Therapeutic Effect of Endogenous Tumor Necrosis Factor on Ascites Meth A Sarcoma

The therapeutic effect of endogenous tumor necrosis factor (TNF) on Meth A ascites fibrosarcoma in mice was investigated. Serum and peritoneal fluid from tumor bearing mice treated with OK-432 and LPS were cytotoxic to tumor cells in vitro. The peak of cytotoxicity in both the serum and peritoneal fluid was found in the fraction corresponding to a molecular weight of approximately 54,000-56,000 on HPLC and the PI was found to be 4.9-5.1 by is oelectric focusing. These results are consistent with previously reported findings on TNF, and indicate that endogenous TNF has a satisfactory life-prolonging effect.

The tumor necrosis factor (TNF) is considered to be one of the clinically most promising anti-cancer cytokines because of its potent and very specific antitumor effect on target cells (Carswell, Old, Kassel, Green, Fiore & Williamson, 1975; Matthews & Watkins, 1978; Niitsu, Watanabe & Urushizaki, 1984).

TNF as an anti-cancer cytokine for the treatment of cancer may be applied in one of the two following ways: 1) by administration of purified TNF or 2) by endogenously inducing TNF in cancer bearing individuals. The antitumor effects of TNF administered exogenously have been examined using crude preparations or serum containings TMF (tumor necrosis serun, TNS) (Carswell et al., 1975; Watanabe, Niitsu, Sone, Neda, Ishigaki & Urushizaki, 1984).

In a previous paper we reported that mice primed with OK-432 and challenged with endotoxin produced a soluble cytotoxic factor in peritoneal fluids (Yamamoto, Nagamuta, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985; Nagamuta, Yamamoto, Usami, Sugawara, Watanabe, Niitsu & Urushizaki, 1985).

This peritoneal cytotoxic factor (PCF) had cytostatic and/or cytotoxic effect not only on mouse tumor cell lines but also on human tumor cell lines without species specificity. Normal cell lines were not affected. Here we report the endogenous production of TNF in tumor bearing mice and its antitumor effects.     

Mechanism of Induction of Endogenous Tumor Necrosis Factor in Ascites of Ovarian Cancer Patients by OK-432, A Streptococcal Preparation

In four ovarian cancer patients with malignant ascites, 10 KE of OK-432 was intraperitoneally administered four times every other day for priming, and 40 KE of OK-432 in a single dose by the same route on day 13 for triggering. The changes in peripheral blood monocytes and intraperitoneal macrophages and the production of tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMC) and ascitic lymphoid cells (ALC) were examined. In two of the four patients in whom TNF was induced in the ascites, the TNF production by PBMC and ALC was noted during priming, and after triggering, an increase in both the number of intraperitoneal macrophages and the TNF production by ALC was noted. In two other patients in whom TNF was not detected in the ascites, the ratio of intraperitoneal macrophages to ALC did not change throughout the whole period, and the TNF production by ALC was not augmented. These findings suggest that the priming administration of OK-432 can induce both intraperitoneal macrophages and peripheral blood monocytes into a primed state, and the triggering administration of OK-432 can increase the number of intraperitoneal OK-432-primed macrophages and induce TNF release from these cells.     

Analysis of antitumor effects of OK-432 against syngeneic mouse fibrosarcoma: combination effect of OK-432 and recombinant lymphokines

OK-432, a streptococcal preparation with potent biological response modifying activities, had the ability to cure mice bearing BAMC-1 (fibrosarcoma) ascites when it was injected intraperitoneally five times, 2, 4, 6, 8, and 10 days after the tumor inoculation. Previously, it was shown that the OK-432 injection on day 2 was indispensable since only a minimal antitumor effect was obtained when an inflammation-inducing agent such as thioglycolate instead of OK-432 was injected on day 2, followed by four OK-432 injections on days 4, 6, 8 and 10. In the present study, the injection of OK-432 on day 2 and a subsequent injection of either IL-2 or IFN-gamma on day 4 or 6 showed a significant antitumor effect, achieving a complete cure in approximately 50% of mice treated, although none of the mice could be cured by a single injection of either OK-432, IL-2, or IFN-gamma on day 2. Interestingly, however, the mice treated with an injection of a lymphokine (IL-2 or IFN-gamma) on day 2 followed by OK-432 on day 4 were not cured either. Peritoneal cells on day 12 in mice treated with OK-432 and either of the lymphokines contained pantropic killer cells, which were Thy-1+ and asialo GM1+ (AsGM1+). Moreover, the antitumor effect of the combined treatment was abolished when mice were pre-treated with anti-AsGM1. No significant antitumor effect was observed in athymic nu/nu mice. Together with our previous findings, these results indicate that lymphokines induced by OK-432 administration may account for some of its therapeutic effects and that these lymphokines may also facilitate the subsequent induction of specific killer cells. These results warrant further investigation on possible effective therapeutic protocols with the combined use of OK-432 and lymphokines.     

Augmentation of interleukin 1 and interleukin 2 production by OK-432

Intraperitoneal (i.p.) administration of OK-432 augmented both interleukin 1 (IL-1) and interleukin 2 (IL-2) production to the rechallenge of OK-432 in vitro. Peritoneal exudate cells (PEC) of mice 8 days after i.p. injection with OK-432 (1 KE/mouse) showed maximum IL-1 production to the restimulation with OK-432 in vitro. OK-432-induced IL-1 was consisted of three molecular weight species (two major peaks: 85 K and 15 K daltons and one minor peak: 67 K daltons) on Sephadex G-100 chromatography. Splenocytes of mice 4 days after i.p. injection with OK-432 (1 KE/mouse) demonstrated maximum IL-2 production to the in vitro rechallenge of OK-432, however, in vivo OK-432 administration failed to enhance ConA-induced IL-2 production in vitro. From gel filtration analysis, OK-432 induced IL-2 had an unique molecular weight (approximately 70 K daltons). From these results, OK-432-induced augmentation of cellular immunity against tumor cells might be due to the activation of so-called lymphokine cascade reaction mediated by IL-1 and IL-2.     

Production of cytotoxic factor into mouse peritoneal fluid by OK-432, a streptococcal preparation

A cytotoxic factor was induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with a streptococcal antitumor preparation, OK-432. No cytotoxic effect on L-929 cells was observed in the peritoneal fluids of mice singly treated with OK-432 or LPS. Various mouse and human tumor cell lines were effectively killed by this peritoneal cytotoxic factor, though normal cell lines were insensitive, which indicates that this factor is not species-specific. The highest level of cytotoxic activity was obtained when LPS was given to mice 5 days after the injection of OK-432. The optimal time for collection of peritoneal fluids for the cytotoxic factor was 2 h following the LPS injection. Interferon activity was found to be negative by the plaque reduction test using L-929 cells with vesicular stomatitis virus. These results suggest that this cytotoxic factor is similar to the tumor necrosis factor (TNF) in the mouse serum.     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.     

Oral administration of a streptococcal agent OK-432 activates peritoneal macrophages in mice

The effect of orally administered OK-432, a streptococcal preparation, on the function of peritoneal macrophages in mice was examined. The administration of OK-432 orally (1 KE or 2 KE, four times every three days) did not affect the numbers of both total peritoneal cells and macrophages recovered five days after the final administration. However, the macrophages exhibited an increase in their spreading ability. Other functions of the peritoneal macrophages including lysosomal enzyme activity, phagocytic activity and interleukin 1 (IL-1) production were also enhanced significantly by the oral administration of OK-432 (1 KE or 2 KE). The production of H2O2, however, was not affected by the same treatment with OK-432. The activation of peritoneal macrophages by orally administered OK-432 reported here may contribute to expansion of the clinical application of this drug.     

Evidence that eosinophil infiltration in the OK-432/fibrinogen-injected Meth-A tumor in mice is mediated by locally produced IL-5

It was previously demonstrated that a single injection of OK-432 (a penicillin-treated freeze-dried Streptococcus) mixed with fibrinogen into cancer tissues induces marked infiltration by eosinophils of the tumor stroma and leads to tumor necrosis. In the present study, we examined mechanisms regulating the local accumulation of eosinophils and the role of infiltrating eosinophils in tumor regression using the OK-432/fibrinogen injected Meth-A fibrosarcoma tumor. After injection of OK-432/fibrinogen into the tumor on the left flank of the BALB/c mice, eosinophil infiltration became obvious in the tumor stroma on day 3 following the accumulation of macrophages and neutrophils, was massive on day 5 and decreased by day 10. After the decrease in the infiltration of eosinophils, the tumor injected with OK-432/fibrinogen diminished markedly in size with ulceration as compared with control. Northern blot analysis revealed that expression of IL-5 mRNA in the tumor tissue was not detected on day 0, was significantly on day 3, reached the maximum on day 5, and thereafter decreased by day 10. Although intraperitoneal injection of rat anti-IL-5 monoclonal antibody in tumor bearing mice prior to OK-432 injection inhibited the infiltration of eosinophils, the antitumor effects of OK-432 persisted. In the blood, neither eosinophilia nor IL-5 activity was recognized during the course of the experiment. These results suggest that intratumoral injection of OK-432/fibrinogen induces local production of IL-5, which in turn recruits eosinophils into the tumor tissue, however, the infiltrating eosinophils do not play an important role in tumor regression.     

Inhibition of autoimmune diabetes in NOD mice with serum from streptococcal preparation (OK-432)-injected mice.

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.     

Simultaneous Intraperitoneal Administration of Ok-432 and Serum Enhances Superoxide Generation from Migrated Polymorphonuclear Leukocytes, with Special Emphasis on the Role of Complements

Superoxide and its derived active oxygen species are responsible for the polymorphonuclear leukocyte (PMN)-mediated tumoricidal activity which is typically shown in the intraperitoneal administration of OK-432, a biological response modifier, for cancer ascites. We examined the effects of intraperitoneal administration of OK-432 with or without syngeneic serum on superoxide generation from PMNs which migrated in the peritoneal cavity using the new method of Cypridina luciferin analog-dependent chemiluminescence for the detection of superoxide. PMNs harvested from rat peritoneal cavity 6 h after the intraperitoneal administration of OK-432 (0.25KE/kg, or 2.5KE/kg) generated larger amounts of superoxide than those harvested after intraperitoneal injection of oyster glycogen (75mg/kg) when stimulated by opsonized zymosan or phorbol myristate acetate. Simultaneous intraperitoneal administration of OK-432 and syngeneic serum induced a greater increase in PMN superoxide generation than OK-432 alone, which was reversed by a complement activation inhibitor (MX-1). Simultaneous injection of OK-432 and heat-inactivated syngeneic serum did not exhibit a significant increase in PMN superoxide generation as compared with OK-432 alone. These results provide pharmacological evidence to the satisfactory therapeutic effects of the intraperitoneal administration of OK-432 with or without serum for patients with cancer ascites, and indicate that complements, in particular C5a, are involved in this enhanced PMN-derived superoxide generation induced by the simultaneous injection of OK-432 and serum.     

Oral administration of a streptococcal agent OK-432 activates alveolar macrophages in mice.

The effect of orally administered OK-432, a streptococcal preparation, on the functions of alveolar macrophages in mice was examined. The oral administration of OK-432 (1, 2 or 4 KE, four times every 3 days) augmented phagocytic activity, lysosomal enzyme activity and interleukin 1 (IL-1) production of murine alveolar macrophages recovered 5 days after the final administration while it did not augment H2O2 production. The number of alveolar macrophages was not affected by the same treatment. These results suggested that oral administration of OK-432 activates alveolar macrophages qualitatively to protect the lung from the metastasis of cancer cells and infectious diseases by pathogenic micro-organisms.     

Adoptive immunotherapy of poorly immunogenic tumors with in vitro sensitized cells generated by intratumoral administration of biological response modifiers

We investigated the efficacy of intratumoral administration of biological response modifiers (BRM) in induction of in vitro sensitized (IVS) cells for adoptive immunotherapy of the poorly immunogenic MCA 102 sarcoma and B16-BL6 (BL6) melanoma. We used the bacterial immunoadjuvant Nocardia rubra cell wall skeleton (N-CWS), and a streptococcal preparation, OK-432, for MCA 102 and BL6, respectively. After C57BL/6 (B6) mice were inoculated subcutaneously (s.c.) with viable MCA 102 or BL6 tumor cells in the foot-pad, mice were injected intratumorally (i.t.) with N-CWS ranging from 10 to 400 gg or OK-432 ranging from 1 to 100 μg. Draining popliteal lymph nodes (LN) were harvested 7 days after i.t. administration of BRM, and LN cells were cultured with irradiated tumor cells in the presence of IL-2 for 11 days. These IVS cells (7.5 × 10⁶ or 2 × 10⁶) were transferred intravenously (i.v.) to B6 mice with 4 day pulmonary metastases established by i.v. injection of viable MCA 102 cells (1 × 10⁶) or viable BL6 cells (3 × 10⁵). The mice were also received intraperitoneally 4 × 10⁴ IU/day of IL-2 for 4 days after adoptive transfer. The transfer of IVS cells from mice immunized by i.t. injection of 100 μg of N-CWS 1 week after inoculation of tumor cells significantly reduced MCA 102 pulmonary metastases, compared with control IVS cells without administration of N-CWS. Moreover, the transfer of IVS cells from mice immunized by i.t. injection of 10 gg of OK-432 3 days after inoculation of tumor cells significantly reduced BL6 pulmonary metastases compared with control IVS cells without administration of OK-432. The administration of N-CWS resulted in no enhancement of in vitro cytotoxicity. Although the administration of 10 gg of OK-432 augmented in vitro cytotoxicity of 1VS cells against BL6, cytotoxic activity was lower than that of IVS cells immunized with N-CWS. The major phenotype was CD8⁺ cells in IVS cells immunized with N-CWS or OK-432. These results suggest that i.t. administration of N-CWS and OK-432 facilitates the production of sensitized T-cells, and this administration route of BRM may be useful in the adoptive immunotherapy of human cancer.     

Combination chemo-immunotherapy of murine solid tumor with OK-432, G-CSF,IL-2, and chemotherapeutics

An experimental tumor model was previously established in which BALB/c mice could survive the otherwise fatal intraperitoneal (i.p.) inoculation of BAMC-1 fibrosarcoma cells if the mice were treated with five sequential i.p. injections of OK-432 (a potent BRM) starting 2 days after tumor inoculation. In the present study, although a single i.p. injection of OK-432 alone on day 2 was not enough to cure the tumor-bearing mice, a combination therapy, an injection of OK-432 (5 mg/kg) on day 2 followed by injection of G-CSF (50 μg/kg) on day 3, could eradicate the tumor cells in more than 80% of the mice. In contrast to this ascites tumor model, solid tumors induced by intradermal transplantation of BAMC-1 tumor cells were resistant to the combined treatments with OK-432 and G-CSF, dying within 60 days. However, when these mice received a combined chemoimmunotherapy with cisplatin, OK-432, G-CSF and IL-2, starting on day 4, the tumors had regressed, and were completely eradicated. When the same treatment was started as late as day 8, no significant therapeutic effect was observed. Even at this advanced stage, however, it was found that a similar chemo-immunotherapy protocol using CAP-D (cyclophosphamide, epirubicin and DWA2114R) in lieu of cisplatin was extremely effective, achieving the eradication of tumors in more than 70% of the mice. These results indicated that the combination therapy with OK-432, G-CSF, IL-2, and chemotherapeutics could achieve a potent anti-tumor effect against the solid tumor, even at the advanced stage. Subsequent experiments using athymic nude mice transplanted with the tumor cells revealed that the same combination therapy showed weak tumor-regressing effects without achieving a complete cure. These results suggest that T-cells are key effectors in this type of chemo-immunotherapy of malignant tumors.     

Effect of immunostimulants and antitumor agents on tumor necrosis factor (TNF) production

OK-432, a lyophilized preparation of Streptococcus pyogenes, showed a priming activity for TNF production in mice, associated with an increase of spleen weight. PSK, a protein-bound polysaccharide preparation from Coriolus versicolor, did not show such activity. Both OK-432 and PSK potentiated the TNF production in mice primed with Corynebacterium parvum (CP) and challenged with Escherichia coli endotoxin (LPS). Cytotoxic antitumor agents of 5-fluorouracil (5-FU), cyclophosphamide (CY) and bleomycin (BLM) suppressed TNF production in mice primed with CP and challenged with LPS. TNF production suppressed by 5-FU, CY and BLM was partially restored by the combined treatment with OK-432 or PSK. These results suggest that the administration of cytotoxic antitumor agents suppresses the intrinsic TNF production in cancer patients, and the combined use of immunostimulants such as OK-432 and PSK is advantageous in restoring TNF production suppressed by cytotoxic antitumor agents.     

Effects of Acetyl-L-Carnitine Oral Administration on Lymphocyte Antibacterial Activity and TNF-α Levels in Patients with Active Pulmonary Tuberculosis. A Randomized Double Blind Versus Placebo Study

Acetyl-L-carnitine (ALC), a drug for the treatment of ageing-related neuroendocrine dysfunctions, was orally administered -2 gm/day for 30 days -to 10 patients with active pulmonary tuberculosis (TBC). Lymphocyte-mediated antibacterial activity and serum levels of tumor necrosis factor (TNF)-U were evaluated before and after treatment, comparing the values with those of 10 TBC patients receiving placebo.

Results show that by day 30, antibacterial activity remained unmodified or increased in ALC-treated subjects, while decreased in the placebo group. No influence of ALC on TNF-U levels was detectable.

These data suggest that the host's immune responses to M. tuberculosis infection can be selectively modulated by drugs acting on the neuroendocrine axis.     

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

PSK and OK-432-Induced Immunomodulation of Inducible Nitric Oxide (NO) Synthase Gene Expression in Mouse Peritoneal Polymorphonuclear Leukocytes and NO-Mediated Cytotoxicity

Abstract

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

Efficacy of in vitro sensitized cells generated by in vivo priming with OK-432 for adoptive immunotherapy of the poorly immunogenic B 16-BL6 melanoma

We investigated the efficacy of the streptococcal preparation OK-432 as an adjuvant for in vivo priming in induction of sensitized cells for adoptive immunotherapy of the poorly immunogenic BI6-BL6 (BL6) melanoma. C57BL/6 (B6) mice were immunized subcutaneously (s.c.) with 3 × 10⁶ viable BL6 tumor cells admixed with various doses of OK-432 ranging from 1 to 100 μg in the foot-pad. Draining popliteal lymph nodes (LNs) were harvested 7 days after immunization and LN cells were further sensitized with irradiated tumor cells in the presence of 60–300 IU/ml of IL-2 for 11 days. These in vitro sensitized (IVS) cells (2 × 10⁶) were transferred intravenously (i.v.) to B6 mice bearing 4-day pulmonary metastases established by i.v. injection of 2–4 × 10⁵ viable BL6 cells. The mice were also received intraperitoneally (i.p.) 4 × 10⁴ IU/day of IL-2 for 4 days after adoptive transfer. Transfer of IVS cells from mice immunized by s.c. injection of tumor cells admixed with 10 μg of OK-432 significantly reduced the numbers of BL6 pulmonary metastases compared with that of control IVS cells without the administration of OK-432 (P = 0.003). These effective IVS cells also significantly prolonged the survival of treated animals (P=0.003). Functional IVS cells required in vitro stimulation with tumor cells. However, addition of OK-432 in the vaccine resulted in no enhancement of in vitro cytotoxicity and no characteristic change of phenotype of IVS cells. These results suggest that in vivo priming of OK-432 facilitates the sensitization of tumor-reactive T-cells. The procedure of in vivo priming with OK-432 may be beneficial in the adoptive immunotherapy of melanoma.     

Characterization of a cytotoxic factor induced in mouse peritoneal fluid by OK-432

A cytotoxic factor (peritoneal cytotoxic factor, PCF) was strongly induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with OK-432. In order to clarify characteristics of PCF, physicochemical and immunological studies were conducted. When incubated with LPS, the macrophages from mice primed with OK-432 induced PCF whereas the lymphocytes did not. These results indicate that PCF is different from lymphotoxin. PCF appears to be quite similar to tumor necrosis factor (TNF) in the serum for the following reasons: The two factors are similar in the mode of cytotoxic action in vitro; both factors have a tumor necrotizing effect when injected into tumor bearing mice; both are produced from macrophages; they are similar in physicochemical characteristics; and the cytotoxic activity of PCF is totally abolished by anti-TNF serum.     

Suppressive effects of the bacterial immunostimulant OK-432 on the incidence of spontaneous thymic lymphoma in AKR mice

The mean survival age of female AKR/J mice was significantly prolonged, the enlargement of thymus was markedly suppressed, and the proliferation of ecotropic and recombinant murine leukemia viruses was inhibited when 2-month-old female AKR/J mice were injected intraperitoneally with attenuated Streptococcus pyogenes, strain Su (OK-432) twice weekly for 8 weeks. However, these effects of OK-432 in 2-month-old female AKR/J mice were not seen in 5-month-old female AKR/J mice. The difference in the effectiveness of OK-432 in these animals probably depends on the difference in the degree of the proliferation of ecotropic and recombinant murine leukemia viruses in the thymus which consequently lead to thymic lymphoma.