Mitogenic proteins of pokeweed. I. Purification, characterization and mitogenic activity of two proteins from pokeweed (Phytolacca octandra).

Saline extracts from the roots of the pokeweed species. Phytolacca octandra were separated by ion-exchange chromatography into three fractions, Po-1, Po-2 and Po-3. Po-1 contained two monomeric proteins with molecular weights of 36,000 and 29,000 and these were partially purified by gel filtration. Po-2 was purified as a single polymeric protein composed of approximately ten 14,000 mol. wt polypeptides and is a new pokeweed mitogen. Po-3 was purified as a single polymeric protein composed of approximately four 31,000 mol. wt subunits, and apart from its polymeric structure closely resembles commercial pokeweed mitogen (PWM). Po-2 and Po-3 were mitogenic for unseparated human peripheral blood lymphocytes but the degree of mitogenic activity in Po-2 preparations was dependent on storage following purification. Purified B cells were not stimulated by either mitogen. Po-3 was a potent mitogen for T cells but preparations of Po-2 required storage before they stimulated T cells. Higher responses were observed in co-cultures of B and T cells than in separated B and T cell cultures. It is suggested that human B and T lymphocytes show synergy in their responses to Po-2 and Po-3.     

Immunoregulation of immunoglobulin production in normal infants and children.

Proportion of T cell subsets, spontaneous and PWM stimulated immunoglobulin production by peripheral blood lymphocytes and concanavalin A- (Con A) stimulated suppressor cell activity on immunoglobulin production by B cells was studied in 37 infants and children, to investigate changes of these parameters with age. Proportion of suppressor/cytotoxic (T8+) T lymphocytes was significantly lower in children below the age of 5 years, while there was no difference in proportion of total T lymphocytes (T3+) and helper/inducer (T4+) T cells. Spontaneous production and secretion of IgG and IgM by lymphocytes from children of all age groups was similar to that found in adults, but lymphocytes of children below the age of 10 years showed a low response to PWM stimulation. The activity of Con A-induced suppressor cells in inhibiting B cells producing immunoglobulins was almost absent in infancy, gradually increased during childhood and reached adult levels in teenagers. A significant association between the proportion of T8+ cells and Con A-induced suppression of B cell proliferation and a relationship between T4+ cells and spontaneous Ig production indicated the increasing maturity with respect to both number and function of peripheral blood lymphocyte subsets with advancing age.     

T and B lymphocytes in peripheral blood of splenectomized subjects]

The report is a part of more extensive studies on the role of the spleen in immune processes. Assessing the ability of lymphocytes T to non-immunological binding of sheep erythrocytes and the ability of B cells to bind immunological complexes through the receptor for C3 component of complement, and the presence of immunoglobulins on the surface of lymphocytes B the number of these cells in peripheral blood was determined in 14 healthy individuals who had been splenectomized. In all these subjects the degree of blastic transformation of lymphocytes in PHA-stimulated 3 day-old white blood cell cultures was determined. It was found that the number of T and B lymphocytes in the peripheral blood of splenectomized and non-splenectomized patients was similiar. Lymphocytes obtained from splenectomized patients had however, an impairment of transformation ability after PHA stimulation. It is suggested that, apart from determination of T and B cell pool in the peripheral blood, an evaluation of their transformation ability after PHA stimulation is necessary for assessment of the immunological state in vitro investigations.     

Brucella fractions behave as nonspecific mitogens and polyclonal B-cell activators for human lymphocytes.

Two lipid-A-free fractions which were extracted from Brucella melitensis and were designated PI and SF stimulated human unsensitized mononuclear cells to proliferate and to secrete immunoglobulins. Both of these effects were observed in cultures of peripheral blood, tonsils, and cord blood lymphocytes. Neither B cells nor T cells alone proliferated in the presence of these fractions, whereas the proliferative response of T cells plus B cells was largely independent of accessory cells. Polyclonal activation was estimated by counting the cells which secreted immunoglobulins of different isotypes into culture supernatants. This phenomenon was strongly T dependent.     

Blastogenesis and Lymphokine Synthesis by T and B Lymphocytes from Patients with Periodontal Disease

Thymus-derived (T) and bone marrow-derived (B) lymphocytes were isolated from human peripheral blood and cultured with various mitogens and antigens. Purified protein derivative of tuberculin stimulated both purified T and B cells from patients with positive skin reactivity to purified protein derivative but did not stimulate nonimmune lymphocytes. Similarly, both T and B lymphocytes from patients with periodontal disease were stimulated to proliferate when incubated with dental plaque, whereas cells from normal individuals without gingivitis were unresponsive. In contrast, one component of plaque, bacterial endotoxins (lipopolysaccharide), minimally stimulated B lymphocytes from both normal or gingivitis patients. T lymphocytes from patients with periodontal disease were also stimulated by plaque antigen to produce chemotactic lymphokine activity (CTX) for human monocytes. B cells purified by the EAC rosetting method nonspecifically produced CTX without concomitant blastogenesis; however, after dissociation of adherent EAC these immune B cells did not spontaneously produce CTX. Lymphokine synthesis by B cells was not dependent on concomitant blastogenesis. Dissociated B cells from periodontitis patients also produced CTX activity after stimulation with dental plaque antigen. Therefore, both T and B lymphocytes, after stimulation with nonendotoxin antigenic components of plaque, proliferated and produced lymphokines, which are presumed to contribute to the pathogenesis of periodontal disease.     

Diminished active T rosette levels and increased spontaneous B lymphocyte blastogenesis in hepatitis B virus positive chronic active hepatitis

The present investigation was designed to detect abnormalities in CMI and the presence of polyclonally activated B cells in patients with HBV positive CAH. We studied the peripheral levels and 3H-thymidine incorporation of three lymphocyte subsets: B lymphocytes, as well as two T cell subsets that are either active or late rosetters with high and low affinity receptors respectively for sheep red blood cells (SRBC). In patients the level of peripheral T active cells was decreased, but they exhibited elevated B cell activation. There was also a significant correlation between the decreased levels of T active cells and increased 3H-thymidine incorporation by B lymphocytes. Taken together, our results are consistent with the hypothesis that patients with HBV positive CAH have a severe impairment of T cell function that may lead to an abnormal B cell activation. The increased B cell activity may account for the presence of circulating immune complexes and the variety of autoantibodies often observed in patients with HBV positive CAH.     

Functional behaviour and immunological phenotype of circulating B lymphocytes in multiple myeloma. Studies with pokeweed mitogen.

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.     

Expression of the costimulator molecules, CD80, CD86, CD28, and CD152 on lymphocytes from neonates and young children

The expression of CD80, CD86, CD28, and CD152 were examined on peripheral blood lymphocytes from adults, neonates (cord blood lymphocytes) and young children (2-20 months of age). There was no difference in the expression of CD80 or CD86 between adult and neonatal B cells, either resting or activated. A higher percentage of resting T cells expressed CD28 in neonates and young children compared to adults. CD28 expression was similar on adult and neonatal T cells activated with PMA and ionomycin. However, CD28 was expressed at greater intensity on a higher percentage of neonatal T cells than adult T cells stimulated with CD3. CD152 expression was lower on neonatal T cells than adult T cells stimulated with PMA and ionomycin and undetectable on neonatal T cells stimulated with CD3. In contrast, intracellular CD152 was equivalent in adult and neonatal T cells stimulated with PMA and ionomycin, suggesting trafficking of CD152 to the cell surface may be differentially regulated in neonatal T cells. Since the T cell response is determined by the balance of signals received from CD28 and CD152, high levels of CD28 expression and lower surface expression of CD152 on neonatal T cells may represent specialisation to promote activation of neonatal T cells.     

Evidence for a spontaneous activity and a weak helper function in cord blood lymphocytes

Human lymphocytes from cord blood (CBL) and adult peripheral blood were studied for suppressive and stimulatory effects. Using a double chamber assay we found that via soluble substance(s) cells from approximately 90% of all cord bloods and about 25% of the tested adults could diminish the proliferative response and the IgM and IgG plaque-forming cell numbers of phytohemagglutinin (PHA) stimulated adult cell cultures. CBL were spontaneously suppressive, whereas cells from 'suppressive adults' required activation with PHA to exert suppression. Co-cultures of T cells and B cells were stimulated with pokeweed mitogen. Cord B lymphocytes were able to secrete not only IgM but also IgG. The Ig response of CBL was weaker than that of adult cells, which argues for an immaturity of cord helper T lymphocytes, and possibly also of cord B lymphocytes.     

In vitro X-ray irradiation of human peripheral blood T lymphocytes enhances suppressor function

We studied the effect of in vitro X-ray irradiation on human peripheral blood T lymphocytes, with regard to their suppressor activity related to the concanavalin A (Con A)-induced suppressor system. To generate suppressor T lymphocytes, purified human T lymphocytes were incubated for 3 days in the first culture, with or without Con A. These lymphocytes were irradiated with various doses of X-ray before, mid or after the culture. After doing a second culture for 6 days, we measured the suppressive influence of these cells on T lymphocyte proliferation rates stimulated with allogeneic mononuclear cells, and B lymphocyte proliferation rates stimulated with pokeweed mitogen. Irradiation (1,000 rad) of cultures to which Con A had not been added induced much the same level of suppressor activity as seen in the cultures with Con A. The suppressor activity gradually increased with lapse of time from the irradiation to the suppressor cell assay. Suppressor T lymphocytes were resistant to X-ray irradiation and independent of DNA synthesis. On the other hand, irradiation-induced enhancement was minimal in cultures incubated with Con A, regardless of the irradiation time. As irradiation of human peripheral blood T lymphocytes was found to induce a suppressor function in vitro, clinical and experimental applications of irradiation in cases of a suppressed T lymphocyte function may be feasible.     

Stimulation of Human Peripheral Lymphocytes via CD3 and Soluble Antigen Abrogates Specific Antibody Production by Reducing Memory B Cell Numbers

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti-CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen-specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen-specific antibodies were obtained after stimulation with anti-CD3 antibodies for 7 days. Addition of a soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen-specific response. The addition of IL-2, IL-4, anti-CD40 or anti-CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen-specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen-specific B cell response could be completely restored only if the soluble antigen was cross-linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.     

脐血来源间充质干细胞可抑制异体T细胞的反应

目的研究脐血间充质干细胞(HUCB-MSCs)对异体T细胞的抑制作用。方法体外培养HUCB-MSCs,流式细胞术测表面标记;取正常人外周血,免疫磁珠分离CD3+T细胞,将分离的CD3+T与HUCB-MSCs 1∶1混合培养5 d,PHA刺激或不刺激,采用3H-TdR掺入法观察T细胞增殖,ELISA方法检测细胞因子,流式细胞术观察细胞凋亡。结果 HUCB-MSCs呈纺锤样的细胞形态,不表达CD14、CD34、CD45、HLA-DR,而表达CD29、CD44、HLA-ABC。HUCB-MSCs抑制PHA引起的T细胞增殖(5 230±550 vs 10 500±800 counts/min,P<0.001);HUCB-MSCs还能抑制异体T细胞分泌IFN-γ(510±60 vs 1 580±100 pg/mL,P<0.001)和TNF-α(590±20 vs 1 180±30 pg/mL,P<0.001),上调IL-4(16.3±8.2 vs 4.1±1.8 pg/mL,P<0.001)和IL-10(105±5 vs 17±2 pg/mL,P<0.001)分泌;HUCB-MSCs不诱导T细胞的凋亡。结论 HUCB-...     

Beta adrenergic receptors in lymphocyte subpopulations

To further evaluate the potential utility of lymphocyte beta adrenergic receptor assays in the study of receptor alterations in human disease, we studied highly purified populations of B and T lymphocytes in peripheral blood to see if differences existed in the concentration or affinity of beta adrenergic receptors and catecholamine-responsive cAMP levels. The mean number of receptors present in particulate fractions of B cells did not differ significantly from the number found in T cells. Similarly, no significant difference in the dissociation constant for (-)[3H]dihydroalprenolol was found. Cyclic adenosine monophosphate (cAMP) accumulation in whole lymphocytes as measured by radioimmunoassay was comparable, although a tendency toward lower basal and stimulated levels in the T cells was evident. The data suggest that differences observed in concentrations of beta adrenergic receptors or catecholamine-responsive cAMP accumulation in lymphocytes from patients with varying illnesses are not likely to be due to differences in the proportions of circulating B and T lymphocytes.     

Differences in effect of isoproterenol stimulation on levels of cyclic AMP in human B and T lymphocytes.

The level of cyclic AMP (cAMP) in human lymphocytes in the presence or absence of isoproterenol stimulation was studied in various lymphocyte subpopulations containing different proportions of B and T lymphocytes. Lymphocytes from thymus and peripheral blood (which contain a majority of T cells) were more sensitive to isoproterenol action than lymphocytes from tonsils or adenoids (where B cells are predominant). Using purified B or T lymphocytes from peripheral blood, tonsils or adenoids, we observed an absence of B lymphocyte response to isoproterenol, whereas T lymphocytes, which had a lower basal c-AMP level than B cells, exhibited in all experiments a significant increase in cAMP content after isoproterenol stimulation. The intensity of the response varied in the different T lymphocyte subpopulations.     

In vitro induction of lymphocyte responsiveness by aStrongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected withStrongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults ofS. vulgaris. In addition,S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by theS. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary forS. vulgaris mitogen-induced transformation of spleen cells. TheS. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells andS. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae ofS. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that theS. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the -globulinaemia, of which IgG(T) is a major component, inS. vulgaris infected horses.     

Immature B cells in fetal development and immunodeficiency: studies of IgM, IgG, IgA and IgD production in vitro using Epstein-Barr virus activation

Epstein-Barr virus has been used as a B cell mitogen to explore the parallels between the B cells found in patients with hypogammaglobulinemia and the immature B cells in fetal tissues. Peripheral blood lymphocytes from 29 cases of late onset hypogammaglobulinemia (common variable immunodeficiency) and from 10 cases of X-linked hypogammaglobulinemia were depleted of T lymphocytes and stimulated with virus in vitro. Immunoglobulin production was measured over a 4-week culture period using inhibition radioimmunoassays for IgM, IgG, IgA and IgD. The results were compared with those seen with fetal liver cells, cord blood lymphocytes and adult lymphocytes. Virus-stimulated cells from fetal sources produced small amounts of IgG and IgA relative to IgM, the ratio of IgM to IgG in the second week being in all cases greater than 10. Similar patterns were seen in 25/29 cases of late onset hypogammaglobulinemia, and in the eight cases of X-linked hypogammaglobulinemia that responded in vitro. In contrast, the ratio of IgM to IgG was always less than 8 in cultures of normal adult peripheral blood or bone marrow lymphocytes, and also in cultures from four cases of hypogammaglobulinemia known independently to have abnormal circulating suppressor cells. Eight cases of selective IgA deficiency showed reduced IgA production; six of these showed a normal ratio of IgM to IgG production. Thus, the B lymphocytes which circulate in many patients with hypogammaglobulinemia are functionally immature.     

Expression of the costimulator molecules, CD40 and CD154, on lymphocytes from neonates and young children

Differential expression of the costimulator molecules CD40 and CD154 on neonatal lymphocytes may be one explanation for limited T-dependent antibody responses in human neonates. CD40 was expressed at similar levels on resting B cells from adults, young children (2-20 months of age) or cord blood. CD40 expression was higher on cord blood B cells compared to adult B cells after stimulation with PMA and ionomycin, but similar on adult and cord blood B cells activated by CD3-stimulated T cells. In contrast to previous reports, cord blood T cells stimulated with PMA and ionomycin expressed adult levels of CD154 initially, but this expression was more transient on cord blood T cells. When adult and cord blood mononuclear cells were stimulated with CD3 mAb, T cells from some cord blood specimens showed different kinetics of CD154 expression compared with adult T cells. However, some cord blood specimens showed adult patterns of T cell CD154 expression. When mononuclear cells were depleted of B cells and monocytes prior to stimulation with CD3 mAb, the MFI and percentage of T cells expressing CD154 increased, with adult and cord T cells showing similar patterns of expression. These results show some differences in expression of CD40 and CD154 between neonatal and adult lymphocytes, but do not directly account for the relative deficiencies of humoral immunity in neonates.     

Aging and Lymphocyte Subsets in the Spleen and Peripheral Blood of the Sprague-Dawley Rat

This study was undertaken to determine the effects of aging on lymphocyte subsets in the peripheral blood and spleens of Sprague-Dawley rats. Rats aged 3, 13 and 26 months were used in the study. Analyses of dual labeled lymphocytes from the 26 month animals show decreases in the numbers of lymphocytes due to decreased cellularity (spleen) or reduced lymphocyte percentages within the total white blood cell population (peripheral blood). In the spleens and blood of the oldest rats, there were reduced numbers of Total T, T helper/ amplifier (T_h/a), virgin T_h, and natural killer (NK) cells. Other changes were observed in the spleen but not peripheral blood. The numbers of T cytotoxic/suppressor cells (T_c/s) B cells, “autoimmune” B cells and NK cells were reduced in the spleen but remained within normal limits in peripheral blood. The data show aging exerts different effects on the peripheral blood and splenic compartments of the immune system. These differences may have teleological significance in relation to immune responses to xenobiotics and neoplastic cells.     

Human thymus cells. Excellent responders but poor stimulators in ''One Way'' mixed lymphocyte reaction.

Human allogeneic 'one-way' mixed lymphocyte reactions between thymus cells and thymus cells were entirely absent. Of twenty-one mixed lymphocyte reactions between peripheral blood lymphocytes as responding cells and thymus cells as stimulating cells, only eleven had a weak but significant reaction. In contrast, a highly significant response was observed in each of eighteen mixed lymphocyte reactions between thymus cells as responding cells and peripheral blood lymphocytes as stimulating cells and in each of eleven mixed lymphocyte reactions between peripheral blood lymphocytes and peripheral blood lymphocytes. These findings indicate that the thymus cells (T lymphocytes) possess excellent proliferative capacity, with little or no stimulating capacity, while peripheral blood lymphocytes (T and B lymphocytes), on the other hand, are good responders, as well as good stimulators, in the mixed lymphocyte reaction.