Satonori Kurashige

A chemiluminescent probe with a Cypridina luciferin analog, 2‐methyl‐6‐phenyl‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one, specific and sensitive for O2− production in phagocytizing macrophages

When a Cypridina luciferin analog (the title compound) was added to a macrophage suspension in Hanks balanced salt solution (control), the system emitted a weak, but detectable light, which was not altered in the presence of Superoxide dismutase. The same system, however, emitted a much stronger light, just after the addition of a trigger, opsonized zymosan. The luminescence was suppressed to the control level in the presence of superoxide dismutase, while it was only slightly influenced, if at all, by NaN3, a scavenger of singlet oxygen and an inhibitor of myeloperoxidase. Some other results obtained also indicate the participation of O2 − in the luciferin analog‐dependent luminescence in macrophages during phagocytosis.

Release of superoxide radicals by mouse macrophages stimulated by oxidative modification of glycated low density lipoproteins

Diabetic patients have high levels of glycated LDL. Although glycated LDLs are implicated in the development of atherosclerosis in such patients, convincing data are lacking. We observed release of superoxide radicals (O2-) from mouse resident peritoneal macrophages stimulated by an oxidized/glycated LDL by using a highly sensitive and specific chemiluminescence method. Oxidized/glycated LDL was achieved by an addition of low concentration of Fe3+ to glycated LDL. Macrophages took up an appreciable amount of the glycated LDL oxidized by iron, leading to the development of foam cells, while they did not take up untreated glycated LDL or the native LDL. These observations clearly indicate that the oxidized/glycated LDL reacts well with macrophages. Since an oxidation of glycated LDL may occur in vivo, the oxidized/glycated LDL might play an important role in atherogenesis.

Effect of Shi-Ka-Ron and Chinese Herbs on Cytokine Production of Macrophage in Immunocompromised Mice

Shi-Ka-Ron is a prescription composed of 8 crude extracts of Chinese herbs. It reduces suppression of cytokine production by peritoneal macrophages in mice Immunocompromised by the anti-tumor agent, cyclophosphamide (CY), in vivo. Although it dose not increase IL-1 production in vitro, it enhances TNF production. We found that Ginseng radix, Lithospermi radix, Astragli radix and Glycyrrhizae radix somewhat reduced suppression of cytokine production in CY treated macrophages. Especially, Glycyrrhizae radix shows an active immune response both in vivo and in vitro. Our results suggested that the mechanism underlying immunomodulation of Shi-Ka-Ron is closely related to cytokine production: each herb stimulating macrophages.

RIBONUCLEIC ACID IN THE IMMUNE RESPONSE

SummaryIn the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.

Purine metabolic enzymes in lymphocytes. IV: Effects of enzyme inhibitors and enzyme substrates on the blastogenic responses of human lymphocytes

Coformycin, which is an inhibitor of adenosine deaminase, significantly inhibited in vitro blastogenic responses of human lymphocytes to both phytohaemagglutinin (PHA) and pokeweed mitogen (PWM), whereas blastogenic responses to bacterial lipopolysaccharide (LPS) were rather enhanced by the addition of coformycin. Blastogenic responses of lymphocytes to PHA and PWM were markedly suppressed by the addition of adenosine, which is a substrate of adenosine deaminase. Allopurinol, which is an inhibitor of xanthine oxidase, inhibited blastogenic responses of human lymphocytes to PHA, PWM, and bacterial LPS. Inosine (a substrate of purine nucleoside phosphorylase) and hypoxanthine (a substrate of xanthine oxidase) showed no or only a small effect on blastogenic responses of human lymphocytes. These results suggest that adenosine deaminase activity is associated with the T‐cell response but not with the B‐cell response and that the impaired T‐cell response in adenosine deaminase deficiency is the result of intracellular retention of adenosine in T cells. The results also suggest that purine nucleoside phosphorylase or xanthine oxidase activity is associated with both T‐ and B‐cell responses.

SEASONAL MODULATION ANTIBODY FORMATION IN RAINBOW TROUT (SALMO GAIRDNERI)

ABSTRACT The humoral immune response in trout was found to be depending on the season of the year, even if the temperature was held constant at 18 ± 1C. When trout were immunized to Aeromonas salmonisida prior to spring, antibody levels were much higher than those in fish immunized prior to winter. The kinetics of agglutinating and cytotoxic antibody formation were also seasonal dependent.

Purine Metabolic Enzymes in Lymphocytes: I. Adenosine Deaminase and Purine Nucleoside Phosphorylase Activities in Mouse Lymphocyte Subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA‐P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleens of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one‐third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA‐P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA‐P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA‐P and Con A, and that of PNP activity was enhanced by PHA‐P but not by Con A.

Effect of Shi-ka-ron on Cytokine Production of Lymphocytes in Mice Treated with Cyclophosphamide

The effect of Shi-ka-ron (CX) on cytokine production of lymphocytes in mice treated with cyclophosphamide was investigated. Shi-ka-ron, a traditional Chinese prescription, consists of 8 crude Chinese herbal extracts. Its main efficacy is to strengthen the body resistance. We observed that each CX component stimulated interleukine-2 and interferon-gamma production of murine splenic lymphocytes both in vitro and in vivo. IL-2 and IFN-gamma production of splenic lymphocytes were also examined in mice treated with CX combined with cyclophosphamide (CY) in vivo. We found that CX not only could increase IL-2 and IFN-gamma production in vitro, but also in vivo at suboptimal concentration.

Enhancing effects of mini-cells prepared from Salmonella typhimurium on anti-tumor immunity in sarcoma 180-bearing mice

SummaryThe enhancing effect of mini-cells of Salmonella typhimurium which do not contain chromosomal DNA on anti-tumor immunity in mice was studied. The growth of sarcoma 180 cells which were subcutaneously transplanted into ICR mice was significantly retarded in mice treated with Salmonella mini-cells at the same time or 7 days after S180 transplantation, while no or only a little growth inhibition was observed in mice treated 7 days prior to S180 transplantation. Treatment with mini-cells inoculation alone did not increase the survival time of mice that had received intraperitoneal transplants of S180 cells. However, a statistically significant increase of survival time was observed in mice treated with a combination of mini-cells and surgical resection of subcutaneous tumors when S180 cells were injected 7 days after the surgical resection. The injection of mini-cells restored macrophage chemotaxis in S180-bearing mice in which macrophage chemotaxis was greatly retarded but lymphocyte activity was not.

Immunopotentiating Activity of Whole Cells and Mini-Cells of Salmonella typhimurium

Salmonella typhimurium strain 9 produces mini‐cells during cell proliferation. Mini‐cells are viable but cannot proliferate since they do not contain chromosomal DNA. Effects of whole cells and mini‐cells of S. typhimurium on the immune responses were investigated, with the following results. Phagocytosis of peritoneal macrophages was enhanced by in vivo stimulation of both whole cells and mini‐cells. Cellular immunity against L1210 cells (mouse leukemia cells) and Sarcoma 180 cells was also enhanced by both whole cells and mini‐cells. Mini‐cells slightly stimulated in vitro blast cell transformation of normal mouse lymphocytes. Whole cells of S. typhimurium induced antibody‐forming cells to produce IgG of higher affinity but mini‐cells did not. Mini‐cells were not directly cytotoxic for normal lymphocytes or L1210 cells.