Blast Transformation Of Human Lymphocytes Research Articles

Influence of Paleobacteria on the Proliferative Activity of Human Lymphocytes In Vitro

When testing the proliferative activity of 14 strains of permafrost microorganisms in the reaction of blast transformation of human lymphocytes in vitro, a strain (Alcaligenes sp.) with mitogen properties was isolated (20-fold increase in the rate of lymphocyte proliferation in comparison with the control). Four strains activated lymphocyte proliferation by 3-9 times in comparison with the control. Three strains produced substances with cytostatic properties and reduced proliferation activity by 33-43% and one strain (Bacillus sp.) almost completely suppressed phytohemagglutinin-induced lymphocyte proliferation. These data indicate that strains with a unique immunobiological potential are concentrated in the population of permafrost microorganisms that have undergone rigorous evolutionary selection.

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 ...

Effects of arsenic on blast transformation and DNA synthesis of human blood lymphocytes

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes were biphasic: the chemicals at very low concentrations enhanced blast transformation and DNA synthesis, whereas higher concentrations inhibited the transformation and DNA synthesis. The concentrations of arsenicals at which the maximum stimulating effect was found were 1 × 10 −5 M, 1 × 10 −6 M or 2 × 10 −6 M, and 0.8 × 10 −6 M or 1 × 10 −6 M for sodium arsenite exposure of 1 h, 3 days and 6 days, respectively; for sodium arsenate, 1 × 10 −5 M, 1 × 10 −5 M, and 2 × 10 −6 M or 5 × 10 −6 M, respectively. Arsenicals must be present for the entire 6 days culture period to produce maximum stimulation of blast transformation of human lymphocytes. The longer exposure of the lymphocytes to arsenicals, the lower the concentrations of arsenicals at which the maximum stimulating effect was found. The stimulating effect of trivalent arsenic (sodium arsenite) was stronger than pentavalent arsenic (sodium arsenate).

Regulation ofin vitro human lymphocyte blast transformation associated with γ-globulin fraction containing a carbohydrate component

Carbohydrate-enriched components were isolated from human serum γ-globulin by chelation of associated transition metals. Similar to the initial γ-globulin preparation, these components stimulatein vitro blast transformation of human lymphocytes from healthy donors and inhibit subsequent phytomitogen-induced lymphocyte blast transformation.

Na, K-ATPase pump in activated human lymphocytes: on the mechanisms of rapid and long-term increase in K influxes during the initiation of phytohemagglutinin-induced proliferation

Functional expression of Na, K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and α-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20–24 h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na, K-ATPase pump during blast transformation of human lymphocytes.

Long-term enhancement of Na,K-ATPase pump during blasttransformation of human lymphocytes is controlled first by translational, then by transcriptional mechanisms

The transition of phytohemagglutinin-activated human lymphocytes from resting state to proliferation is accompanied by a long-term increase in ouabain-sensitive Rb(K) influx which is closely related to a cyclosporin A-sensitive step of G0/G1/S progression. At least two distinct phases of the up-regulation of cation pump has been revealed: the initial stage (5–20 h) which is cycloheximide-inhibitable and actinomycin D (α-amanitin)-unaffected, and the later stage (after 20 h) which is cycloheximide- and actinomycin D (α-amanitin)-inhibitable. Thus, the enhanced Na,K-ATPase pump during the cell progression from quiescence to proliferation is controlled both at translational and transcriptional levels.

Influence of ganglioside GM3 and its breakdown products on lymphoblastic transformation and T‐suppressor activity

The influence of ganglioside GM3 and some of its breakdown products on phytohemagglutinin-induced blast transformation of human lymphocytes and concanavalin-A-induced T-suppressor activity was studied. The structures of two major hydrolysis products of GM3 were established by negative-ion fast-atom-bombardment mass spectrometry as neuraminyllactosylsphingosine (NeuLacSph) and neuraminyllactosylceramide (NeuLacCer). Both substances were shown to be potent inhibitors of mitogen-induced lymphoblastic transformation whereas their acetylation products NeuAcLacSphAc and GM3 did not affect the proliferative response of lymphocytes to phytohemagglutinin. On the other hand, only GM3 and NeuLacSph were able to enhance concanavalin-A-induced T-suppressor activity. On the basis of these data, it is suggested that the effects of GM3 and its breakdown products on lymphoblastic transformation and T-suppressor activity must rest on different mechanisms and that N-deacylation of GM3 appears to be an essential step in conversion of the ganglioside into an inhibitor of lymphocyte blast transformation.

Inhibitory effects of carcinogenic tryptophan pyrolysis products on phytohemagglutinin-induced blast transformation of human lymphocytes

In order to evaluate the effect of carcinogenic heterocyclic amines on the stimulus-reaction system of human cells, we examined the effect of 5 kinds of carcinogenic heterocyclic amines including 2-amino-3-methylimidazo [4,5- f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2- a3',2'- d)imidazole (Glu-P-1), 2-amino-dipyrido[1,2- a:3',2'-( d]imidazole (Glu-P-2), 3-amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole (Trp-P-1) and 3-amino-1-methyl-5 H-pyrido[4,3- b] indole (Trp-P-2) on human lymphocyte (4 × 10 5 cells/ml) blast transformation induced by 0.125% phytohemagglutinin (PHA). Both Trp-P-1 and Trp-P-2 exhibited an inhibitory effect on the lymphocyte proliferation stimulated by PHA at the final concentration of 1.0–5.0 and 1.0–10 μM, respectively. The concentrations of Trp-P-1 and Trp-P-2 causing 50% inhibition were 3.1 and 5.1 μM, respectively. However, the other carcinogenic heterocyclic amines examined did not show any inhibitory effect on lymphocyte mitogenesis even at a final concentration of 10 μM. Viabilities of lymphocytes were more than 90% ( n = 3 × 5) when 5 kinds of carcinogenic heterocyclic amines (0.1 ml) were respectively added at the final concentration of 10 μM to the lymphocyte culture medium (4 × 10 5 cells/0.9 ml).

Effect of the inhibitory material from male genital tract on natural killing activities

A male inhibitory material (HuSePl-Frl) was separated from normal human seminal plasma by gel filtration on Sephadex G-100. This material has high avidity for cell membranes of human red blood cells (RBC). In vitro, PHA-M-induced blast transformation of human lymphocytes is inhibited by small quantities of HuSePl-Frl. This inhibition was also evident when RBC coated with HuSePl-Frl is added to mitogen stimulated lymphocytes in cultures. Small quantities of HuSePl-Frl are inhibitory to natural killer (NK) cell activation. The modulating role of the HuSePl-Frl in preventing the spermatozoal rejection during fertilization is discussed.

The Synthesis of Folate‐binding Protein in Lymphocytes during Transformation

The blast transformation of human lymphocytes exposed to phytohaemagglutinin was associated with an increase in the intracellular concentration of a folate-binding protein. The protein was measured by a radioimmunoassay and by immunofluorescent microscopy using an antiserum raised against purified folate-binding protein prepared from chronic myelogenous leukaemia cells. Because it has been previously shown that protein-bound folate cannot function as a cofactor or substrate, the synthesis of this binder during some phase of the replicative cycle may serve to modulate folate-dependent RNA and DNA synthesis by controlling the concentration of free folate.

Increase of UV-induced DNA repair synthesis during blast transformation of human lymphocytes

UV-induced DNA repair synthesis, measured as unscheduled DNA synthesis, was studied in human peripheral lymphocytes in various phases of the cell cycle. Mitogen transformation of the lymphocytes was effected with phytohemagglutinin (PHA), and the stage in the cell cycle was determined by measuring the Feulgen DNA content and the dry mass in individual cells by cytophotometry. The initial rate of repair was determined by autoradiography after UV-light irradiation (19.2 J/m 2) and incubation of the cells for 30 min with [ 3H]thymidine. When the cells progressed from the G0 to the G1 phase there was a 3-fold increase in the grain count. The correlation between the grain count and the dry mass indicated an increase in the initial rate of repair during the progression of cells from G0 to G2 phase. G2 cells were more heavily labelled than those in G1, but there did not seem to be any difference between these two phases as regards the relationship between grain count and DNA content. The results indicate that the initial rate of UV-induced DNA repair may differ in various phases of the lymphocyte cell cycle.

Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response.

Ability of Fab-fragments of normal rabbit and human γ-globulins to inhibit blast transformation of human lymphocytes induced by phytomitogens

Monovalent and bivalent Fab fragments from normal human and rabbit γ-globulins were shown to suppress blast transformation of human lymphocytes induced by phytohemagglutinin and concanavalin A. The pepsin F(ab')2 fragments obtained from highly purified rabbit antidinitrophenyl antibodies possess similar properties. An inhibitory action of the fragments was observed when they were added to the culture both simultaneously with and 24 and 48 h after the mitogen. The results may mean that suppression of lymphocyte transformation by fragments of γ-globulins is not due to their composition with the mitogens for receptors on the target cells; activity of the Fab fragment is evidently determined by structures located outside the combining site of the antibody.

IN VITRO STIMULATION OF HUMAN LYMPHOCYTES BY BORDETELLA PERTUSSIS

Bordetella pertussis (B.p.) induces blast transformation of human lymphocytes; whole killed B.p. are more efficient than extracts obtained by sonication. Similar responses were obtained with each of the four strains used in the Danish pertussis vaccine. B.p. with low amounts of Protective Antigen and Histamine-Sensitizing Factor also induced lymphocyte transformation, but were less toxic to the lymphocytes at high concentrations. The supernatants of B.p. cultures were purified with respect to Lymphocytosis Promoting Factor; evidence is presented that these purified fractions possess T-lymphocyte mitogenic activity. Lymphocytes from all normal humans were stimulated by B.p., including cells from cord blood. Cells from childbearing women, obtained immediately after delivery, showed a general depression of lymphocyte transformation including the response to B.p. Children with whooping cough had a lower lymphocyte response to B.p. than healthy children. A highly significant correlation was observed between the responses to B.p. and to E. coli in the adults and newborn examined. It is concluded that the major part of the lymphocyte transformation induced by B.p. is non-specific.

Enhancement of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCMC) by an Interphase Material (IPM) extracted from a non-pathogenic strain of mycobacteria

Interphase Material (IPM) extracted from M. smegamtis has been previously shown to protect mice against experimental tumors, to activate macrophages as measured by in vitro growth inhibition of neoplastic cells, and to induce nonspecific blast transformation of human lymphocytes and murine B cells.

A calcium transport system for mammalian cells

Several decades ago, Heilbrunn showed that the microinjection of a small amount of inorganic calcium into a muscle cell caused contraction of the muscle (1). Since then it has been shown that not only are steady state conditions of many types of cells affected by intracellular calcium concentration (2), but specialized responses evoked by specific stimuli and associated with movement of calcium into the cell (3) can be induced directly by increasing intracellular calcium. For example, calcium ionophores cause insulin secretion by pancreatic islet cells (4), histamine release from basophils (5) and blast transformation of human lymphocytes (6). These findings suggest that one of the necessary steps in the chain of events initiated by the physiological stimulus (e.g. release of histamine from basophils when specific antigen reacts with cell-bound IgE) is a local increase in calcium ion concentration. The physiological role of calcium has been studied most extensively in heart and skeletal muscle. It is now well established that the concentration of free calcium ions inside cells is very low compared to extracellular fluid (7,8). A minimum system for achieving this is a limited cell membrane permeability to calcium and an active transport system for pumping calcium out of the cell. In this review we will present the thesis that mammalian heart and skeletal muscle have in addition a calcium transport system (CTS) which is located external to the electric membrane, is oriented opposite to the cell membrane calcium pump (i.e. pumps toward the cell) and establishes a microdomain immediately external to the electric membrane in which calcium concentration is maintained higher than in the rest of the extracellular space. Since this system is assembled on the outside of the cell, from components circulating in the blood plasma, we have had a unique opportunity to isolate and characterize them. Thus we were able to avoid extraction from the cell membrane structure, the difficult first step facing students of other transport systems.

Lectin-induced blast transformation of human lymphocytes.

Phytohaemagglutinins1 comprise a class of leguminous plant proteins exhibiting differing specificities for the binding of carbohydrate residues. Despite their shared ability to induce specific precipitation of various polysaccharides, glycoproteins, blood group substances and cellular membranes, only a few phytohaemagglutinins are known to be capable of inducing blast transformation of small lymphocytes. Among such phytomitogens are the lectins from Phaseolus vulgaris2, Lens culinaris3, Wistaria floribunda4 and Canavalia ensiformis5,6. The use of concanavalin A (con A) from Canavalia ensiformis in the detection and preliminary characterization of carbohydrate-containing polymers has been established7. Con A also has been shown to be capable of distinguishing between transformed and normal cells; in vitro agglutination of transformed cells occurs under conditions which produce little agglutination of most normal cells8. A similar preference of con A for tumour cells is exhibited in vivo, resulting in diminished survival of mouse tumour cells (to 1% at 100 mg kg−1 i.p.) (H. Lin, W. R. Bruce and M. J. Walcroft, submitted for publication). The efficacy of con A as a therapeutic agent, however, has been limited by its toxicity and therefore it is of interest to study: (1) other lectins which might manifest similar biological activities but with diminished toxicity; (2) means of chemical modification which might lower con A toxicity without concomitant loss of activity.