Interaction and migration of T- and B-lymphocytes in immune responses during carcinogenesis induced by methylcholanthrene]

The (CBA X C57BL) F1 mice were injected intramuscularly with methylcholantrene (MCA) in a dose of 0.3 mg, and their T- and B-cells ability to cooperate in the immune response against sheep red blood cells, and also migration of these cells from the thymus and the bone marrow to the spleen were studied. The MCA immunosuppressive action proved to be associated with the inhibition of migration and cooperation of T- and B-lymphocytes in the immune response. A conclusion was drawn that the immunosuppressive effect developing during the carcinogenesis was complex and it was realized at various stages of immunogenesis.     

Effect of combined administration of hydrocortisone and EDTA on the number of the antibody-producing cells in the spleen of mice immunized with sheep erythrocytes]

Experiments on mouse hybrids (CBA X C57BL)F1I indicated that injection of hydrocortisone in a dose of 1 mg/mouse 24 hours after the immunization with sheep red blood cells against the background of multiple EDTA injection resulted in a relative reduction of the plaque-forming cells in the spleen--more than 6-fold in comparison with control, and more than 3-fold in comparison with the effect of hydrocortisone or EDTA alone. This may possibly be the consequence of a more intensive hydrocortisone incorporation under conditions of prolonged hypocalciemic action of EDTA complexon.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Haemolysis and artifactual lung damage induced by an euthanasia agent

The artifactual development of endothelial necrosis, pulmonary congestion, and oedema that has been reported in dogs, cats, and monkeys when the animals were killed with 1-1.5 ml/kg body weight of the euthanasia agent T-61 was reproduced in adult ewes of the merino breed. This species also exhibited a marked pulmonary congestion with intra-alveolar haemorrhages, septal oedema, and a diffuse cellular damage of the alveolar septa when the recommended dose of 0.3 ml/kg body weight was administered after forcing blood flow through one lung by clamping the contralateral hilum. The red coloration of the damaged lung areas may be due to haemolysis, another aspect of T-61 induced cell damage in this species. The degree of haemolysis increases with increasing blood concentration of the agent and approximates complete haemolysis at a T-61 blood concentration of 5%. The blood concentration dependent degree of haemolysis in sheep suggests a similar relationship between blood concentration of the agent and degree of pulmonary tissue damage.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Action of benzodiazepines on the immune response

The action of benzodiazepine receptor agonists--diazepam and tazepam--on the immune response was studied in CBA mice. It has been shown that benzodiazepine at low doses of 0.5 and 1 mg/kg stimulates and at a dose of 8 mg/kg suppresses the rosette-forming reaction to immunization with 5 X 10(8) sheep red blood cells. Diazepam and tazepam fail to change the immune response if the immunization dose is 5 X 10(6). The dose-dependent effect of diazepine (ranging from stimulation to inhibition) may be caused by different benzodiazepine receptors involved in the process. It is suggested that a certain intensity of the immune response is needed for the manifestation of the immune modulating action of benzodiazepine.     

Effect of the antineoplastic antibiotic bruneomycin on lymphocyte population ratio in mice

Data on the quantitative ratio of the populations of T- and B-cells in the lymphoid organs of mice at various periods after oral administration of bruneomycin are presented. It was shown that in contrast to the total number of T- and B-cells in the spleen and mesenteric lymph nodes amounting to the minimal values at the early periods (days 1-3) after the antibiotic injection, their relative content remained rather high. Complete recovery of the number of T- and B-lymphocytes in the above organs was observed only by the end of a month period. Study on the kinetics of the immune response to sheep red blood cells showed that reactivity of the antibody producers in the experiments with bruneomycin increased 1.5-2 fold as compared to the control.     

The inhibitory effect of T-2 toxin on tolerance induction of delayed-type hypersensitivity in mice.

Mice injected intravenously with 1 X 10(9) sheep red blood cells (SRBC) showed no delayed-type hypersensitivity (DTH) response to SRBC and were unresponsive to DTH induction by sc injection of an optimal dose of SRBC. However, when treated with T-2 toxin, a mycotoxin, 2 days after the iv injection, mice became to show significant DTH response and to be responsive to the DTH induction by the sc injection. When the spleen cells of the mice receiving the iv injection were transferred to unsensitized syngeneic recipients, the DTH response of the recipients to SRBC was suppressed. However, the suppressor activity of the spleen cells was decreased by T-2 toxin treatment. By the iv injection, cell population of the spleen was increased and that of the thymus decreased. In contrast, by T-2 toxin treatment 2 days after the iv injection, cell population of the spleen was not increased and that of the thymus was markedly decreased. The ratio of theta-bearing cells was increased in the spleen by the iv injection. However, such increase was not observed after the T-2 toxin treatment. The ratio of Ig-bearing cells in the spleen was not changed by the iv injection and the T-2 toxin treatment after the iv injection. T-2 toxin seems to interfere with generation of suppressor cells for the DTH response.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Experimental Mycoplasma pulmonis infection of rats suppresses humoral but not cellular immune response

Humoral antibody response to sheep red blood cells and cellular immune response to bovine serum albumin were studied in Mycoplasma pulmonis infected, adult, male Sprague-Dawley rats. The hemagglutinating antibody response to sheep red blood cells was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection. Antibody titers during all days postinfection were depressed significantly (p less than 0.05) in infected rats as compared to noninfected controls. Cellular immune responses were evaluated by a delayed hypersensitivity response. Rats were sensitized at 0, 3, 5, 7, 14, 21 or 28 days postinfection with bovine serum albumin and challenged with heat aggregated bovine serum albumin 7 days later. Cell-mediated immune responses in infected rats were not significantly different at any point from controls. These results indicate that M. pulmonis infection in rats suppresses the humoral antibody response to sheep red blood cells, but not the cellular immune response to bovine serum albumin.     

Simultaneous detection of T-, B- and D-rosette forming lymphocytes and null cells in humans]

For the identification of T-, B-, and D-rosette-forming lymphocytes and the "null" cells in the human peripheral blood a simultaneous reaction of rosette-forming cells with the use of zymosan-complement, sheep red blood cells, fixation and staining of smears in extraction of the lymphocytes tested with the aid of verographin may be recommended.     

Characteristics of bunyavirus- and togavirus-induced nonspecific suppressors inhibiting delayed hypersensitivity

Some properties and mechanisms of action of nonspecific suppressor cells, inhibiting delayed hypersensitivity to sheep red blood cells and activated in vivo in experimental tick-borne encephalitis and Tahyna virus infections in mice, have been studied. These nonspecific suppressor cells have been identified as T-lymphocytes in experiments with the use of antisera to T- and B-lymphocytes. The function of the suppressor cells can be realized without their proliferation and is mediated by a soluble factor whose formation requires the synthesis of protein. In respect to hydrocortisone, the above-mentioned suppressor cells are subdivided into 2 subpopulations: hydrocortisone-resistant in the thymus and hydrocortisone-sensitive in the spleen.     

E1Lymphocytopenia in pseudo-lupus-erythmetaosus syndrome]

Seven female patients with pseudo-lupus erythematosus (LE)-syndrome had markedly reduced lymphocyte counts in their peripheral blood during the active phase of the disease. One patient, we were able to study during the active phase of her disease, had a diminuation of spontaneous rosettes-forming (T-)lymphocytes, using neuraminidase-treated sheep red blood cells for the test. In this case the percentage of surface-immunoglobulin-bearing (B-)lymphocytes determined by an indirect immunofluorescence technique was augmented. In comparison with 20 normal controls 6 other patients did not show any alterations in the relation of B- and T-lymphocytes in the peripheral blood. 5 patients had circulating lymphocytotoxic antibodies in their serum. A specificity of these antibodies for T-lymphocytes could not be realized.     

Combinative ligand-receptor interactions: epinephrine depresses RAW264 macrophage antibody-dependent phagocytosis in the absence and presence of met-enkephalin

Macrophages have been shown to possess cell surface receptors for opiates and catecholamines. The abilities of these ligands to affect RAW264 macrophage antibody-dependent effector activity directed against sheep red blood cells were tested. Phagocytosis was measured by the uptake of 51Cr labeled erythrocytes and optical microscopy. Cytolysis was measured by 51Cr-release assays. Met-enkephalin increased specific antibody-dependent phagocytosis in a dose-dependent fashion; the optimal dose was found to be 10(-8) M. Epinephrine diminished phagocytosis in a dose-dependent manner exhibiting a maximal inhibition at 10(-4)-10(-5) M. This inhibition can be blocked by propranolol. The combined effects of simultaneous treatment with met-enkephalin and epinephrine were measured. At the several doses tested, the combined effects of these two ligands on the amount of phagocytosis were equivalent to or more inhibitory than epinephrine alone. Thioglycolate-elicited murine peritoneal macrophages demonstrated similar responses to epinephrine, met-enkephalin, and their combination. Therefore, in vitro models more closely approximating in vivo neuroregulation of macrophage function demonstrate phagocytic inhibition.     

Spontaneous haemolytic activity of Atlantic halibut (Hippoglossus hippoglossus L.) and sea bass (Dicentrarchus labrax) serum

The spontaneous haemolytic (SH) activity of sera was compared in groups of cultured halibut and sea bass. The optimum assay temperature was determined for each species and different red blood cell donors were tested. The effects of heat inactivation, storage temperature and of different agents like EDTA, EGTA, yeast cell components and bacterial LPS were compared. Halibut sera gave optimum lysis with sheep red blood cells (RBC) at 16 degrees C whereas sea bass sera showed optimum lysis with rabbit RBC at 37 degrees C. The haemolytic activity of halibut sera was inactivated at 45 degrees C while sea bass sera were inactivated at 56 degrees C. The haemolytic activity of halibut sera was significantly reduced during short-term storage at -80 degrees C, whereas the sea bass sera maintained fairly good activity after 1-year storage at -80 degrees C. EGTA and EDTA inhibited the spontaneous haemolytic activity of sera from both the species. Zymosan and MacroGard from yeast cells also inhibited the haemolytic activity of the sera of both species, whereas LPS had a very slight effect. Considerable variation in haemolytic activity was observed within both the halibut and sea bass groups studied.     

Effect of 57Co-bleomycetin on the immune response induced in mice by ram erythrocytes

The effect of 57Co-bleomycin on the immune response induced by sheep red cells was studied on mice. It was found that the immune response was not suppressed when the labeled antibiotic was used in a single dose of 5-20 mg/kg. By the 5th day the level of 57Co-bleomycin in the skin, thymus and blood was 3-10 times higher than that of 57CoCl2 and in the spleen it was 1.3-1.5 times higher.     

Calcium, carbonic anhydrase and gastric acid secretion

Previous data concerning the action of calcium (Ca) on gastric acid secretion (GAS) indicated that calcium ions increase GAS elicited by gastrin released through a vagal mechanism, and also by a direct effect on parietal cells. Our research showed that the stimulating effect of calcium on gastric acid secretion can be antagonized by verapamil administration, which reduces gastric acid secretion . In the present study we followed the effect induced by administration of calcium and Ca-chelating agents (disodium EDTA) on gastric acid secretion and on carbonic anhydrase (CA) activity. We selected two groups of healthy volunteers: Group I (n=21) received a single i.v. dose of CaCl2 (15 mg/kg b.w.), whereas Group II (n=22) received a single i.v. dose of disodium EDTA (5 mg/kg b.w.). We determined blood calcium before and after treatment, gastric acid secretion at 2 hours. erythrocyte CA II activity, and CA IV activity in membrane parietal cells, which were isolated from gastric mucosa obtained by endoscopic biopsy. Assessment of carbonic anhydrase activity was achieved by the stopped-flow method. In Group I calcium administration increased blood calcium, HCl output, CA II and CA IV activity as compared to initial values. In Group II, disodium EDTA reduced blood calcium, HCl output, CA II and CA IV activity as compared to initial values. The results demonstrated that increased blood calcium and GAS values after calcium administration correlated with the increase of erythrocyte CA II and parietal cell CA IV activity, while disodium EDTA induced a reversed process. Our results also show that cytosolic CA II and membrane CA IV values are sensitive to calcium changes and they directly depend on these levels. Our data suggest that intra- and extracellular pH changes induced by carbonic anhydrase might account for the modulation of the physiological and pathological secretory processes in the organism.     

Participation of hematopoietic stem cells in the process of immune response formation

The endogenous colony formation and immunogenesis in mice of CBA and C57BL strains immunized with various doses of sheep red blood cells (SRBC) were studied. An absolute or relative increase of antibody forming cells under the action of growing SRBC dose from 2.10(7) to 2.10(8) as well as a decrease following 2.10(9) dose administration were noted. The number of endogenous hemopoietic spleen colonies augmented depending on increased antigen dose. Strain differences in the number of endogenous spleen colonies remained. It is suggested that the stimulation mode of hemopoietic lines is based on a specific RES blockade by SRBC resulting in enhanced proliferative effect of stem cells.     

High Susceptibility of Strain A Mice to Endotoxin and Endotoxin-Red Blood Cell Mixtures.

Heppner, Gloria (University of California, Berkeley), and David W. Weiss. High susceptibility of strain A mice to endotoxin and endotoxin-red blood cell mixtures. J. Bacteriol. 90:696-703. 1965.-Strain A mice were shown to be considerably more susceptible to lethal effects of endotoxin lipopolysaccharide (LPS) than mice of several other strains. Complexes of sublethal quantities of LPS and sheep red blood cells were synergistically toxic for strain A mice. Separate administration of sheep red blood cells and heat-killed salmonellae, in either order and as long as 24 hr apart, also proved to be synergistically lethal for strain A mice, but not for R(III) animals studied comparatively. Sheep red blood cell lysates possessed the ability of the intact cells in forming lethal combinations for strain A mice with killed salmonellae. Strain A red blood cell-killed salmonellae complexes were also lethal for strain A mice, but less so then complexes made with sheep red blood cells. A x R(III) F(1) hybrid animals showed the same resistance characteristics as the resistant R(III) parental strain. Possible explanations for these findings are suggested, and their relevance to an immunological mode of action of endotoxin lethality is discussed.