK cells were CD3+CD56(-)-dominant. About half of CD3+CD56+ PWM-stimulated LAK cells was CD8+. These results suggest that more efficient adoptive immunotherapy is possible by using high-dose PWM-stimulated LAK cells with more potent cytotoxicity. Interleukin-1 beta and tumor necrosis factor alpha were significantly increased in the culture media after 24-h incubation with 1 micrograms/ml of PWM. Secretion of interferon-gamma was not enhanced by this concentration of PWM within 24 h. Therefore, PWM is considered to activate monocytes or macrophages to produce these cytokines in advance, influencing the proliferation and the cytotoxicity of LAK cells.
Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; CD56 Antigen; CD8 Antigens; Cell Division; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Humans; Immunity, Cellular; In Vitro Techniques; Interferon-gamma; Interleukin-1; Killer Cells, Lymphokine-Activated; Lymphocyte Subsets; Muromonab-CD3; Pokeweed Mitogens; Receptors, Antigen, T-Cell; Tumor Necrosis Factor-alpha
PubMed: 1379578
DOI: 10.1111/j.1349-7006.1992.tb00136.x -
Developmental Immunology 1992Normal values for percentages of lymphocyte subpopulations and functional responses to mitogen stimulation in infancy are not well established. In the present study,... (Comparative Study)
Comparative Study
Normal values for percentages of lymphocyte subpopulations and functional responses to mitogen stimulation in infancy are not well established. In the present study, lymphocyte subpopulations were examined in umbilical cord blood samples and in peripheral blood samples drawn before 7 and 24 months of age (mean age 10.4 months) from a healthy population of infants born in Tucson, Arizona. Results indicate significant increases occurred from birth to later infancy in the percentages of total T cells (CD3), T-cell subsets (CD4, CD8) and B cells (CD20). The CD4/CD8 ratio and the functional responses to ConA and PWM mitogens significantly decreased from birth to later infancy. PHA responsiveness did not show a significant change. Results from cross-sectional analyses (n = 271) were supported in a smaller longitudinal subset (n = 37). There were no detectable ethnic- or gender-related differences in cord blood or samples obtained in later infancy. The normal values established in this study will be useful in studies of immune-system maturation and in the clinical evaluation of newborns, infants, and toddlers suspected of either acquired or congenital immune-deficiency states.
Topics: Cell Count; Concanavalin A; Female; Hispanic or Latino; Humans; Infant; Infant, Newborn; Lymphocyte Subsets; Male; Phytohemagglutinins; Pokeweed Mitogens
PubMed: 1627949
DOI: 10.1155/1992/64292 -
Clinical and Experimental Immunology May 1984To gain insight into possible determinants of in vivo polyclonal B cell activation seen in rheumatoid arthritis (RA), we enumerated immunoglobulin secreting cells...
To gain insight into possible determinants of in vivo polyclonal B cell activation seen in rheumatoid arthritis (RA), we enumerated immunoglobulin secreting cells appearing in cultures of peripheral blood mononuclear cells that were stimulated with pokeweed mitogen (PWM) or a newly described polyclonal B cell activator, bacterial peptidoglycan. Peptidoglycan, the major constituent of the cell wall of gram positive bacteria, has properties which warrant its consideration in the pathogenesis of RA; including the ability to induce rheumatoid factor production as well as a RA like syndrome in experimental animals. RA patients as a group had similar immunoglobulin secreting cell responses in PWM stimulated cultures compared to arthritis controls and showed moderately depressed responses compared to healthy volunteers. However, their in vitro responses to peptidoglycan were markedly depressed when compared to those of both control groups. Of note, severely reduced peptidoglycan-induced responses were seen in 26 of 55 rheumatoid patients who demonstrated intact PWM-induced responses. These impaired responses to peptidoglycan were not due to (1) aberrant kinetic response; (2) shift in the dose-response pattern; (3) decreased cell survival in culture or (4) the inability of peptidoglycan to activate RA cells. Cell fractionation studies indicated that peptidoglycan reactive B cells were present in the blood of some patients but their reactivity was abrogated by suppressor T cells. These studies provide evidence of aberrant in vitro polyclonal B cell activation in patients with RA and provide a basis for further investigation of peptidoglycan as an immunopathogenetic agent in this disease.
Topics: Adult; Aged; Antibody-Producing Cells; Arthritis, Rheumatoid; B-Lymphocytes; Cells, Cultured; Female; Humans; Immunoglobulins; Lymphocyte Activation; Male; Middle Aged; Peptidoglycan; Pokeweed Mitogens; Staphylococcus aureus
PubMed: 6610510
DOI: No ID Found -
Clinical and Experimental Immunology Oct 1973Methods based on [H]thymidine incorporation and morphology were used for further studies on the effect of hydrocortisone on the transformation of human lymphocytes by...
Methods based on [H]thymidine incorporation and morphology were used for further studies on the effect of hydrocortisone on the transformation of human lymphocytes by phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Result obtained with both methods showed inhibition of PHA stimulation in cultures preincubated with 0·01–10 μg/ml of hydrocortisone. [H]thymidine incorporation due to PWM in vertical tube cultures was depressed by 1·0 μg/ml of hydrocortisone in cultures harvested at 48 hr, but not in similar cultures incubated for 3, 4 and 5 days. In vertical cultures 10 μg/ml of hormone depressed the level of uptake in most experiments. By contrast [H]thymidine incorporation was enhanced in horizontal PWM cultures by 0·1 and 1·0 μg/ml of hydrocortisone, and 10 μg/ml caused stimulation or a return to the normal PWM level. Microscopic and time-lapse observations on living cells showed that typical PWM blasts first appeared on the third day of incubation and after 5 days were numerous in corticosteroid-treated as well as in untreated cultures. Ten micrograms per millilitre of hydrocortisone increased the fragility of PWM blasts in both kinds of cultures and caused degeneration of variable numbers of blasts in 5-day-old vertical tube cultures. The depression of [H]thymidine uptake in vertical cultures was thought to be due to a combination of enhanced toxicity of corticosteroid hormone in deep cultures, and loss of incorporated [H]thymidine due to increased cellular fragility. Results obtained with the isotopic and morphologic methods indicated that the transformation of B lymphocytes by PWM is relatively resistant to the action of hydrocortisone .
Topics: Cells, Cultured; Humans; Hydrocortisone; Lectins; Lymphocyte Activation; Lymphocytes; Macrophages; Mitogens; Motion Pictures; Thymidine; Tritium
PubMed: 4762017
DOI: No ID Found