Action of cytogenin on lymphoid cells and their cytokine production.

Action of cytogenin on macrophages and T cells was investigated. Phagocytosis of yeast and production of PMA-elicited superoxide anion by macrophages taken from mice given cytogenin po were augmented. Cytogenin enhanced productions of IL-1 alpha by macrophages and IFN gamma and GM-CSF by spleen cells although it did not enhanced production of TNF alpha by macrophages and IL-6 by macrophages and spleen cells. Macrophages stimulated with cytogenin caused to stimulate proliferation of purified T cells in Intercell cultures in which each cell population was cultured without contact. Results suggest that cytogenin primarily activates macrophages to produce monokines such as IL-1 alpha and it causes to stimulate proliferation and differentiation of T cells resulting in production of lymphokines such as IFN gamma and GM-CSF.

Cytostatin, a novel inhibitor of cell adhesion to components of extracellular matrix produced by Streptomyces sp. MJ654-NF4. I. Taxonomy, fermentation, isolation and biological activities.

Cytostatin has been identified as a novel inhibitor of cell adhesion to components of extracellular matrix (ECM) in cultured broth of Streptomyces sp. MJ654-NF4. Though cytostatin did not inhibit EL-4 cell adhesion to ECM components such as laminin and fibronectin; it inhibited the adhesion of B16 melanoma cells to laminin and collagen type IV but not to fibronectin. It exhibited antimetastatic activity on B16 melanoma cells in mice. The cytotoxicity of cytostatin are also reported.

T cell activation by conagenin in mice.

Conagenin (CNG), a low molecular immunomodulator, enhanced incorporation of [3H]thymidine into T cells activated by concanavalin A but did not to non-activated T cells. The culture supernatants of activated T cells treated with CNG enhanced incorporation of [3H]thymidine into cytokine dependent cell lines, CTLL-2 and IC-2 cells. This indicates that CNG exclusively acts on activated T cells and stimulates them to promote DNA synthesis and to produce lymphokines, which may include T cell growth factors and hematopoietic growth factors. These activities were also observed with T cells taken from mice given CNG.

Effect of conagenin in tumor bearing mice. Antitumor activity, generation of effector cells and cytokine production.

Antitumor effects and function of T cells in tumor bearing mice given conagenin (CNG), a low molecular immunomodulator, were investigated. The administration of CNG, once a week for 4 weeks, was the most effective schedule in inhibiting growth of IMC carcinoma, a syngeneic tumor. In this regimen, cytotoxic T lymphocytes and natural killer activities in spleens of CNG treated mice were maintained at higher levels than those of non-treated mice. Lymphokine production by splenic T cells was also enhanced in cultures, whereas monokine production by macrophages, which was increased in accordance with tumor growth, was reduced by CNG administration. The antitumor effect of CNG was not observed in mice given anti-asialo GM1 serum and in athymic mice. Results shown in this report suggest that CNG exerts its antitumor effects through activation of T cells and enhancement of generation of antitumor effector cells.

Delaminomycins, novel extracellular matrix receptor antagonist. IV. Structure-activity relationships of delaminomycins and derivatives.

Delaminomycins A, B, C and their derivatives were prepared and investigated biological activities of them. Among these compounds, spiro compounds (A2, B2 and C2) showed stronger inhibitory activity than natural products (A1, B1 and C1) on B16 melanoma cells adhesion assay and Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, A1, B1 and C1 showed more potent inhibitory activity than spiro compounds (A2, B2 and C2). On the other hand, as to C-5' substituents of pyrrolidine ring, the order of inhibitory activity was R = OH > R = OCH3 > R = H on Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, the order of inhibitory activities were R = H > R = OCH3 > R = OH. Inhibitory activities of A4 which was lacked pyrrolidine ring were reduced on B16 melanoma cells adhesion assay and on cytotoxicity against tumor cells in vitro in comparison with those of A1.

Delaminomycins, novel nonpeptide extracellular matrix receptor antagonist and a new class of potent immunomodulator. I. Taxonomy, fermentation, isolation and biological activity.

In order to develop a new class of immunomodulator we have searched for a low molecular weight inhibitors of cell adhesion to components of extracellular matrix (ECM), fibronectin (FN), laminin (LM) and collagen type IV (CL), and succeeded to find a group of novel antibiotics, named delaminomycins. Delaminomycins were isolated from the mycelium and cultured broth of Streptomyces albulus MJ202-72F3. It was purified by use of centrifugal partition chromatography (CPC), preparative reverse phase HPLC and Sephadex LH-20 and was obtained as a white powder. Delaminomycins with inhibitory activity for cell adhesion to ECM components suppressed immune responses in vitro and in vivo and exhibited antimicrobial activity on Gram-positive bacteria.

Biological activities of IC201 ((3S,8E)-1,3-dihydroxy-8-decen-5-one), a low molecular weight immunomodulator produced by Streptomyces.

IC201 was found in cultured broth of Streptomyces cirratus as an antitumor antibiotic which was effective in retarding growth of the established solid tumor of Ehrlich carcinoma by treatment starting 8 days after tumor inoculation. It retarded growth of the established solid tumor of IMC carcinoma but had no cytotoxicity at 100 micrograms/ml. IC201 treatment kept NK cell activity of tumor-bearing mice at normal level and stimulated cytostatic activity of peritoneal macrophages. It stimulated phagocytosis of yeast and phorbol myristate acetate-elicited superoxide production by peritoneal macrophages. The addition of IC201 to P388D1 cell cultures enhanced release of interleukin 1 (IL-1) into cultured supernatant but it affected lipopolysaccharide-induced IL-1 production. Although the addition to macrophage-depleted cultures did not show any stimulatory effect, mixed lymphocyte culture reaction was augmented in cultures using spleen cells as stimulator cells taken from mice given IC201. Results indicate that IC201 primarily activates macrophages and the activation may cause modulation of immune responses.

Induction of antitumor resistance to mouse leukemia L1210 by spergualins.

Spergualin and its analog, 15-deoxyspergualin showed a marked antitumor effect against L1210 by intraperitoneal and oral administrations. After treatment with these substances 40- or 60-day survivors (cured mice of L1210) were resistant to reinoculation of L1210 cells. They were resistant only to L1210. The antitumor effector cells in these mice were determined to be T cells. NK activity of spleen cells was also enhanced by spergualins. The antitumor activity of 15-deoxyspergualin was markedly reduced in immuno-deficient mice. IL (interleukin)-2 production, but not IL-1, was enhanced in supernatant of mixed lymphocyte cultures by treatment with 15-deoxyspergualin. The mechanism of action of 15-deoxyspergualin on the immune system was discussed.

Production and characterization of monoclonal strain-specific antibodies against prototype strains of Rickettsia tsutsugamushi.

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain-specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti-Gilliam, four anti-Karp and five anti-Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain- or type-specific polypeptides and do not show any cross-reaction among strains.

Augmented mitotic response of human peripheral lymphocytes in the presence of lymphocytes from nude mice: detection of a small number of functional human lymphocytes in the explanted host.

In the present paper, authors showed a method to detect a small number of functional human peripheral lymphocytes (HPL) in the presence of lymphoid cells of nude mouse (n-ML). HPL was mixed with n-ML to give a total number of 5 X 10(4) cells and cultured in vitro in the presence of PHA for 5 days. HPL responded will to PHA while n-ML did not, as assessed by 3H-TdR incorporation in the presence of the mitogen. Synergistic response between these two cell population was observed. Five--ten folds enhancement was shown by mixing 5 X 10(3) HPL and 4.5 X 10(4) n-ML (10% of HPL) and 500 HPL became detectable by comixing with n-ML, while 5,000 HPL was the lowest limit to be detected in a single cell culture with this mitogen. The results seem to indicate that this method could detect functional HPL as small number as one percent of the cell mixture. On the other hand, xenogeneic mixed lymphocyte reaction was observed when the percentage of HPL exceeded 40%. Mitomycin-C treatment of either HPL or n-ML revealed that a major part of cells incorporating 3H-TdR was HPL which could be activated by the stimulation with xenogeneic murine nucleated cells and that a smaller part of murien B cells were also stimulated by the HPL.

A histopathological study of lymphoid tissue reaction to metastatic nasopharyngeal carcinoma in nude mice.

In attempts to heterotransplant human NPc into nude mice, using seven cultured cell lines, we have succeeded in growing a carcinoma simplex, composed of Epstein-Barr virus-determined nuclear antigen-positive and Epstein-Barr virus genome-positive cancer cells, at the injected site with two of the cell lines. These originated from a spindle-cell carcinoma (Chinese NPC-204) and from a combined-cell carcinoma (Chinese NPC-501), respectively. During the first few passages, wandering macrophages were prevalent and increased in number in response to the presence of the tumours. In conjunction with a gradual decrease in the number of wandering macrophages in the medullary sinuses, diffuse hyperplasia of lymphocytes occurred in regional lymph nodes. As a result of the release of lymphocytes and macrophages into the peripheral lymph nodes, the spleen underwent extensive change, as manifested by the collapse of the splenic cords and the formation of septa studded with granulomas. Under these conditions of immunosuppression, lymphatic metastases were observed during the periods between the 11th and 14th generations and the 24th and 30th generations with NPC-204 and between the 9th and 14th generations with NPC-501. The neighbouring lymph nodes, like the spleen, were often studded with epithelioid-cell granulomas, formed by the aggregation of macrophages around nuclear debris in the subcortical areas. We assume that the clumps of debris are the remnants of metastatic cancer cells which were probably killed by macrophages or by sensitized lymphocytes. If the lymph nodes contain a barrier of granulomas, they are not invaded by tumour cells from the cortical sinuses, except in the rare case of retrograde metastasis from the hilus. It would appear that macrophages can replace T lymphocytes, which are found in very small numbers in the nude mice used in this study, in killing tumour cells and, furthermore, in protecting the lymph nodes from the spread of metastases. Metastasis cannot occur in these nude mice when their lymphoreticular system, especially that of the spleen, is working in a stable balance.