Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

Induction of immune interferon in mice treated with a bacterial immunopotentiator,OK-432

Summary When a bacterial immunopotentiator, OK-432, was injected to intact DDI mice, a viral inhibitor with the properties of immune interferon (IF) was induced in the circulation. The maximum titer of antiviral activity (10,240 units/ml) was observed 24 h after intraperitoneal (IP) injection of 50 KE OK-432/kg body weight. The possibility that the induction of immune IF may depend upon the action of thymus-derived (T) lymphocytes and macrophages was inferred from experiments with thymus-defective nude mice and DDI mice treated with either X-rays or immunosuppressive agents.     

Inhibition of in vitro LAK generation by OK-432

Summary The present study was designed to investigate the in vitro effect of OK-432 on interleukin-2-(IL-2) induced lymphokine activated killer (LAK) generation, and especially to test whether OK-432 can substitute for IL-2 or act in synergism with IL-2 for activation of cytotoxic lymphocytes. Surprisingly, our results showed that the addition of OK-432 to 4-day LAK activation cultures significantly inhibited both the generation of cytotoxic effectors to the natural killer (NK) resistant Daudi cell line and the proliferative responses of lymphocytes in a dose dependent manner. The inhibition of activation was total at 0.5 KE/ml of OK-432, a dose which was still effective in augmenting NK activity against K562. The addition of penicillin G potassium (PCGk), which is contained in OK-432 at a concentration of 134,700 units/mg of dried cocci, to the LAK culture system also inhibited LAK generation at equivalent concentrations as contained in the OK-432 preparation. This inhibition of LAK generation by OK-432 was significantly eliminated by dialysis of OK-432. These results indicated that the inhibition of LAK generation was partly due to PCGk contained in the OK-432 preparation, and that OK-432 did not act synergistically with IL-2 in standard LAK activation systems.     

Mechanism of antitumor effect on mouse hepatocellular carcinoma by intratumoral injection of OK-432, a streptococcal preparation

Intratumoral (i.t.) injection of OK-432, a streptococcal preparation, into implanted tumors of mouse hepatocellular carcinoma (MIH-2) showed antitumor effect including tumor eradication. Intraperitoneal administration of same dose OK-432 did not exhibit tumor suppressive effect. In vitro cytotoxic test suggested that direct cytotoxic effect of OK-432 was not associated with antitumor activity by i.t.-OK-432 treatment. It was also found that Toll-like receptor 4 signaling was not involved in i.t.-OK-432 treatment. Three mice out of five, which had shown tumor eradication by i.t.-OK-432 treatment did not reject re-challenge of MIH-2 cells. Splenocytes from i.t.-OK-432 treated mice did not produce IFN-gamma by stimulation with MIH-2 cells in vitro, but produced abundant IFN-gamma by stimulation with OK-432. Immunofluorescence microscopy demonstrated that CD4+T cells, but not CD8+T cells, infiltrated to i.t.-OK-432 treated tumor tissue produced IFN-gamma. Tumor-infiltrating CD4+T cells from i.t.-OK-432 treated tumor tissue produced IFN-gamma by in vitro stimulation with OK-432 higher than those from untreated tumor tissue. IFN-gamma directly induced apoptosis of MIH-2 cells in vitro. Collectively, i.t.-OK-432 treatment induced priming of CD4+T cells to antigenecity of OK-432, and repetitive i.t.-OK-432 treatment induced IFN-gamma production from OK-432-sensitized CD4+T cells in tumor site, leading to apoptosis of MIH-2 cells susceptible to IFN-gamma.     

Mechanism of immunotherapeutic activity of OK-432 in the treatment of peritoneal carcinomatosis

Summary In this report the mechanism of therapeutic activity of OK-432 for the treatment of peritoneal carcinomatosis was investigated by correlating effector-cell augmentation with therapeutic activity in rats bearing MADB-106 carcinomatosis. Tumor cells were injected i.p. and the treatment with OK-432 was initiated 5 days later with 0.5, 1, 5 or 10 KE/animal of OK-432 injected i.p. semiweekly. Significant therapeutic activity was observed at all doses examined with greater prolongation of survival noted at the higher doses of OK-432. Animals treated with 0.5 KE/animal had a prolongation of the median survival time from 14 days for saline-treated animals to 17 days for the OK-432 treated animals (P<0.0008), while animals treated with higher doses had much longer periods of survival, some animals being tumor-free at 185 days. In the same studies, natural killer (NK) cell, lymphokine-activated killer cell, cytotoxic T lymphocyte, and macrophage tumoricidal/cytostatic activities were measured 7 days and 14 days following tumor injection (2 days and 9 days after initiation of immunotherapy). OK-432 had immunostimulatory activity in most of the assays of immune function examined and this correlated with host survival, including augmentation of peritoneal and peripheral blood cytotoxic T lymphocyte activity on day 14, peritoneal and alveolar macrophage activity on day 7 and day 14, as well as natural killer cell activity on day 14. These results suggest that the therapeutic doses are also immunomodulatory doses for the effector cells mentioned above. We suggest, therefore, that immunological monitoring may help to optimize treatment protocols for the treatment of peritoneal and perhaps pleural effusions with OK-432.By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. This research was sponsored by the National Cancer Institute, DHHS, under contract no. N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Antitumor effect of the streptococcal preparation OK-432 in a murine model of ovarian cancer

Summary The antitumor effects of the streptococcal preparation OK-432 were analyzed in a murine ovarian teratocarcinoma (MOT) model. Administration of OK-432 i.p. prevented tumor outgrowth in 75% of mice challenged with 10³ MOT cells i.p. 24 h previously. Treatment was less successful in mice challenged with 10⁴ or 10⁵ cells, preventing tumor growth in 25% of the former and only 5% of the latter group. Tumor-challenged mice cured by injections of OK-432 were not rendered resistant to a subsequent challenge with 10³ MOT cells 75 days after initial treatment. Only the i.p. route of administration was effective as i.v. OK-432 did not prolong survival of tumor-challenged mice. An antitumor response was detected as early as 24 h after i.p. treatment. This correlated temporally with an influx of neutrophils into the peritoneal cavity. Peritoneal cells obtained between 6 and 24 h after treatment were capable of lysing MOT targets in vitro. A single cell cytotoxicity assay demonstrated that peritoneal neutrophils, elicited by i.p. injection of OK-432, could bind to and lyse MOT targets. These data indicate that OK-432 is effective against small tumor cell inocula in this murine model of ovarian cancer and, furthermore, that the neutrophilic response into the peritoneal cavity plays a role in tumor rejection.     

In vivo immunological antitumor effect of OK-432-stimulated dendritic cell transfer after radiofrequency ablation

Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy.     

Enhancement of the photodynamic antitumor effect by streptococcal preparation OK-432 in the mouse carcinoma

 Biological response modifier antitumor effects are enhanced by the activation of the host defense mechanisms. We have investigated the antitumor effect of photodynamic therapy (PDT) and/or local administration of a biological response modifier, the streptococcal preparation OK-432, on transplanted NR-S1 mouse squamous cell carcinoma. Hematoporphyrin oligomers (20 mg/kg body weight) were used to photosensitize PDT. A pulsed Nd:YAG dye laser, tuned at 630 nm, was used as the light source. The laser power was 15 mJ cm⁻² pulse⁻¹, and the irradiation time was 40 min. The photosensitizer was injected intraperitoneally 48 h before laser irradiation. Where used, OK-432 was injected into the tumor either 3 h prior to PDT or immediately afterwards. The antitumor effects were evaluated 48 h after each protocol by (a) estimating the area of tumor necrosis (%) in hematoxylin/eosin-stained specimens, and (b) bromodeoxyuridine immunohistochemistry. Furthermore, the tumor sizes were evaluated 3, 7 and 10 days after each protocol, and the survival time after each protocol was evaluated as well. The anti-tumor effect of PDT was enhanced by administration of OK-432 3 h before PDT, whereas the administration of OK-432 immediately after PDT did not potentiate a PDT antitumor effect. Treatment with OK-432 alone had little effect on tumors. Photodynamic therapy in combination with local administration of OK-432 3 h before PDT is considered to be a useful treatment modality. Received: 23 July 1999 / Accepted: 31 May 2000     

Induction of antitumor L3T4-positive T cells by OK-432 at tumor sites in mice

We have previously reported the development of antitumor effector cells by day 12 after tumor implantation using a murine malignant ascites model with BAMC-1 tumor, which could be cured completely by five consecutive i.p. injections of OK-432 starting on day 2. In contrast, the OK-432 treatment with the same protocol failed to cure the tumor-bearing athymic mice, though it could suppress tumor growth temporarily. The results suggest that T cells may play a critical role in achieving a therapeutic effect. The present study was designed to clarify the nature of the antitumor effector cells induced by OK-432 in euthymic mice. The number of tumor cells in the pertioneal cavity of OK-432-treated euthymic mice increased gradually up to day 12 and dropped suddenly on day 14, while in the athymic mice the tumor cells transiently decreased in the first 7 days then started to expand drastically on day 8. The timing of the appearance of the effector cells was examined by adoptive-transfer experiments. The peritoneal exudate cells (PEC) obtained from BAMC-1 bearing euthymic mice on various days during the treatments with OK-432 were passively transferred intraperitoneally on the respective days (synchronous transfer) or on day 7 (convergent transfer) to BAMC-1-bearing athymic mice, which were treated similarly with OK-432. More than 85% of the recipient athymic mice survived when an adoptive transfer was made on and after day 7. These results indicated that the effector cells developed before day 8 in euthymic mice. The effector cells detectable on day 7 in the PEC represent plastic- or nylon-wool-column-nonadherent cells, which could cure the tumor-bearing athymic mice. Furthermore, the effector cells were destroyed when the nylon-wool-column-nonadherent cells were treated with an anti-L3T4 antibody and complement whereas the same treatment with anti-Lyt2 antibody had no effect. These L3T4⁺ cells did not possess asialo-GM1 antigen. Although the exact mechanism of action of the effector cells is yet to be clarified, the induction of human equivalents of this type of effector cell would be a good parameter indicative of clinical effects induced by OK-432 or other biological response modifiers in an individual cancer patient.     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis

OK-432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK-432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pretreatment of OK-432 reduced the lethality of lipopolysaccharide (LPS)-induced endotoxic shock in D-(+)-galactosamine-sensitized C3H/HeN mice. OK-432 did not affect the TNFalpha production in blood, but it did decrease the susceptibility to TNFalpha. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK-432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK-432 against the endotoxemia and bacteremia may depend on an up-regulation of clearance of LPS and bacteria and the augmented resistance to TNFalpha.     

OK-432 stimulates primary production and activity of murine natural killer cells

Although many immunostimulants have been shown to increase the lytic activity of natural killer (NK) cells in the periphery, little is known about their effects on NK cells in the bone marrow, the primary site of NK production. In the experiments reported here, we tested OK-432, a pharmaceutical preparation of Streptococcus pyogenes, for its effects on both the primary production and lytic activity of NK cells in C57BL/6J mice. NK activity in bone marrow cells (BMC) and spleen cells (SC) was significantly increased following intravenous administration of OK-432, peaking on day 2 in BMC and on day 3 in SC. Concomitantly, there were marked changes in the cellularity in the two compartments. Bone marrow cellularity fell significantly on day 1 post-OK-432 and then gradually returned to normal, whereas spleen cellularity rose rapidly and remained elevated. As a consequence, the total NK activity (per femur or per spleen) was significantly increased at 48-96 h after administration of OK-432. The target specificity was unchanged. The phenotype of NK cells in BMC as determined by cytotoxic depletion was unchanged by OK-432, but splenic NK activity shifted to a 'less mature' phenotype, intermediate between that of normal BMC and SC. Cytokinetic studies using 3H-TdR revealed an increase in the production of NK cells in the bone marrow following administration of OK-432. Proliferating NK cells also appeared in the spleen. Whether these were recently produced NK cells from the bone marrow that still retained the ability to proliferate or mature NK cells that were stimulated into cell cycle cannot be determined from these experiments. These data are the first to directly demonstrate the modulation of the primary production of NK cells by an immunologically active drug.     

Immunohistochemical analysis of inflammatory infiltrates around the injection site of a streptococcal preparation,OK-432, in rat tongue

On the assumption that neutrophils around the injection site of OK-432, a heat- and penicillin-treated lyophilized preparation of the Su strain ofStreptococcus pyogenes, enhance immunologic response through the production of Interleukin-1 (IL-1), OK-432 was injected into rat tongue, and specimens from the tongue were immunohistochemically investigated at various intervals after the injection, to clarify the process of inflammatory and immune responses at the injection site. Neutrophils and mononuclear cells appeared around the OK-432 injection site after 1 hour, increased to their maximum level at 24 hours, and then decreased from the 3rd to the 7th day. IL-1 was detected on neutrophils 3 hours after the injection, and OX-08-positive cells (suppressor/cytotoxic T cells and the majority of natural killer cells) remarkably increased. OX-39-positive cells (IL-2 receptor) appeared after 12 hours. These results suggest that neutrophils around the injection site of OK-432 at early phases of inflammation play a role in the expression of BRM function through IL-1.     

Mechanism of polymorphonuclear leukocytes accumulation examined using inhibitors of complement and arachidonic acid cascade in rats treated with OK-432

When the streptococcal preparation OK-432 was intraperitoneally injected for the treatment of carcinomatous peritonitis, antitumor polymorphonuclear leukocytes (PMNs) accumulated in the peritoneal cavity. We examined the mechanism of this PMN accumulation using an in vivo system in rats. FUT-175, EDTA and K76 inhibited C5a generation by OK-432 in vitro, but EGTA, prednisolone and inhibitors of arachidonic acid cascade did not. In in vivo experiments, EDTA, FUT-175, antirat C3 serum and K76 reduced the accumulation of PMNs onto filter membranes, when these reagents were reacted with OK-432 for 3 h through filter membranes placed on the turned rat peritoneum. EGTA failed to inhibit PMN accumulation. Prednisolone, indomethacin, OKY046 and AA861 inhibited PMN accumulation in a dose-dependent manner. These inhibitions of PMN accumulation were confirmed by histological examination. It was concluded that complement-derived chemotactic factor C5a generated by OK-432 induced PMN accumulation in association with chemotactic arachidonic acid metabolites.     

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide,IL2-cultured lymphocytes andin vivo IL2 against advanced murine plasmacytoma

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.     

Orally administered streptococcal preparation,OK-432 augments the antitumor immunity of patients with gastric or colorectal cancer

A streptococcal preparation, OK-432, was orally administered at a dose of 5 KE to patients with gastric or colorectal cancer for 7–14 days before their operations, and its immunomodulatory effects on peripheral blood lymphocytes (PBL), regional node lymphocytes (RNL) and tumor infiltrating lymphocytes (TIL) were assessed. The group treated with OK-432 included 8 gastric and 6 colorectal cancer patients, and the control group included 8 gastric and 8 colorectal cancer patients. The NK cell activity of PBL was significantly augmented by the oral administration of OK-432, and the proportions of Leu 7+ and Leu 11+ cells in PBL also increased. The responses of PBL and TIL to autologous tumor extracts in the presence of interleukin-2 were enhanced after the oral administration of OK-432. The proportion of OKT8+ cells in PBL increased after treatment with oral OK-432, whereas the proportion in RNL significantly decreased. These results indicate that oral OK-432 affects NK and T cells and may augment the antitumor immunity of patients with gastrointestinal cancer.     

Mechanism of action of a streptococcal preparation (OK-432) in prevention of autoimmune diabetes in NOD mice. Suppression of generation of effector cells for pancreatic B cell destruction

We have previously reported that treatment with a streptococcal preparation (OK-432), one of the biologic response modifiers, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding rats used as animal models of insulin-dependent diabetes mellitus (IDDM). We studied the mechanism by which OK-432 inhibited development of IDDM in NOD mice. In female NOD mice diabetes spontaneously developed from 10 to 15 wk of age and the cumulative incidence of diabetes amounted to 74.7% at 30 wk of age. NOD mice, however, never developed diabetes over the observation period of up to 45 wk of age when they were i.p. injected with 0.1 mg of OK-432 weekly from 4 to 15 wk of age. OK-432 treatment in younger age had a stronger inhibitory effect on the development of diabetes. Diabetes was transferred to young, irradiated mice by spleen cells of nontreated, adult mice, but barely transferred by those of OK-432-treated mice. Furthermore, these spleen cells of OK-432-treated mice did not suppress transfer of diabetes by diabetic mice spleen cells. Treatment with cyclophosphamide promoted development of diabetes in nontreated NOD mice due to removal of suppressor cells. However, cyclophosphamide did not show the promotive effect in OK-432-treated mice. Taken together, these results indicate that OK-432 treatment prevented development of diabetes mainly by suppression in generation of the effector cells for pancreatic B cell destruction. The same OK-432 treatment did not suppress the immune response to exogenous AG such as xenogeneic SRBC and allogeneic tumor cells. The study suggests that BRM in the natural environment such as streptococci may suppress induction and progression of autoimmunity and be useful for the immunotherapy of human IDDM.