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Clinical and Experimental Immunology Jul 1985The in vitro T-cell dependent B-cell activity was analysed in 34 patients with myasthenia gravis and in 16 controls by culturing 1 X 10(6) peripheral blood lymphocytes...
The in vitro T-cell dependent B-cell activity was analysed in 34 patients with myasthenia gravis and in 16 controls by culturing 1 X 10(6) peripheral blood lymphocytes for 8 days with or without pokeweed mitogen (PWM) and measuring the amounts of IgG and IgM released into the culture supernatant. Increased production of IgG in the unstimulated cultures was found in 20 patients, 8 of whom also produced increased amounts of IgM. Upon PWM stimulation the patients produced normal amounts of IgG. The IgM production, however, was decreased in the patient population compared to the controls. Only one of the controls produced more IgG than IgM, either unstimulated or during PWM stimulation. In contrast 10 patients produced more IgG than IgM in the unstimulated cultures and 15 patients did so during PWM stimulation. The 'spontaneous' production of immunoglobulins was not measurable before 4 days of culture and was not the result of in vivo activated B-cells. We suggest that the increased 'spontaneous' immunoglobulin production and the decreased production of IgM during PWM stimulation may be a result of abnormal T-cell activity. The abnormalities found were not related to disease activity, the presence of multiple autoantibodies in the serum, steroid therapy or previous thymectomy. Besides these abnormalities, patients not treated with immunosuppressive drugs also showed decreased PWM induced IgG synthesis.
Topics: Adult; Aged; Autoantibodies; B-Lymphocytes; Female; Humans; Immunoglobulin G; Immunoglobulin M; Immunosuppressive Agents; In Vitro Techniques; Kinetics; Lymphocyte Activation; Male; Middle Aged; Myasthenia Gravis; Pokeweed Mitogens; T-Lymphocytes; Thymectomy; Time Factors
PubMed: 3876181
DOI: No ID Found -
PloS One 2012Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin...
Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations.
Topics: Animals; B-Lymphocytes; Cell Proliferation; Cells, Cultured; Drug Evaluation, Preclinical; Drug Synergism; HEK293 Cells; Humans; Ligands; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Pokeweed Mitogens; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptors
PubMed: 22238657
DOI: 10.1371/journal.pone.0029806 -
Journal of Anatomy Jan 1985Human peripheral blood lymphocytes were cultured with phytohaemagglutinin or pokeweed mitogen for various intervals up to 17 days and studied by scanning electron...
Human peripheral blood lymphocytes were cultured with phytohaemagglutinin or pokeweed mitogen for various intervals up to 17 days and studied by scanning electron microscopy. Activated lymphocytes in 3 day cultures were large irregular cells characterised mainly by an abundance of very fine microvilli, which were much thinner, shorter and more densely packed than the microvilli on uncultured lymphocytes. Cells intermediate in size and surface morphology between these and unstimulated lymphocytes were numerous in 1 day cultures. Some motile cells and large cells with finger-like or conical microvilli were also present. Cell counts showed that after 6 days in phytohaemagglutinin culture small villous cells resembling normal healthy lymphocytes were progressively more numerous, suggesting that most of the activated cells reverted to small lymphocytes. Very large cells were also present and displayed a heterogeneous surface morphology of villi, ridges, blebs and ruffles. A few of these cells had typical monocytoid features but others were predominantly villous. In pokeweed mitogen cultures there was extensive cellular degeneration affecting all cell types after 6 days. In unstimulated cultures the lymphocytes had fewer microvilli or were smooth-surfaced. There was extensive lymphocyte degeneration after 3 days and eventually typical monocytes were the predominant cells. Large, villous cells, which could be activated lymphocytes, were occasionally encountered in unstimulated cultures.
Topics: Cell Movement; Cells, Cultured; Humans; Lymphocyte Activation; Lymphocytes; Microscopy, Electron, Scanning; Microvilli; Monocytes; Phytohemagglutinins; Pokeweed Mitogens
PubMed: 4066474
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1993Mechanisms for increased levels of quinolinic acid (QUIN) following systemic immune stimulation were investigated. In gerbils, systemic administration of pokeweed...
Mechanisms for increased levels of quinolinic acid (QUIN) following systemic immune stimulation were investigated. In gerbils, systemic administration of pokeweed mitogen (PWM) increased plasma and cerebrospinal fluid QUIN levels, while plasma kynurenic acid levels were decreased and cerebrospinal fluid kynurenic acid levels were unchanged. PWM also increased the QUIN concentrations of brain and systemic tissues. In slices of spleen, lung, liver, duodenum, and kidney, PWM caused marked increases in [13C6]QUIN formation from L-[13C6]tryptophan (but not from [13C6]anthranilic acid). PWM also increased QUIN excretion in the urine and enhanced the formation and excretion of [13C6]QUIN following an intraperitoneal injection of L-[13C6]tryptophan. Indoleamine-2,3-dioxygenase activity was increased in the brain, kidney, lung, spleen, and duodenum while hepatic L-tryptophan-2,3-dioxygenase activity was reduced, data consistent with in vitro L-kynurenine formation from L-tryptophan. Kynurenine-3-hydroxylase activity was increased in the duodenum, lung, and spleen, but not in the brain, kidney, or liver. Kynureninase activity was increased in the brain, lung, and duodenum, but not in the spleen, kidney, or liver. 3-Hydroxyanthranilate-3,4-dioxygenase activity was unchanged in the brain, lung, and liver. No change in kynurenine aminotransferase activity was observed in the brain or lung, while liver kynurenine aminotransferase activity was reduced. We conclude that increased activities of kynurenine pathway enzymes in various tissues following systemic immune stimulation, in conjunction with macrophage infiltration of the affected tissue, provide a mechanism to account for increased concentrations of QUIN.
Topics: Animals; Duodenum; Female; Gerbillinae; Hydrocortisone; Immunization; Kidney; Kinetics; Kynurenine; Liver; Lung; Pokeweed Mitogens; Quinolinic Acid; Spleen; ortho-Aminobenzoates
PubMed: 8340378
DOI: No ID Found -
Frontiers in Immunology 2020Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite...
Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the synthesis of nicotinamide adenine dinucleotide (NAD). As a precursor for NAD, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation [e.g., lipopolysaccharide (LPS) or pokeweed mitogen (PWM)] in macrophages, microglia, dendritic cells, and other cells of the immune system. NAD serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair, and the activity of various enzymes such as the NAD-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here, we describe Quin staining in the brain, spleen, and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 h) and prolonged (48 h) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD synthesis from tryptophan. Further, we propose that the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer cells.
Topics: Animals; Biomarkers; Cell Movement; Gerbillinae; HSP90 Heat-Shock Proteins; Hippocampus; Immunity; Indoleamine-Pyrrole 2,3,-Dioxygenase; Inflammation; Kynurenine; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred C57BL; NAD; Pokeweed Mitogens; Poly(ADP-ribose) Polymerases; Quinolinic Acid; Rats; Spleen; Tryptophan
PubMed: 32153556
DOI: 10.3389/fimmu.2020.00031 -
Clinical and Experimental Immunology Oct 1981J chain synthesis was investigated by in vitro pokeweed mitogen (PWM) stimulated peripheral blood lymphocyte (PBL) cultures in eight patients with selective IgA...
J chain synthesis was investigated by in vitro pokeweed mitogen (PWM) stimulated peripheral blood lymphocyte (PBL) cultures in eight patients with selective IgA deficiency and compared with that of normal persons. In normals, all IgM-containing cells always had the J chain but only in a portion of IgG- and IgA-containing cells was J chain detectable. The percentage of J chain-positive cells amongst IgG or IgA cells increased during culture, reached a peak at days 5-6 or 6-7, respectively, and then decreased. IgA-deficient patients had very few IgA-containing cells and an increased number and percentage of J chain-positive IgG cells, except for one patient, who had a significant number of IgA-containing cells without IgA secretion into the culture supernatants. Measurement of Ig in culture supernatants by radioimmunoassay revealed that lymphocytes from seven patients secreted significantly smaller amounts of IgG and IgM than did the normal controls, in addition to the defect in IgA production. These results suggested the presence of some ontogenetic relationship between J chain-positive IgG cells and the precursors of IgA-producing cells, and some functional immaturity of most IgG-producing clones seen in patients with selective IgA deficiency.
Topics: Cells, Cultured; Dysgammaglobulinemia; Humans; IgA Deficiency; Immunoglobulin G; Immunoglobulin J-Chains; Immunoglobulin M; Leukocyte Count; Lymphocyte Activation; Lymphocytes; Pokeweed Mitogens
PubMed: 6802535
DOI: No ID Found -
Clinical and Experimental Immunology Aug 1991Using modified ELISA and spot-ELISA, which permit the parallel determination of heavy chain subclass and the presence of covalently linked J chain, we analysed IgA found...
Cellular origins of human polymeric and monomeric IgA: enumeration of single cells secreting polymeric IgA1 and IgA2 in peripheral blood, bone marrow, spleen, gingiva and synovial tissue.
Using modified ELISA and spot-ELISA, which permit the parallel determination of heavy chain subclass and the presence of covalently linked J chain, we analysed IgA found in cell culture supernatants or secreted by individual cells from peripheral blood, spleen, bone marrow, gingiva and synovial tissue, with respect to its polymeric or monomeric IgA form (pIgA, mIgA) and IgA1 or IgA2 subclass. The ELISA for determination of J chain in tissue culture supernatants was specific and highly sensitive (detection limit in pg). The results demonstrated that IgA1-producing cells predominated in the tissues examined, and that J chain could be detected in association with the majority of IgA1 and IgA2 secreted by individual cells. With respect to the frequency of cells secreting polymeric, J chain-containing IgA, only 20-30% of cells from the bone marrow were engaged in the synthesis of PIgA. In other tissues the frequency of cells secreting pIgA1 and pIgA2 was considerably higher. Peripheral blood mononuclear cells secreting pIgA2 were easily inducible during stimulation with T cell-dependent pokeweed mitogen, whereas Epstein-Barr virus-transformed cells secreted preferentially mIgA1. When the frequencies of pIgA-, pIgA1- or pIgA2-secreting cells (determined by spot-ELISA technique) from different tissues were correlated with the proportion of pIgA to mIgA (and IgA subclasses) secreted in tissue culture supernatants, data obtained suggest that many individual IgA-producing cells could be engaged in simultaneous secretion of mIgA and pIgA.
Topics: Adult; Bone Marrow; Bone Marrow Cells; Gingiva; Humans; Immunoglobulin A; Immunoglobulin J-Chains; Lymphocytes; Pokeweed Mitogens; Spleen; Synovial Membrane
PubMed: 1907532
DOI: 10.1111/j.1365-2249.1991.tb05730.x -
Clinical and Experimental Immunology Nov 1985In vitro IgA synthesis induced by pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (STA), and combinations of STA and PWM (STA/PWM) was studied in lymphocytes of...
In vitro IgA synthesis induced by pokeweed mitogen (PWM), Staphylococcus aureus Cowan I (STA), and combinations of STA and PWM (STA/PWM) was studied in lymphocytes of IgA deficient (sIgAd) blood donors. Cultures of T-depleted (non-T) cells with autologous or allogeneic control T, irradiated T (T), or T4 cells suggested abnormalities in non-T cell fractions in most (12/22) sIgAd donors. T cell abnormalities in themselves, detectable in six donors, did not appear to account for the failure of IgA synthesis. Repeat studies in 10 donors indicated fluctuations in in vitro IgA synthesis in four. IgA synthesis induced by STA/PWM combinations was observed in only one of eight sIgAd donors. Our findings suggest that in some donors defects leading to failure to produce IgA may not be constant and support the hypothesis of a maturation arrest in IgA+ B cells in sIgAd donors.
Topics: B-Lymphocytes; Cells, Cultured; Dysgammaglobulinemia; Humans; IgA Deficiency; Immunoglobulin A; Immunoglobulin A, Secretory; Immunoglobulin G; Immunoglobulin M; Pokeweed Mitogens; Staphylococcus aureus
PubMed: 3878748
DOI: No ID Found -
European Spine Journal : Official... Apr 2011The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the...
The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 μg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-β1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1β and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-β1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1β expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.
Topics: Adult; Aged; Cell Differentiation; Cell Proliferation; Coculture Techniques; Cytokines; Diskectomy; Female; Humans; Immunoglobulin G; Intervertebral Disc; Leukocytes, Mononuclear; Lymphocytes; Male; Mesenchymal Stem Cells; Middle Aged; Pokeweed Mitogens
PubMed: 21181480
DOI: 10.1007/s00586-010-1662-9 -
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