Effects of astragali radix extract on carcinogenesis, cytokine production, and cytotoxicity in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine.

We studied the effects of astragali radix extract, a Chinese herb and one of eight components in Shikaron, on carcinogenesis, natural killer (NK) cell activity, and the cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus 10 mg/kg/day or more of Astragalus extract (7, 2, and 3 mice among 15 mice in 10, 20, and 40 mg/kg/day group, respectively, vs. 14 of 15 mice treated with BBN alone). Astragalus extract prevented the cytotoxic activity of lymphocytes against YAC-1 cells from the depression by BBN. It also protected the production of interleukin-2 and gamma-interferon of lymphocytes from the depression by BBN. These results, including our previous findings, suggest that the Astragalus extract exerts an anticarcinogenic effect in carcinogen-treated mice through activation of cytotoxic activity and the production of cytokines.

Anticarcinogenic effects of shikaron, a preparation of eight Chinese herbs in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine.

We studied the effects of Shikaron, which is composed of 8 Chinese herb extracts, on carcinogenesis and the cytotoxic activity and cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus Shikaron 200 mg/kg/day (5 of 20 mice, 25%), than in mice treated with BBN alone (16 of 20 mice, 80%). Shikaron protected the cytotoxic activity of lymphocytes against YAC-1 cells from suppression by BBN. Cytotoxic activity against P-815 cells significantly increased in mice treated with BBN plus Shikaron, as compared with normal mice and BBN-treated mice. Shikaron also protected production of interleukin-2 and interferon-gamma by lymphocytes from suppression by BBN. These findings strongly suggest that Shikaron exerted an anticarcinogenic effect in carcinogen-treated mice through activation of NK and LAK cells and cytokine production by T lymphocytes.

Immunological characteristics of the effector cells induced by a combination therapy with cyclophosphamide and allogeneic lymphocytes.

A significant inhibition of tumor growth was observed when sarcoma 180 (S180)-bearing ICR strain mice were treated by a combination therapy, with a low dose of cyclophosphamide (CY) and an inoculation of allogeneic lymphocytes collected from C57BL/6 mice. The growth-inhibitory effect was significantly increased by an inoculation of a relatively lower dose of allogeneic lymphocytes (1 x 10(5) cells) and CY. The effector cells induced in the mice treated with CY and allogeneic lymphocytes expressed the Lyt 1.2, Lyt 2.2, IL-2R antigens on their membrane surface and did not express the H2KbDb (donor H-2) antigen, and they showed a specific cytostatic activity against S180 cells. These results strongly suggested that a combination therapy with a low dose of CY with an inoculation of allogeneic lymphocytes augmented an induction of specific cytotoxic T lymphocytes in the tumor-bearing recipient mice.

Effects of Lentinus edodes, Grifola frondosa and Pleurotus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butyl-N-butanolnitrosoamine.

ICR mice were treated with a carcinogen, N-butyl-N'-butanolnitrosoamine BBN), every day for 8 consecutive weeks and the effects of oral administration of edible mushrooms on the induction of urinary bladder carcinoma and on the activities of macrophages and lymphocytes were studied. Bladder carcinoma were found in all 10 mice (100%) treated with BBN alone, while we observed carcinoma only in 9 of 17 mice (52.9%), in 7 of 15 mice (46.7%) and 13 of 20 mice (65.0%) treated with Lentinus edodes, Grifola frondosa and Pleurotus ostreatus, respectively. Chemotactic activity of macrophages was suppressed in mice treated with BBN alone but maintained almost the normal level in mice treated with BBN plus Lentinus, Grifola or Pleurotus. Lymphocytes collected from mice treated with BBN plus each mushroom showed almost normal blastogenic response against concanavalin A, although those from mice treated with BBN alone completely retarded their response. Cytotoxic activity of lymphocytes against Yac-1 cells was also maintained at a normal level in mice treated with BBN plus each mushroom. Whereas in mice treated with BBN alone significant depression of NK cell activity occurred. Significantly higher cytotoxic activity against P-815 cells was observed in lymphocytes from mice treated with BBN plus each mushroom than that in lymphocytes from normal mice or mice treated with BBN alone.

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.

Immunological properties of TtT/M-87 cell line established from murine pituitary tumor-associated macrophages.

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-alpha and IL-1 alpha, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-gamma. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.

Effect of vitamin B complex on the immunodeficiency produced by surgery of gastric cancer patients.

We studied the effect of vitamin B complex (vitamin B1, B6 and B12 complex) on the immune responsiveness in gastric cancer patients who underwent surgery. The depression of blastogenic responses to both PHA and PWM was observed 2 weeks after surgery in half of the patients treated with Vitamedin but the degree was significantly less than that in the control patients without vitamin B treatment whose lymphocyte responses were depressed. Moreover, the blastogenic responses were induced by vitamin B administration 2 or 4 weeks after surgery in 5 of the 8 stage III-IV patients whose lymphocytes had not responded prior to surgery. Four weeks after surgery, the patients without vitamin B treatment showed only a tendency of recovery of their lymphocyte responses, whereas the recovery of blastogenic responses in the patients treated with vitamin B was significant. Essentially similar results were obtained with skin reactions to PHA and PPD. These results suggest that the administration of vitamin B1, B6 and B12 complex is useful for the protection against and the recovery of immune dysfunction produced by surgery in cancer patients.

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 Hank's 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.

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.

Synergistic anti-suppressor effect of mini cells prepared from Salmonella typhimurium and mitomycin C in EL 4-bearing mice.

The subcutaneous growth of EL4 cells was significantly accelerated when they were injected together with spleen cells collected from mice which had received EL4 cells SC 14 days previously, and all mice died within 18 days after receiving this mixture; 80% of mice which received a mixture of EL4 and spleen cells collected immediately after EL4 graft survived over 40 days. Spleen cells collected 14 days after EL4 graft suppressed the blastogenic responses of normal spleen lymphocytes to concanavalin A, pokeweed mitogen, and lipopolysaccharide of Escherichia coli in a mixed culture system. Acceleration of tumor growth was retarded when EL4 cells were injected together with spleen cells from EL4-bearing mice treated with both Salmonella typhimurium mini-cells and mitomycin C, and 60% of mice survived over 40 days. Blastogenic responses of normal spleen lymphocytes mixed with spleen cells from EL4-bearing mice treated with both mini-cells and mitomycin C were restored almost to control levels. The results suggest that combination treatment with mini-cells and mitomycin C synergistically inhibits the induction of suppressor cells in EL4-bearing mice.

Depression of resistance against Salmonella infection in sarcoma 180-bearing mice and its restoration by Salmonella mini-cells.

The reduction of resistance against Salmonella infection was resulted from the reduction of macrophage activities in Sarcoma 180-bearing mice. Mean survival day of S180-bearing mice was markedly reduced to 1.4 +/- 0.5 days after the challenge with 1 X 10(8) of Salmonella typhimurium LT2 cells in contrast with 6.8 +/- 1.9 days of normal mice. Eighty percent of S180-bearing mice died within 14 days after the challenge with 1 X 10(6) of LT2 cells, whereas all of normal mice survived more than 30 days after the challenge with 3 X 10(6) or less of LT2 cells. Chemotactic and O2- producing activities of macrophages collected from S180-bearing mice which were treated with Salmonella mini-cells restored to normal level. Bactericidal activity of macrophages was also restored by the mini-cell treatment. The restoration of the resistance to Salmonella was accompanied with increases of macrophage activities.

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response.

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.

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 spleen 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.