Thymidine Uptake Of Lymphocytes Research Articles

Comparison of antiproliferative effects of 1-histamine-2 receptor antagonists, cimetidine, ranitidine, and famotidine, in gastric cancer cells

In the immune system, histamine is known to suppress cytotoxic T-lymphocytes and mitogen induced lymphocyte thymidine uptake, down-regulate some cytokines, and activate suppressor T-lymphocytes, and in the gastrointestinal system, histamine was reported to have trophic effects on gastrointestinal epithelial cells. Enhanced rates of cell proliferation by histamine are implicated in the pathogenesis of carcinogenesis. This study was designed since there is a lack of comparative data about the cell proliferations of histamine-2 receptor antagonist (H2-RA), cimetidine, ranitidine, and famotidine, in gastric cancer. KATO-III and AGS cell lines were used in this experiment. The concentrations of the histamine and cimetidine were 10 −5, 10 −8M, respectively and those of ranitidine and famotidine were 10 −6-10 −9M, respectively. Cell proliferation after drug treatment was evaluated by direct cell counting, [ 3H]thymidine incorporation, and MTT assay. Activities of ornithine decarboxylase (ODC), a rate limiting enzyme in polyamine synthesis, were measured after each drug treatment. Protein kinase A, a cAMP-dependent protein kinase system, was assayed using [α- 32p]ATP. Histamine showed statistically significant cell proliferating effects in a dose-dependent manner ( P < 0.01), the maximal effect in 10 −5M concentration. ODC activities were increased in accordance with the increment of cell numbers after histamine treatment. Cimetidine reversed the histamine-stimulated cell proliferation significantly, the maximal effect in 10 −5M concentration ( P < 0.01). Although ranitidine showed the tendency to attenuate the cell proliferation dose-dependently, but without statistical significance, famotidine did not show such an effect at all. cAMP-dependent protein kinase activities were significantly increased following 10 −5M histamine treatment, also reversed significantly by cimetidine co-administration ( P < 0.01). Beneficial clinical outcomes could be anticipated from cimetidine treatment in patients with gastric cancer by anti-proliferating effects against gastric cancer cells. These effects of H2-RA are likely to be mediated by specific interactions at the H2-receptor.

Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi.

The study attempted to identify immunologic markers for progression of Leishmania donovani chagasi infection to disease in children in an area endemic for visceral leishmaniasis (VL). [3H]thymidine uptake of lymphocytes stimulated with L. donovani chagasi antigen from children with asymptomatic infection (25,286 +/- 11,648) and from children with self-healing subclinical infection (15,511 +/- 4681) was greater (P = .001) than that observed with lymphocytes from children who progressed to classic VL (4811 +/- 2984). The interferon-gamma (IFN-gamma) levels from asymptomatic and subclinically infected children (74 +/- 90 units/ml) were higher (P = .02) than those observed in children who progressed to VL (7 +/- 8 units/ml). Absence of lymphocyte blastogenesis and IFN-gamma production were associated with progression of infection to classic VL. In the presence of these markers, children should be closely followed to identify signs and symptoms that would permit early initiation of therapy.

Effects of aspirin-like drugs on mitogen-stimulated DNA synthesis of lymphocytes from pregnant rats and offspring.

Previous studies have shown that salicylates and protein-calorie malnutrition compromise immunological responses in humans and experimental animals. The present study compared the effects of prenatal normal and low protein diets, with and without aspirin-like drug treatments, on lymphocyte blastogenesis measured by tritiated thymidine uptake for DNA synthesis in splenic lymphocytes from pregnant rats and their offspring following stimulation with the mitogens concanavalin A, phytohemagglutinin, and pokeweed mitogen. Aspirin treatment was associated with increased lymphocyte thymidine uptake for blastogenesis in pregnant rats fed the normal protein control diet and their offspring. The phytohemagglutinin-stimulated increase detected in offspring lymphocytes could not be statistically guaranteed. A low protein diet alone and a normal protein diet combined with salicylamide treatment was associated with decreased blastogenesis in pregnant rats but not in their offspring. Salicylamide or aspirin combined with a low-protein diet decreased blastogenesis in both dams and their offspring. Aspirin combined with a normal protein diet did not adversely affect blastogenesis in either pregnant rats or their offspring. This study suggests that low dietary protein and aspirin-like drugs may independently decrease lymphocyte blastogenesis of pregnant rats and in combination they may also reduce lymphocyte blastogenesis in offspring. The significance of increased lymphocyte blastogenesis in both mothers and offspring following aspirin treatment of pregnant rats fed a normal protein diet is unclear.

Sensitivity of human lymphocytes to acetaldehyde: Comparison between alcoholic and control subjects

The action of acetaldehyde (ACH), the first metabolite of ethanol, was studied on human lymphocytes stimulated in vitro by phytohemagglutinin (PHA) or Concanavalin A (Con A). ACH caused a dose-dependent decrease of [ 3H]thymidine uptake in lymphocytes from both alcoholic and control subjects. The area under the curve of [ 3H]thymidine incorporation as a function of ACH concentration was determined for each subject and referred to as the lymphocyte sensitivity index. Indexes for alcoholic subjects were found to be higher than those for controls, indicating a lower sensitivity to ACH of lymphocytes from alcoholics. We also found a wide range of sensitivity indexes within the same group. These results are consistent with the current hypothesis that not everybody is at equal risk to develop alcohol related disorders.

Serum deoxythymidine kinase correlates with peripheral lymphocyte thymidine uptake in chronic lymphocytic leukemia.

The serum thymidine kinase (S-TK) and proliferative activity of the leukemic cells were determined in 27 untreated patients with chronic lymphocytic leukemia (CLL). A significant positive correlation between S-TK and proliferation expressed as a proliferative index (PI) was found (r = 0.70, p less than 0.001). Additionally, PI (r = 0.59, p less than 0.01) and S-TK (r = 0.47, p less than 0.05) correlated to peripheral blood lymphocyte count. When different variables and combinations of variables were studied in order to define their capacity for discriminating between progressive and indolent CLL, S-TK activity and PI proved to be powerful indications. In longitudinal studies, both S-TK and PI paralleled disease activity. A model where a combination of S-TK and PI gives information of the degree of localized disease is proposed.

Lymphocyte suppressive peptides from fibrinogen are derived predominantly from the A alpha chain.

Abstract Cellular immune responses can elicit local deposition of fibrin at the site of immunologic reactions, as well as the formation of intravascular fibrin in disseminated reactions. The subsequent physiologic proteolysis of fibrinogen and fibrin by plasmin results in small peptides that suppress lymphocyte functions in vitro and in the immune response in vivo. The intramolecular origin of lymphocyte suppressive activity and the proteolytic events responsible for the release of active peptides have been analyzed. Plasmic peptides from the isolated B beta and gamma constituent chains of fibrinogen did not inhibit mitogen-driven responses of human peripheral blood mononuclear cells. In contrast, plasmic digests of the A alpha chain, but not the intact A alpha chain were suppressive. Advanced plasmic digests of fibrinogen and the A alpha chain were suppressive at similar concentrations, suggesting that biological activity is derived predominantly from the A alpha chain. Limited plasmic digests of fibrinogen were fractionated to yield a heat-precipitable 250,000 dalton fragment X and heat-soluble proteolytic products containing fragments derived from the carboxyl-terminal region of the A alpha chain including a 42,000 dalton major A alpha chain derivative. Neither fragment X nor derivatives produced by its additional plasmic proteolysis were suppressive. In contrast, the heat-soluble fraction from limited plasmic cleavage was suppressive, and this activity was enhanced 10-fold by additional plasmic cleavage of this fraction. The isolated 42,000 dalton A alpha chain fragment was devoid of activity, but plasmic digestion of this derivative generated peptides of less than 8000 daltons that inhibited mitogen-stimulated thymidine uptake by lymphocytes. Two synthetic peptides corresponding to A alpha 220-230 and B beta 43-47, peptides with known vasoactive activities, suppressed lymphocyte thymidine uptake at very high concentrations. Based on their maximal yield from plasmic digests of fibrinogen, these two peptides would account for only 1% of the immunosuppressive activity of fibrinogen derivatives. In summary, the results indicate that the suppressive activity of fibrinogen is predominantly derived from the 42,000 dalton carboxyl terminal region of the A alpha chain of the molecule and is not attributable to the known vasoactive peptides. Initial proteolytic release of this region from the core of fibrinogen does not result in suppressive activity, but additional cleavage releases small peptides with the lymphocyte inhibitory function.

Captopril enhances in vitro human lymphocyte thymidine incorporation.

Angiotensin-converting enzyme activity is present in human sarcoid as well as murine schistosome granulomas. Inhibition of angiotensin-converting enzyme by captopril results in decreased granuloma size in animals. Since captopril is used clinically to treat hypertension, its effect on human lymphocyte thymidine incorporation was determined. It was found that 5 X 10(-5) M and 10(-4) M captopril enhances lymphocyte thymidine uptake induced by 25 micrograms/ml phytohaemagglutinin-P. This suggests that captopril can alter immunological reactivity in man and that angiotensin-converting enzyme may have an immunological function.

Immunological status of newborn infants.

Cord blood analysis of 21 infants indicated a deficient immunological status of newborn babies as revealed by the content of T lymphocytes and bactericidal activity of leucocytes; however,3H thymidine uptake of lymphocytes following PHA stimulation was normal. Serum IgG levels were high at birth as compared with IgM and IgA. The hemoglobin, total protein and albumin levels in plasma were mostly within normal limits.

Depression of monocytes and lymphocytes by stress-related humoral factors and anaesthetic-related drugs.

The in vitro effects of six anaesthesia-related drugs and five stress-related serum factors on monocyte-mediated cytolysis and thymidine uptake in mitogen-(PHA)-stimulated lymphocytes have been studied. Thiopentone depressed both the monocyte and lymphocyte function in a dose-dependent way. However, at thiopentone concentrations which may be present in the serum after a single intravenous anaesthesia induction dose, the monocyte depression was moderate and depression of the lymphocytes was not observed. The other drugs tested, fentanyl, morphine, pancuronium, diazepam and bupivacaine, did not alter the cellular functions significantly. Prostaglandin-E2 in concentrations of 10(-6) and 10(-7) M markedly depressed monocyte-mediated cytolysis. Cortisol, catecholamines and serotonin did not alter this function. However, a synergistic depressive effect of the combination of prostaglandin-E2 and cortisol was observed. The proliferative response of PHA-stimulated lymphocytes was depressed by cortisol in concentrations of 2200 nmol/l and 1100 nmol/1 Again, there was a marked synergistic effect of the combination of cortisol and prostaglandin-E2, while prostaglandin-E2 alone, catecholamines and serotonin did not influence the PHA-response. A possible explanation for the depression of monocyte-mediated cytolysis and lymphocyte-thymidine uptake during and after surgery under general anaesthesia may be the combined effect of endocrine and local stress factors.

Influence of fibrinogen fragments on human lymphocyte thymidine uptake

Fragments of human fibrinogen were tested for ability to supress activation of human blood lymphocytes by the lectin phytohaemagglutinin (PHA). Measurement of cellular 14C-thymidine uptake was used as an indicator of DNA synthesis and cell proliferation. The tested fragments were obtained by plasmin digestion or by CNBr fragmentation of fibrinogen. Inhibition of uptake, with good doseresponse relationship, was demonstrated for the plasmic fragment PL-3 and for the CNBr fragment Hol-DSK. Digests of the latter with trypsin and plasmin gave the same effects as the intact fragment. However, reproducibility was poor with PL-3. Hol-DSK gave better reproducibility, but with pronounced interbatch variations. Other fragments of fibrinogen were tested, but gave no effect in the studied concentrations. The reason for the poor reproducibility is discussed.

気管支喘息児のリンパ球に関する研究

Recently, researches on the mechanism of bronchial asthma have been focused on immunological aspects, including lymphocytic responses.In this paper the lymphocytic re sponses and serum IgE level were studied on asthmatic children visited the out-patient clinic of Kansai Medical University Hospital, and they were compared with those of non-asthmatic children. The studies were carried out in several directions, such as in the patients of pre- and post- immunotherapy, in the patients with varied severities (mild, moderate, severe) of asthmatic attacks. Lymphocyte studies were also do ne on the lymphocytic responses to mitogens, and the following results were obtained.1. There was no significant difference in the lymphocyte populations between controls and asthmatic children as well as between mild, moderate and severe groups.2. There was no correlation between elevated IgE and immunotherapy using house dust extracts.3. The tritiated thymidine uptake of lymphocytes stimulated with PHA and Con. A decreased in severe group, compared with mild or moderate group. However, pokeweed mit ogen did not show any significant influence on thymidine uptake of lymphocytes.4. In vitro study of lymphocytic DNA synthesis, Epinephrine inhibited the DNA synthesis of lymphocytes obtained from the control children, but it showed a decreased inhibition in th e case of lymphocytes from the severe group.5. The results of this present study support the conclusions that in asthmatic children nonspecific suppressor T-cells decrease only in its functional activity and β-adrenergic cyclic AMP system is damaged.

Evidence that calmodulin may be involved in phytohaemagglutinin-stimulated lymphocyte division.

Phytohaemagglutinin-stimulated and non-stimulated incorporation of [3H]thymidine into human peripheral blood lymphocytes is inhibited by the calcium antagonist PY 108–068 and by the calmodulin antagonists trifluo-perazine and N-(6-aminohexyl)-5-chloro-l-naphthalene sulphonamide (W7). It is argued that calmodulin may be involved in both non-stimulated [3H]thymidine uptake in lymphocytes and also in the lymphocyte response to phytohaemagglutinin.

In vitro effects of cyclosporin A on lymphocyte subpopulations. 1. Suppressor cell sparing by cyclosporin A.

The fungal metabolite, cyclosporin A, is a potent immunosuppressive compound. Experiments were performed in vitro with both human and nonhuman primate peripheral blood lymphocytes to study the effect of this agent on suppressor cell activity. Cyclosporin A did not affect the generation or function of concanavalin A-induced suppressor lymphocytes as measured by their ability to suppress thymidine uptake of lymphocytes in secondary cultures. No evidence of suppressor cell induction was noted by incubation of lymphocytes with only cyclosporin A. We conclude that, although cyclosporin A does not generate or induce suppressor cell lymphocytes, it does spare them, while inhibiting other subpopulations. This effect may create an imbalance in the immune system which results in profound suppression.

Mechanism of Immunosuppression of Progesterone on Maternal Lymphocyte Activation during Pregnancy

Abstract Lymphocyte reactivity was measured in human mixed lymphocyte (MLC) and mitogen-stimulated cultures in the presence of various steroids. When present during the entire culture period at concentrations between 1 and 20 µg/ml progesterone, estradiol, and testosterone blocked 3H-thymidine (3H-TdR) incorporation. Estriol and 16 α-hydroxy progesterone were without suppressive effects. The kinetics of steroid mediated suppression of thymidine incorporation and DNA synthesis was investigated at various times during lymphocyte activation. When progesterone was present only at the beginning of culture, subsequent DNA content, but not thymidine incorporation was suppressed. However, if added late during the activation process, progesterone reduced thymidine incorporation independent of cellular DNA content. If present during the entire culture period both were suppressed. Cortisol, but not progesterone irreversibly inhibited lymphocyte thymidine incorporation if added during the first 12 hr of culture. A reversible blockade of lymphocyte thymidine uptake could be observed with progesterone even during the final hours of mitogen or MLC cultures. Thus, effects of steroid hormones on cellular DNA synthesis and thymidine incorporation appear to be independent processes. Cell fractionation experiments demonstrated that progesterone blocked the uptake of 3H-TdR into the lymphocyte nucleoside pool thereby reducing its availability for incorporation into DNA. This study is consistent with the hypothesis that lymphocyte proliferation at the maternal fetal-interface during pregnancy may be under specific regulation by progesterone. The relatively high local concentrations of the hormone produced by trophoblast are immunosuppressive whereas the substantially lower peripheral steroid levels are entirely inadequate to suppress systemic immunity. We suggest that at least one important role of progesterone in maintaining pregnancy is its regulation of cellular immunity in the maternal-fetal bed.

The effects of heavy metals on [ 3H]thymidine uptake in lymphocytes

The effects of lead, cadmium, mercury, and zinc were determined on the response of mouse splenic lymphocytes cell cultures to stimulation by the mitogens phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Mice were exposed for 30 days to the metal salt in the drinking water. Splenic lymphocytes were cultured at 15 and 30 days to determine the response to the mitogens by measuring the uptake of [ 3H]thymidine. In vitro response was measured after adding the metal salts directly to the cell culture. In vivo exposure to high concentrations of lead, cadmium, or mercury for 30 days resulted in significantly ( p < 0.05) decreased responses to both mitogens. Cadmium (1.42 m m) and mercury (0.50 m m) produced these decreases after 15 days of exposure. Cadmium and mercury also produced dose-dependent inhibition in vitro, with the response to PHA being more sensitive. The results suggest that the metals may affect the lymphocyte directly by altering the synthesis of cellular DNA and thereby influence production of antibodies. Lymphocyte membrane ATPase activity was measured to ascertain the metal effects. All metals used caused a dose-dependent inhibition of ouabain-insensitive ATPase in isolated lymphocytes.

Cell-Mediated Immunity during Natural Measles Infection

Natural measles causes prolonged depression of cell-mediated immunity yet little is known as to how the infection influences lymphocyte function. Therefore, we studied the properties and function of lymphocytes during and after measles. The number and proportion of circulating thymus-derived lymphocytes was low during the acute stage of measles, and at this time 37% of these cells showed positive immunofluorescent staining for measles virus after stimulation with phytohemagglutinin. 7% of B cells were shown to contain virus but their numbers did not alter during the infection. Acute-phase lymphocytes, when stimulated, yielded infective virus and half were killed on incubation with autologous serum and complement. In acute measles the increase in [(3)H]-thymidine uptake of lymphocytes when stimulated with an optimal dose of PHA was normal in media with 10% fetal calf serum and low in media containing 10% autologous serum: the mean values were 56.8+/-34.1 and 23.7+/-25.9 cpm x 10(3) per 10(6) lymphocytes, respectively. Stimulation of acute-phase lymphocytes by Candida antigen was also low in media containing autologous serum averaging 1.2 x 10(3) cpm per 10(6) lymphocytes. On recovery 4-6 wk later this rose significantly to 18.9+/-19.8. The mean migration index of leukocytes to heat-killed candida cells in acute measles was 0.84+/-SD 0.08, and this fell significantly to 0.75+/-SD 0.08 4 wk later. Thus, depletion of T cells, an inhibitor of lymphocyte proliferation in the serum and a possible defect in antigen processing, interacts to depress cell-mediated immunity in measles.

Ontogeny of human cell-mediated immunity: age-related variation of in vitro infantile lymphocyte transformation.

Thymidine uptake of unstimulated and of phytohemagglutinin (PHA)-, streptokinase-streptodornase (SK-SD)- and candida extract (candida)-stimulated lymphocytes of normal infants less than 20 months old was evaluated. Thymidine uptake of unstimulated and PHA-stimulated young infants' lymphocytes was avid, resembling that of newborn cells. Over periods of weeks to months, infantile lymphocytes demonstrated transition in unstimulated and PHA-induced thymidine uptake from response patterns like cord blood cells to ones more characteristic of adult lymphocytes. Candida- and SK-SD-induced thymidine uptake of lymphocytes from very young infants was likewise apparently quite avid, resembling cord blood cells. However, factoring out high unstimulated thymidine uptake by conversion of data to stimulation indexes clarified differing paces of acquisition of transformation responsiveness of the two naturally acquired infectious antigens. Specific lymphocytes reactivity to SK-SD was quite low in all groups of infants compared with candida-induced transformation, which in some infants acquired adult proportions.

Arthritis of mice induced by Mycoplasma pulmonis: humoral antibody and lymphocyte responses of CBA mice.

Peak arthritis occurred 14 days after intravenous injection of Mycoplasma pulmonis and persisted in some mice at low levels for 84 days. A marked lymphocytosis occurred during the first week of infection with only a slight increase in polymorphonuclear leukocytes. Complement-fixing antibodies appeared in low titer 3 days after infection and moderate levels persisted for 84 days. The metabolic-inhibiting and mycoplasmacidal antibody responses were absent or minimal. M. pulmonis appeared to be mitogenic for mouse lymphocytes as evidenced by (i) increased uptake of [3H]thymidine for normal lymphocytes exposed to various concentrations of nonviable M. pulmonis antigen, and (ii) a 13-fold increase in [3H]thymidine uptake in lymphocytes taken from mice 3 days after infection with M. pulmonis in the absence of added antigen. Lymphocytes taken from infected mice transformed significantly more at all time periods than control lymphocytes when exposed to M. pulmonis antigen. This response was maximal at 3 days and minimal at 21 to 35 days after infection. Lymphocytes sensitized to M. pulmonis did not transform when exposed to M. arthritidis antigen or vice versa. M. pulmonis infection had no effect upon the mitogenic responses of lymphocytes to phytohemagglutinin or lipopolysaccharide. There was no statistically significant correlation between persistence of arthritis and degree of humor antibody or lymphocyte responses. However, persisting arthritis was associated with a higher incidence of mycoplasma isolations.

Humoral and cellular immunity in asthma

Parameters of humoral and cellular immunity have been measured in 91 asthmatic patients. Mean serum levels of IgG and IgE were raised. IgG levels were higher in those with a family history of asthma. IgE levels were higher in those with a past history of atopic eczema, but intrinsic and extrinsic asthma could not be differentiated on the basis of IgE levels. Thirteen of 74 patients failed to respond to tetanus immunization, while only 1 failed to respond to Salmonella typhi H antigen. Tetanus nonresponders had a raised mean serum IgA level, reduced spontaneous lymphocyte tritiated thymidine uptake, and reduced thymidine uptake in fetal calf serum. Eight of 87 patients failed to mount delayed hypersensitivity reactions to a battery of five intradermal antigens. The tritiated thymidine uptake of lymphocytes stimulated with phytohemagglutinin was normal in autologous serum, but reduced in fetal calf serum. The data support the hypothesis that asthma may be associated with immunodeficiency states.

Immunopathology of oral leukoplakia.

The lymphocyte transformation test was performed with autologous saline homogenates of leukoplakia. A negative correlation was established (P<0.05) between the (14)C thymidine uptake of lymphocytes in vitro and the non-pyroninophilic mononuclear cell infiltration in biopsies. A depression of lymphocyte transformation was revealed in patients with carcinoma or carcinoma in situ and some with epithelial atypia, as compared with those showing only hyperkeratosis and to a less extent those with acanthosis. A corresponding depression was not found when lymphocytes were stimulated with phytohaemagglutinin, Candida albicans or Herpes simplex antigens. The Pyroninophilic cell count was raised in biopsies with carcinoma or carcinoma in situ and in some with epithelial atypia or acanthosis. These result suggest that in leukoplakia the carcinomatous transformation may be associated with some immunological changes.