Mitogen-induced Blast Transformation Research Articles

The effect of calyx infusion of hibiscus sabdariffa l. On t-cells-mediated immune response in mitogen-induced blastogenesis of human lymphocytes in vitro

Free radical production and disturbance in the redox status may modulate the expression of a variety of immune and inflammatory molecules. Antioxidants could inhibit the proliferation of T lymphocytes induced by mitogens. The aim of this study was to investigate the effect of the infusion of dried calyx of Hibiscus sabdariffa L. on T-cell-mediated immune response. We examined the effect of the infusion on spontaneous and mitogen-induced blast transformation of human lymphocytes in vitro. Peripheral blood lymphocytes from heparinised venous blood of healthy subjects (n=17) were examined. Five and 10 mg ml-1 phytohemagglutinin (PHA) and 5 mg ml-1 pokeweed mitogen (PWM) were added. Depending on the concentration, calyx infusion of H. sabdariffa decreased mitogen-induced blastogenesis in normal subjects. It did not affect, however, the level of spontaneous proliferation of human lymphocytes in vitro. The infusion of hibisci flos may alleviate over-stimulated condition of the immune system in hypersensitivity ...

Effect of autologous sera on mitogenic stimulation of peripheral blood lymphocytes of mini-pigs chronically intoxicated with alcohol

There are no statistically significant differences in the stimulation index for purified lymphocytes of control mini-pigs and mini-pigs with chronic alcohol intoxication. Autologous sera of control and experimental animals strongly suppress mitogen-induced blast transformation of lymphocytes without death of these cells. There are no statistically significant intergroup differences in the absolute number (per mm3) of T lymphocytes in peripheral blood.

Decreased serum dipeptidyl peptidase IV activity in major depression

It has been recently shown that severe depression is characterized by immune dysfunctions such as blunted mitogen-induced blast transformation, which is linked to interleukin-2 (IL-2) mechanisms, and to autoimmune responses. In order to explore one of the putative pathophysiological mechanisms underlying both factors, we have measured the predexamethasone and postdexamethasone serum dipeptidyl-peptidase IV (DPP IV) activity in depressed inpatients and normal controls. This enzyme is an important mediator of IL-2-related blast proliferation, and it may play a role in autoimmunity. We found significantly lower DPP IV levels in major depressives as compared with healthy controls, and melancholics exhibited significantly lower enzyme activity than minor depressives. There was a significant negative correlation between serum DPP IV activity and the severity of illness. However, we were unable to detect any significant relationships between DPP IV on the one hand, and mitogen-induced blast transformation, soluble IL-2 receptor accumulation in PHA culture supernatant, total number of leukocytes and lymphocytes, T lymphocytes, CD4 + and CD25 + cells, on the other. Men exhibited significantly higher serum DPP IV levels than women.

A further exploration of the relationships between immune parameters and the HPA-axis activity in depressed patients

In order to investigate the relationship between the immune apparatus and the hypothalamic-pituitary-adrenal (HPA)-axis activity in depressed patients, we measured in vitro lymphocyte responses to the mitogens Phytohaemagglutinin (PHA), Pokeweed (PWM) and Concanavalin A (Con A) and 8 a.m. baseline cortisol values in plasma, free cortisol excretion in 24 h urine (UFC), basal and post-dexamethasone beta-endorphin values. Major depressed patients with melancholia/psychotic features exhibited a significantly lower mitogen-induced blast transformation as compared to minor and simple major depressed patients. The lymphocyte responses to the three mitogens were significantly inversely related to baseline cortisol values and postdexamethasone beta-endorphin values. The proliferative capacity of lymphocytes to stimulation with PHA and PWM was significantly and positively related to UFC excretion. Up to 45% of the variance in the immune-responses to the mitogens was explained by the baseline cortisol, post-dexamethasone beta-endorphin and UFC values.

Phosphotyrosine phosphatases are involved in reversion of T lymphoblastic proliferation

Resting T lymphocytes can be activated by mitogens or antigens to become T blasts, which revert spontaneously both in vivo and in vitro in extended cultures to secondary, memory T lymphocytes. We have studied the role of phosphotyrosine phosphatases (PTPase) in the reversion of lymphoblasts in extended, phyto-hemagglutinin-stimulated cultures of human T lymphocytes. Membrane-associated PTPase activity is high in resting T cells, but decreased during mitogen-induced blast transformation. When the blasts were reverting to lymphocytes, the PTPase activity increased more than twofold concomitantly with an elevated surface expression of CD45. When T blasts from phytohemagglutinin-activated cultures were kept in the presence of sodium orthovanadate, an inhibitor of PTPase, they maintained their lymphoblastic proliferation and did not revert to resting lymphocytes. This was accompanied by retention of a 48-kDa phosphotyrosine-containing protein. Our data indicate an important role for PTPase in the transition of lymphocytes from an activated to a resting stage.

Effect of luteotrophic hormone (LH) and thymosin on mitogen-induced blast transformation of Balb/c mouse lymphoid cells

The present study investigates the effect of a pituitary hormone (LH) and a thymic factor (Thymosin Fraction 5 = TF5) on in vitro and in vivo spleen cell proliferative response of 2, 6 and 8-month-old Balb/c mice. In vitro experiments showed that the addition of LH at various concentrations (0.5, 5, 50 or 500 ng/ml) to cultures increased significantly the proliferative response to some known mitogens (ConA, PHA, PWM). The LH stimulation was further enhanced by subsequent addition of TF5 to these cultures. However, the addition of LH to LPS cultures resulted in a blockage of the cell growth which persisted after the addition of TF5. In vivo experiments showed that injection of LH (5 or 50 ng/ml) to 2, 6 and 8-month-old Balb/c mice had a significant increase on blast transformation of lymphoid cells following their incubation with ConA, PHA and PWM, and a significant decrease when they were incubated with LPS. The physiological significance of these findings which point out an intimate connection between immune and endocrine functions is discussed.

Increased concanavalin A-induced suppressor cell activity in humans with occupational lead exposure

E-rosette-forming cells (E-RFC), mitogen-induced blast transformation, OKT4+, OKT8+ cells, and their ratio were found to be normal in 10 subjects chronically exposed to lead with blood levels of 40-51 micrograms%. However, concanavalin A (Con A)-induced suppressor cell activity (SCA) in these subjects was significantly greater than in normal matched controls. The clinical relevance of this observation is not clear, but it may have some bearing on the various immunologic defects described in lead exposure.

Transglutaminase in cell proliferation and transformation.

Transglutaminase (TGase) activity was reduced in intact mitogen-stimulated human peripheral blood lymphocytes (PBL) when compared to intact resting PBL. Moreover, a treatment of the same quiescent immunocompetent cells with purified liver TGase and Ca2+ completely suppressed the mitogen-induced blast transformation. A decrease in TGase activity in neoplastically transformed seminal vesicle epithelial cells with respect to their normal parent counterpart was also observed. Our data support the notion of a possible implication of TGase in cell proliferation and transformation.

Effects of lentinan on cytotoxic functions of human lymphocytes.

The in vitro effects of lentinan on natural killer (NK), antibody-dependent cell-mediated cytotoxicity (ADCC), lectin-dependent cell-mediated cytotoxicity (LDCC) and mitogen-induced blast transformation were studied in patients with solid tumors and chronic lymphocytic leukemia (CLL). NK activity was measured against 51Cr-labelled K-562 targets, ADCC against antibody-coated chicken red cells. LDCC and natural cell-mediated cytotoxicity (NCMC) was assessed using 3H-thymidine prelabelled HEp-2 targets. Mitogen (PHA- and Con A-) induced blast transformation was measured by thymidine incorporation. Blastogenesis and LDCC was not influenced by lentinan. 1 microgram/ml lentinan increased NCMC of tumor-bearing subjects. The most prominent enhancement of NK and ADCC activity was seen in CLL patients, where a dose-related increase was seen (from 0.01 to 1 microgram/ml).

1,25-Dihydroxyvitamin D-induced suppressor cells in uremic versus normal lymphocytes.

1,25-Dihydroxyvitamin D3 (DHD) has been shown to suppress mitogen-induced blast transformation. This inhibition is abolished by prior elimination of adherent cells. Chronic renal failure is an immunodeficiency state on the one hand and is associated with abnormalities in vitamin D metabolism on the other. The effect of DHD on the induction of suppressor cells in uremic vs. normal peripheral blood mononuclear cells was investigated. Study groups included 16 chronically uremic patients and 16 age- and sex-matched controls. DHD induced suppressor cell activity in normal lymphocytes. However, no suppressor cell activity was observed in lymphocytes from the uremic patients preincubated with DHD. The origin of the responder cells (normal or uremic) did not affect the outcome. The results would suggest that monocyte-adherent suppressor cells from uremic subjects are either incapable of binding DHD or fail to mount a normal post-receptor intracellular chain of events culminating in suppressor activity.

Potential mechanisms of minoxidil-induced hair growth in alopecia areata

In vivo, topical minoxidil therapy is associated with changes in the follicular epithelium, tissue and blood lymphocyte populations, lymphocyte blastogenic response to mitogens, and perifollicular vasculature. Biopsy specimens taken from areas of terminal hair regrowth show a dose-dependent increase in hair follicle length, a decrease in tissue lymphocyte populations associated with a simultaneous increase in peripheral blood lymphocyte counts, and reopening of previously closed lumina of perifollicular vessels. Responder lymphocytes show pretreatment-increased concanavalin A and phytohemagglutinin-induced blastogenesis, which decrease toward control values after treatment. In vitro, at concentrations approximating the range of tissue levels in patients treated topically with the 5% solution, minoxidil affects both epithelial cells and lymphocytes in tissue culture. Cultured murine epithelial cells show increased cell proliferation and delayed senescence. Cultured human lymphocytes show suppression of mitogen-induced blast transformation. Differential effects on responder, nonresponder, and control lymphocytes are seen. Minoxidil may induce hair regrowth in alopecia areata by a synergistic stimulatory effect on follicular epithelium and a suppressive effect on lymphocyte-mediated immunologic phenomena. A contributing role for its vasodilatory properties must also be considered.

Immunosuppression-induced defective lymphocyte proliferation to murine cytomegalovirus is prolonged following the cessation of immunosuppression.

Defective murine cytomegalovirus (MCMV) antigen-specific proliferation, induced by treatment of MCMV-infected mice with either antilymphocyte globulin (ALG), prednisolone, or both agents, was eventually restored following the cessation of immunosuppression. At 100 and 278 days following the end of immunosuppressive therapy splenic lymphocytes from infected and subsequently immunosuppressed mice responded significantly in vitro to soluble MCMV antigen after having lost this response immediately upon initiation of immunosuppression. Circulating specific antibodies and mitogen-induced blast transformation were comparable between infected mice and infected mice that also were immunosuppressed. At 278 days following the cessation of immunosuppression splenocytes from infected mice that had been treated with ALG yielded greatly increased background proliferation. Nylon-wool adherence was used to obtain enriched populations of T cells, and B cells and monocytes from MCMV-infected mice. While T cells alone did not respond in vitro to MCMV antigen, recombining B cells and monocytes with the T cells reconstituted in vitro proliferation. Defective lymphocyte proliferation to MCMV for an extended period of time following the end of immunosuppressive therapy indicated a prolonged inability to respond to an active MCMV infection. Identification of the cellular basis for the proliferation defect might lead to the development of effective immunotherapy.

Barbiturate inhibition of lymphocyte function. Differing effects of various barbiturates used to induce coma.

The present studies evaluated the effect of phenobarbital, pentobarbital, and thiopental at concentrations comparable to those attained during therapeutic barbiturate-induced coma, on in vitro mitogen-induced lymphocyte activation. Lymphocytes from normal volunteers were incubated for 72 hours in culture medium containing mitogen (phytohemagglutinin) and a range of concentrations of the barbiturates (5 to 833 microgram/ml). Three parameters of lymphocyte activation (mitogen-induced blast transformation, 3H-thymidine incorporation, and cell proliferation) were all suppressed by the barbiturates. The suppression was dose-dependent. The greatest suppression was caused by the short-acting barbiturate, thiopental. Lymphocyte responses were much less affected by the long-acting barbiturate, phenobarbital. The intermediate-acting barbiturate, pentobarbital, was also intermediate in its ability to inhibit lymphocyte activation. The two-to threefold difference between the effects of thiopental and pentobarbital on lymphocyte function may have direct clinical relevance, since it is primarily these two agents that are employed to induce therapeutic "barbiturate coma." Since lymphocyte suppression appears to be much more marked in the presence of thiopental, these observations support a role for the other barbiturates in programs of induced coma.

A nuclear antigen associated with cell proliferation and blast transformation.

A nuclear antigen associated with cell proliferation (proliferating cell nuclear antigen-PCNA) and blast transformation is recognized by autoantibodies in the sera of some patients with systemic lupus erythematosus. This autoantibody is a precipitating antibody and also reacts in immunofluorescence, staining the nucleoplasm of proliferating and blast-transformed cells. The autoantibody was used as a reagent to determine the distribution of PCNA in a synchronized continuous B lymphoid cell line (WiL-2) and in mitogen-induced blast-transformed lymphocytes. In WiL-2 cells, PCNA was detected as speckled nucleoplasmic staining in G1, S, and G2 phases of the cell cycle. In addition, during late G1 and early S phases, PCNA was also detected in the nucleolus. During mitogen-induced blast transformation of lymphocytes, PCNA was noticed in the nucleolus before the initiation of DNA synthesis and later became nucleoplasmic with disappearance of nucleolar staining. These studies demonstrate that the relationship of PCNA to proliferation and blast transformation may be associated with events related to DNA synthesis in these cells.

Immediate loss of cell-mediated immunity to murine cytomegalovirus upon treatment with immunosuppressive agents.

Splenic lymphocytes from cytomegalovirus-infected mice lost their in vitro proliferative responses to cytomegalovirus antigen within 3 h after in vivo treatment with antilymphocyte globulin and prednisolone. The response was inhibited when the agents were administered separately or together, and inhibition persisted through a 2-week course of immunosuppression. Circulating specific antibodies were depressed by multiple injections of antilymphocyte globulin alone or with prednisolone, but not by prednisolone alone. Mitogen-induced blast transformation was immediately depressed by immunosuppression with both agents. Although the response to lipopolysaccharide returned briefly, it declined with continuing treatment. Cytomegalovirus infection augmented the depressive effect of immunosuppression on the lipopolysaccharide proliferative response. Prednisolone treatment of infected animals did not affect the concanavalin A response, and lipopolysaccharide stimulation decreased more slowly and to a lesser extent than it did in mice treated with antilymphocyte globulin or both agents. Loss of specific cell-mediated immunity and simultaneous depression of humoral immunity indicated that immunosuppression immediately created an inability to respond to an active cytomegalovirus infection.

Steroid-induced inhibition of nucleoside uptake in isolated mouse thymocytes

In view of the evidence suggesting a possible effect of high concentrations of steroids on membrane properties, we have investigated the effect of several steroid molecules on the uptake and incorporation of [ 3H]uridine in isolated mouse thymocytes. Our results demonstrate that the sex steroids, the estrogenic compound, diethylstilbestrol, and several non-hormonal steroid molecules induce a marked inhibition of nucleoside uptake. This effect, which occurs only at concentrations above 10 −6M, is almost instantaneous but transient and does not therefore appear to be mediated through specific receptor occupancy. Since sex steroids have been shown to inhibit mitogen-induced blast transformation at concentrations close to 10 −5M, we suggest that this membrane effect of sex steroids may partly explain their immunosuppressive effects.

Endocytosis and exocytosis of phytohemagglutinin (PHA) cell surface receptors of human lymphocytes during blast transformation

The ultrastructural distribution of T lymphocyte surface membrane receptors for phytohemag-glutinin (PHA) during blast transformation is examined using PHA covalently coupled to ferritin (PHA-Fe). Human peripheral blood lymphocytes from normal donors were enriched for T cells by nylon wool elution and cultured in vitro with PHA-Fe at a concentration known to cause maximal stimulation of DNA synthesis as measured by [ 3H]thymidine incorporation. Over the course of a 72 h incubation period, cell samples were harvested at regular intervals and examined by transmission electron microscopy. Within several minutes of culture at 37 °C the majority of the ferritin (Fe)-labeled PHA surface receptors on almost all cells undergo rapid endocytosis; some Fe label remains at the cell surface. After several hours, endocytotic vesicles containing Fe-labeled receptors coalesce and undergo condensation. Within 36–48 h, most endocytotic vesicles transform into multivesicular bodies (MVBs). After 48–72 h, 70–80% of the cells had the ultrastructural appearance of blast transformation as characterized by increased size, euchromatic nuclei, nucleolonema and polyribosomes. In 40 % of the blast cells the Fe-labeled MVBs are exocytosed to the cell surface; cytoplasmic MVBs in the remaining portion of the blasts and non-blast lymphocytes do not appear to undergo exocytosis. Although endocytosis and exocytosis of lymphocyte surface receptors during mitogen-induced blast transformation are observed, the role and significance of receptor redistribution to cell activation remains unclear.

Lymphocyte Mitogen Reactivity and Enumeration of Circulating B- and T-Cells During Feline Leukemia Virus Infection in the Cat23

Mitogen-induced blast transformation of peripheral blood lymphocytes and quantitative changes in circulating T- and B-cells were studied serially in cats inoculated with feline leukemia virus (FeLV). Concanavalin A-induced blast transformation sharply declined beginning at 5 weeks post inoculation (Pl) in FeLV-infected cats when compared to age-matched uninfected control cats. Similar but less consistent changes were seen in responses to pokeweed mitogen-induced stimulation. In most infected kittens this defect persisted until they died from thymic lymphosarcoma, 15-24 weeks Pl. An early lymphopenia, due primarily to a decrease in circulating B-cells, occurred in infected cats 5-8 weeks Pl. Following a return of total and B-lymphocytes to control values, infected cats developed increased numbers of T-cells at 16 or more weeks Pl, which correlated with circulating lymphoblastic lymphocytes bearing T-cell markers. These results correlated neoplasia arising in a thymus-derived lymphocyte population with mitogenic hyporeactivity in the preneoplastic period and suggested that FeLV-induced immune alterations may be a necessary antecedent of leukemogenesis in the cat.

Modulation of lymphocytic responses by factors in human plasma. I. The effects on normal, stimulated lymphocytes of various normal plasmas in microplate culture.

Mitogen-induced blast transformation of human peripheral blood lymphocytes was studied using a microplate culture technique. Cells and plasmas from normal individuals were interchanged to that sources of variation in the degree of transformation could be evaluated. Also evaluated was the normal range of plasma effects. It was found that the cells of different individuals vary considerably in their responses to mitogens in a given plasma, but the hierarchy of responses is fairly consistent from plasma to plasma. Furthermore, plasmas vary from donor to donor, but also settle into a fairly consistent hierarchy regardless of the cells employed. Finally, new hierarchies are established when materials from the same donors are collected after an interval of a week. It was shown that special, nonrandom interactions may occur which are not attributable simply to the average response of a given cell and the average response to the plasma used. The range of variation to be expected in a normal system, established here, should permit comparison of plasma effects in a more interpretable framework than previously was possible.