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PubMed1986

Separation of human B lymphocytes on Helix pomatia A haemagglutinin into two major fractions differing in responsiveness to T-dependent mitogen (pokeweed mitogen) or antigen (tetanus toxoid).

A B-cell fraction consisting of 70% of cells carrying the B-cell-associated B1 antigen, 15-20% of M1+ non-B cells, and less than 3% of T cells was prepared from the peripheral blood of healthy human donors, previously vaccinated with tetanus toxoid (TT). As assessed by immunofluorescence after treatment with neuraminidase, approximately 40-50% of the B cells had surface structures binding to Helix pomatia A haemagglutinin (HP). The cells were separated into three fractions by affinity chromatography on HP conjugated to Sepharose (P, non-retained cells; EI, cells eluted with 0.1 mg/ml N-acetyl-D-galactosamine (D-GalNac); EII, cells eluted with 1 mg D-GalNac/ml). The majority of B cells in fraction EII were HP+ and were rich in cells expressing the B2 differentiation antigen. Sixty per cent of the B cells in this fraction also expressed the major HP-binding glycoprotein, gp 150. In the presence of autologous T cells, these B cells were strongly responsive to activation by either pokeweed mitogen (PWM) or antigen (TT), as reflected by differentiation into plasma cells, secretion of polyclonal IgG and IgM, or IgG anti-TT antibodies. In contrast, fraction P, which contained more than 90% HP-B cells, and which was partially depleted of B2+ cells, responded poorly or not at all to both PWM and TT. Fraction EI was a mixed fraction that responded in an intermediate fashion. When the preparations were depleted of contaminating non-B cells carrying the monocyte or large granular lymphocyte associated M1 antigen, their response to the two stimulating agents did not alter. The results suggest that HP+ B cells differ from HP-B cells in their responsiveness to T-cell signals. Fractionation on unsolubilized HP offers a simple and efficient way of separating B cells into at least two subsets differing in their responsiveness to T-cell-derived differentiation and maturation signals.

Animals↗

PubMed1989

T-cell phenotypes after stimulation of human mononuclear cells by pokeweed mitogen or pokeweed mitogen bound to erythrocytes.

Stimulation of human peripheral blood mononuclear cells (PBMC) by pokeweed mitogen bound to erythrocytes (E-PWM) has been found to result in an increased blast cell formation, lymphocyte proliferation, and enhanced immunoglobulin production, compared to stimulation of PBMC by PWM. Using flow cytometric analysis we compared the T helper/inducer (CD4+) to T suppressor/cytotoxic (CD8+) cell ratios of PBMC after stimulation by PWM and by E-PWM. E-PWM was found to induce significantly lower CD4+:CD8+ ratios on days 6 and 9 of the culture than PWM did. This effect was due predominantly to a relative increase in CD8+ T cells after stimulation of PBMC by E-PWM, compared with stimulation by PWM. However, the increase in T suppressor/cytotoxic cells on days 6 and 9 after E-PWM stimulation was accompanied by a simultaneous relative decrease in CD4+/2H4+ T cells (T suppressor-inducer cells) and by a relative decrease in CD8+/Leu-15+ T cells (CD4+/2H4+ independent T suppressor cells) on day 9, compared with stimulation by PWM. These results suggest that the greater immunoglobulin production after stimulation of PBMC by E-PWM than by PWM may be the result of a relative lack of suppression on fully differentiated B cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

In vitro secretion of bovine immunoglobulins during pokeweed mitogen or pokeweed mitogen and antigen activation of lymphocytes.

Activation of bovine peripheral blood mononuclear cells (PBM) towards immunoglobulin (Ig) synthesis and secretion was examined in vitro using pokeweed mitogen (PWM) or PWM plus sheep red blood cells (SRBC). Bovine PBM were composed of 7-30% B cells, 25-56% T cells and 1-4% macrophages. Cell density and PWM concentrations were critical parameters for obtaining reproducible maximum lymphocyte proliferation and polyclonal B cell activation. Inclusion of aminopterin, a folic acid antagonist, reduced cellular proliferation and viability but had no apparent effect on the time of appearance of peak proliferation. Co-culture of PBM with SRBC and PWM generated anti-SRBC specific antibodies.

Animals↗

PubMed1984

In vitro induction of B cell differentiation in canine mononuclear blood cells: bacterial lipopolysaccharide modulates the action of pokeweed mitogen.

Pokeweed mitogen (PWM) was shown to induce the generation of plasmacytoid cells (PC) from canine peripheral blood mononuclear cells in vitro. PC were detected by immunofluorescence staining of cytoplasmic immunoglobulin. Optimal culture conditions for PC formation were established and the range of the PC response in normal dogs assessed. Addition to PWM of bacterial lipopolysaccharide enhanced PC formation in most instances. This occurred in the absence of increased cell proliferation and without altering the time course of the response. Mitogen-induced PC generation may provide a useful tool for delineating the capacity of canine blood cells to mount a humoral immune response.

Animals↗

PubMed1979

Mitogenic proteins of pokeweed. II. The differentiation of human peripheral blood B lymphocytes stimulated with purified pokeweed mitogens (Po-2 and Po-6) from pokeweed, Phytolacca octandra.

Purified pokeweed mitogens, Po-2 and Po-3, extracted from Phytolacca octandra, stimulated plasma cell formation in cultures of human peripheral blood lymphocytes. Plasma cell formation did not occur in cultures of purified B cells but was dependent on T-cell help. High T-cell numbers, however, suppressed plasma cell formation in mixed B- and T-cell cultures stimulated with Po-2. T-cell helper function was exerted across a major histocompatibility barrier and was not dependent on T-cell proliferation. Soluble helper factor(s) from activated T cells were not demonstrated. Po-3 was an effective B-cell stimulant only at concentrations between 0.1 and 1.0 micrograms/ml. In contrast, relatively high concentrations of Po-2 (50--100 micrograms/ml) were required to induce B-cell differentiation. More plasma cells were generated in Po-2-stimulated cultures than in Po-3-stimulated cultures and this was thought to reflect the more aggregated state of Po-2.

B-Lymphocytes↗

PubMed1979

Inactivation of the mitogenic property of pokeweed mitogen by erythrocytes.

In whole blood culture, pokeweed mitogen (PWM) is unable to stimulate human lymphocytes. This is because the mitogenic property of PWM is inactivated by erythrocytes, presumably due to absorption. The inactivation was observed with as few as 5 x 10(6)/ml of human erythrocytes. Therefore, when PWM is used to study the functions of human lymphocytes, especially of suppressor T lymphocytes, erythrocytes should be removed from lymphocyte preparations for accurate analysis.

Absorption↗

PubMed1985

Distribution of Ig classes and IgG subclasses among human B cells activated by Nocardia opaca-delipidated cell mitogen or by pokeweed mitogen.

The relative proportions of cells synthesizing the three major Ig classes or one of the four IgG subclasses in cultures stimulated with pokeweed mitogen (PWM) or Nocardia-delipidated cell mitogen (NDCM) were investigated. In cultures of human peripheral blood mononuclear cells (PB MNC) stimulated with PWM, the number of IgG-containing cells (CC) was higher than the number of IgM-CC, and a substantial number of IgA-CC was found. Conversely, in NDCM-stimulated PB MNC cultures IgM-CC outnumbered IgG-CC and only few IgA-CC were detected. In those cultures, the removal of T cells resulted in an increase in the number of IgM-CC concomitant with a decrease in the number of IgG-CC. A substantial number of cells containing simultaneously IgG or IgA in addition to IgM could be found in PWM-stimulated cultures. These cells were virtually absent in NDCM-stimulated cultures. The relative proportions of IgG subclass-CC were IgG1-CC greater than IgG2-CC greater than IgG3-CC greater than or equal to IgG4-CC in PWM-stimulated and IgG2-CC greater than IgG1-CC greater than IgG3-CC greater than or equal to IgG4-CC in NDCM-stimulated cultures. The removal of T cells from NDCM-stimulated cultures did not result in major alteration of this distribution. The role of T cells and of the genomic order of the Igh-C genes in their phenotypic expression triggered in vitro by PBA is discussed.

B-Lymphocytes↗

PubMed1988

Anti-pokeweed mitogen antiserum inhibits and enhances blastogenesis of mononuclear cells induced by pokeweed mitogen.

The interaction between pokeweed mitogen (PWM) and peripheral blood mononuclear cells (PBMC) was investigated using rabbit anti-PWM antiserum (anti-PWM) and 125I-PWM. Incubation of PBMC with PWM in the presence of anti-PWM resulted in an inhibition of the mitogenic effect of PWM. Anti-PWM predominantly blocked the interaction of PWM with monocytes, which is essential for optimal stimulation of lymphoid cells with PWM. Addition of anti-PWM to PBMC at several time-points after incubation with PWM showed inhibition of mitogenic activity when anti-PWM was added within 8 hours. However, enhancement of PWM-induced blast cell formation was found when anti-PWM was added after 48 hours. Further analysis revealed that the inhibition of PWM stimulation was mediated by the F(ab')2 part of anti-PWM IgG. On the other hand F(ab')2-anti-PWM was not able to enhance the effect of PWM. Incubation of PBMC with 125I-PWM and anti-PWM simultaneously, decreased the binding of PWM to both lymphocytes and monocytes. In contrast, addition of anti-PWM 48 hours after the incubation of PBMC with PWM resulted in an increased binding of PWM to monocytes. These results show that anti-PWM can modulate the lymphocyte reaction to PWM and suggest two possible mechanisms by which PWM can stimulate PBMC, both of which are dependent on the interaction of PWM with monocytes.

Antibodies↗

PubMed2001

The effects of pokeweed mitogen (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation and embryo development in vitro.

The effects of two commonly used cell culture mitogens, pokeweed (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation in vitro (IVM) and preimplantation embryo development in vitro were evaluated by randomized complete block experimental design with three treatments. Effects were measured by quantifying subsequent embryo development. Oocyte maturation was adversely affected by PWM-containing medium as indicated by a decrease in cleavage rate and subsequent embryo development to morula and blastocyst stages. Embryo developmental competence was also adversely affected by PWM. Development in PHA-containing medium was significantly better (P<0.05) than in the PWM treatment, although there was no difference (P>0.05) when compared to Control. We conclude that there are no beneficial effects in adding mitogenic agents to culture medium to enhance in vitro embryo production and development.

Animals↗

PubMed1993

Human colorectal carcinoma-specific glycoconjugates detected by pokeweed mitogen lectin.

Pokeweed mitogen (PWM) lectin, known to bind branched poly-N-acetyllactosamines, has a highly selective affinity for human colorectal carcinomas. We performed light microscopic (LM) histochemistry with PWM lectin on paraffin sections of human colorectal tissues. In histological sections, normal mucosae and adenomas with mild dysplasia exhibited negative reaction (0/10, 0/13, respectively) with or without neuraminidase pre-digestion, whereas adenomas with moderate dysplasia showed a small increase in PWM lectin reactivity after neuraminidase digestion (4/23). In contrast, we observed a high incidence of positive reactivity in colorectal carcinoma without neuraminidase pre-digestion (38/44). After digestion with neuraminidase, there was increased reactivity of colorectal carcinomas in situ (7/12) and invasive carcinomas (13/32). These results imply that human colorectal carcinomas consistently contain substantial amounts of PWM-reactive branched poly-N-acetyllactosamine glycoconjugates structures. We also compared the staining patterns of PWM lectin and monoclonal antibodies (MAb) directed to Lewis X (LeX) or Lewis Y (LeY) antigen. PWM lectin reactivity was largely confined to the apical membranes of carcinoma tissues. MAb-LeX or MAb-LeY immunoreactivity was seen on the apical membranes and in the cytoplasm of both adenomas and carcinomas. Therefore, histochemical studies with this lectin should be useful for identification of carcinoma tissues and analysis of glycoconjugates associated with colorectal carcinoma.

Adenoma↗

PubMed1977

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.

Antibody Specificity↗

PubMed1983

[Immunocompetence of newborn calves: evaluation of the proliferative response of lymphocytes in vitro to 3 nonspecific mitogens (concanavalin A, phytohemagglutinin, pokeweed mitogen) during the 1st 3 months of life].

A micromethod technique was used to evaluate in vitro sensitivity of the peripheric bovine lymphocytes obtained from a newly born calf, up to 3 months of age to different non-specific mitogens: Phytohemagglutinin (PHA) Concanavaline A (Con A) and Pokeweed Mitogen (PWM). The results obtained show that the calf lymphocytes respond to the 3 mitogens by a considerable cellular proliferation. The blastogenic response was found at various levels during the first 3 months of life, and appeared to stabilize at levels similar to the adult bovine. Highly sensitive variations were noted in the lymphocyte reactivity, notably with PHA and Con A. These results seem to indicate the existence of periods of T cell immunodeficiency, not only during the first few days after birth, but throughout the first months of the calves' life. It may also be indicative of the interest of immunostimulant therapy during this period, which needs further investigation.

Aging↗

PubMed1985

Gamma interferon enhances mitogenic responses induced by pokeweed mitogen.

Interferon (IFN)-induced suppression of the lectin-stimulated lymphoproliferative response was studied comparatively with human IFN-alpha, IFN-beta and IFN-gamma, using an equal unit of their antiviral activity ranging from 31.25 to 1000 IU/ml. Both IFN-alpha and IFN-beta inhibited phytohemagglutinin (PHA) and pokeweed mitogen (PWM)-stimulated lymphoblastogenic response similarly in a dose-related fashion, but the IFN-gamma effect was far less. Indeed, the PWM-stimulated lymphocyte blastogenesis in the cultures incubated for 7 days was enhanced in the presence of IFN-gamma at a concentration of 62.5 IU/ml. The enhancing effect was found to be highest at the lowest concentration of IFN-gamma examined. The IFN-gamma induced enhancement of lectin-stimulated blastogenesis was found mainly in the PWM cultures incubated for 7 days but less in cultures incubated for 5 days or in PHA cultures incubated for 3 days, suggesting that the observed effect might be caused by the activation of interleukin production.

Humans↗

PubMed1992

The effect of chronic immunostimulation of the nonlactating bovine mammary gland with interleukin-2, pokeweed mitogen, and lipopolysaccharide.

Interleukin-2, pokeweed mitogen, and lipopolysaccharide were evaluated for their ability to accelerate involution and to stimulate local cellular defenses in the nonlactating bovine mammary gland. Twelve cows were divided into three treatment groups of 4 cows each to receive interleukin-2, pokeweed mitogen, or lipopolysaccharide. One day after drying off, 3 mammary quarters of each cow were infused with 100 micrograms of immunostimulant daily for 21 d; the remaining control quarter received PBS. Secretion samples were collected weekly to determine bacteriologic status, total SCC, and differential cell counts. On d 21, cows were killed, and tissues were collected for microscopy. Overall, SCC were higher in immunostimulated quarters, but only those infused with interleukin-2 were significantly elevated over controls. By wk 3, the percentage of neutrophils decreased in interleukin-2 and pokeweed mitogen quarters over pretreatment values, percentage of macrophages increased in interleukin-2 quarters, and percentage of lymphocytes increased in pokeweed mitogen and lipopolysaccharide quarters. Percentage of alveolar lumina was reduced, and connective tissue stroma increased, in all immunostimulated quarters compared with those of controls, suggesting accelerated involution. Involution was greatest in quarters treated with interleukin-2. Leukocyte infiltration was greater in immunostimulated quarters than in control quarters. Similarly, concentrations of Ig-producing plasma cells were greater in immunostimulated quarters than in control quarters. Quarters infused with interleukin-2 exhibited the greatest concentration of plasma cells, followed by quarters treated with pokeweed mitogen and lipopolysaccharide; IgG1 plasma cells predominated, followed by IgG2, IgA, and IgM. Interleukin-2 accelerated involution and stimulated local antibody production more than did the two mitogens, suggesting a potential role for this cytokine as a general immunostimulant at drying off.

Animals↗

PubMed1996

Efficient production of IgG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide, pokeweed mitogen, and interleukin 4.

Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), and Staphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed for in vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulation of human IgG production was a culture medium containing 20 micrograms/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.

Antibodies, Monoclonal↗

PubMed1978

[Action of various mitogens (phytohemagglutinin, cocanavalin A, pokeweed mitogen) on a purified human B- and T-lymphocyte population mediating spontaneous rosette formation].

The activity of various most widely used "polyclonal" mitogens, such as PHA, Con A and PWM on human peripheral blood lymphocyte subpopulations, after separation into the two main functional classes (B and T), was investigated. For separating the lymphocyte subclasses, the E-rosette formation technique has been followed, using the gradient centrifuged pellet and supernatant respectively. For the mitogens' activity evaluation, micro-culture system has been performed and 3H thymidine uptake has been assessed. The PHA has been shown to be much more active on the T lymphocyte subclass, though the mitogen cannot be kept very selective. Very much higher activity on the T subclass shows the Con A, which could be considered selective, if we hypothesize an at least partial receptor competition at lymphocyte membrane level between the mitogen and sheep erythrocytes. The PWM has been found very little active and not specific for either lymphocyte subclass.

Adult↗

PubMed1988

Characteristics of the mitogenic activity found in the supernatant of pokeweed mitogen stimulated human mononuclear cells.

Human peripheral blood mononuclear cells were stimulated with pokeweed mitogen (PWM), washed, and cultured in fresh mitogen-free culture medium; a second set of peripheral blood mononuclear cells was used to assess the mitogenic activity of supernatants of these cultures. The effects of this stimulation resembled those of a normal PWM stimulation, including the formation of blast cells, proliferation of lymphoid cells, and immunoglobulin synthesis. Our findings led us to perform experiments to investigate the nature of this mitogenic factor. The molecular weight of the mitogenic activity was found in the region of 700 kD. Additional experiments indicated that human alpha 2-macroglobulin was not responsible for the high molecular weight of the mitogenic factor. Protein digestion or precipitation with trichloroacetic acid only partially abolished the mitogenicity of the mitogenic supernatant. Comparison of the molecular weight of the mitogenic factor with that of the PWM in culture medium showed that the mitogenic activity in the supernatants could be caused by PWM associated with a high molecular weight component.

B-Lymphocytes↗

PubMed2000

Mitogen immunotherapy for HIV infections exemplified by phytohemagglutinin and pokeweed mitogen.

The ideal treatment for HIV infections would supplement highly active antiretroviral therapy (HAART) started at the time of initial diagnosis with an agent that can facilitate cure in the briefest time possible to avoid adverse effects of extended HAART such as drug toxicity, noncompliance, and shortened telomere-induced replicative senescence of CD8 cells. As a crucial part of the regimen, the agent must be able to initiate killer reactions against the HIV, replenish CD4 cells, rejuvenate CD8 cells, block HIV invasion of CD4 cells, activate latent HIV-sequestering host cells, stimulate effective immune reactions against malignancies, generate effective immune responses against conventional and opportunistic infections, circumvent the treatment- and immune response-evading capacities of HIV mutations, and reconstitute immune and hematopoietic competences. Over the past decade, the L4 isolectin of PHA has been presented as a mitogen capable of meeting these goals as they have become defined, but its potential has not been recognized sufficiently to expedite its evaluation. Pokeweed mitogen (PWM) offers the major advantage of being up to 500 times more potent than PHA and has much smaller molecular weight (22,500 to 38,000 daltons versus approximately 125,000 daltons). Given by injection, it has demonstrated curative effects for metastatic malignancies in dogs and cats unassisted by ancillary therapy. Unlike PHA, PWM does not block the attachment of HIV gp 120 to CD4 membrane receptors, but it can obstruct CD4 cell invasion by binding with the CD4 receptors themselves. An expeditious manner of applying PWM would be to give minute doses of an ultra potent root extract capable of inducing a benign plasmacytoid lymphoblastic leukemoid reaction that reverses spontaneously after about 10 weeks. This might prove curative given in conjunction with HAART or possibly even without it. The preferred route of administration for the mitogens in humans would be intravenous although oral administration might prove more advantageous, particularly with PWM. These mitogens might be used singly or, if necessary, in combination after the toxicities have been carefully delineated in humans. Economic forces might restrict treatment availability solely to orally administered PWM therapy in poverty-stricken undeveloped countries if this were shown to be curative. These proposals originating from the authors long concerned with mitogen management of various disorders including HIV infections but only peripherally involved with their clinical applications, are offered for consideration by investigators perceptive enough to see the wisdom of exploring the curative possibilities.

Adjuvants, Immunologic↗