Administration Of OK-432 Research Articles

Refractory Hepatic Lymphorrhea Successfully Treated by Percutaneous Transhepatic Lymphography and Intraperitoneal Administration of OK-432 Following Pancreaticoduodenectomy for Intraductal Papillary Mucinous Neoplasm of the Pancreas

消化器外科手術後に発生するリンパ漏は時に栄養障害，免疫能の低下を招き，早期に対処しなければ重篤化することがある．今回，我々は膵頭十二指腸切除術（pancreaticoduodenectomy；以下，PDと略記）後に発生した難治性リンパ漏が肝リンパ漏であると診断し，当初はオクトレオチドの持続皮下注およびエチレフリンの持続静注の保存的治療を行ったが軽快せずpreshock状態に陥ったため，直接肝リンパ漏を閉塞する目的で経皮経肝リンパ管造影（percutaneous transhepatic lymphography；以下，PTLと略記）を行いリンパの漏出が半減し，その後に選択的OK-432の腹腔内投与を行い著効した症例を経験した．PD後のリンパ漏に対してPTLとOK-432の腹腔内投与による治療例の報告はなく，術後難治性リンパ漏の治療において，それが肝リンパ漏に起因する場合には本法は有効な治療選択肢の一つになりうると考えたため報告する．

Treatment of bronchial fistula after extraplural pneumonectomy using flexible bronchoscopy with the administration of OK432, fibroblast growth factor basic and fibrin glue sealant.

Treatment options for bronchial fistula (BF) after pneumonectomy are often limited and carry significant morbidity and mortality. The patient underwent right extrapleural pneumonectomy for malignant pleural mesothelioma had BF without macroscopic fistula found by bronchography. We treated this minor BF using bronchoscopy with the administration of OK-432, fibroblast growth factor basic, and fibrin glue sealant. Two weeks after this treatment, we confirmed the improvement of the fistula by bronchography. Bronchoscopic therapy for BF was useful for a small, early fistula without infection.

Successful treatment of hepatic lymphorrhea by percutaneous transhepatic lymphangiography followed by sclerotherapy using OK-432

BackgroundConventional lymphangiography cannot detect leakage sites of hepatic lymphatic vessels. Percutaneous transhepatic lymphangiography can be used to visualize leakage sites, and once the leakage site has been confirmed, effective sclerotherapy can be performed.Case presentationA rare case of intractable hepatic lymphorrhea due to injury of the hepatoduodenal ligament following pancreaticoduodenectomy is reported. Drainage of massive ascites from the drainage tube continued after surgery. Percutaneous transhepatic lymphangiography visualized the intrahepatic lymphatic vessels and the leakage site at the hepatic hilum. An 8-Fr drainage catheter was inserted adjacent to the leakage point under fluoroscopic computed tomography guidance. Repeated sclerotherapy using intraperitoneal administration of OK-432 (picibanil) through the catheter was performed, which exposed the leakage site, and control of the ascites was finally achieved.ConclusionsTo the best of our knowledge, this is the first successful case of detection of a leakage site using intrahepatic lymphangiography, followed by sclerotherapy using OK-432.

OK-432 Administration Inhibits Murine Allergic Rhinitis at the Induction Phase, through the Macrophage Activation with TLR2 Signaling Pathway.

OK-432, a preparation of a low-virulence strain (Su) of Streptococcus pyogenes (Group A) killed by a penicillin and lyophilized, is a stiff inducer of Th1 cytokines, and exerts anti-cancer effects in tumor-bearing mice. OK-432 has been reported to consist of many bacterial components, such as peptidoglycan, M-protein, etc. However, it is yet to be ascertained which bacterial component induces T helper 1 (Th1) responses. For the last decade, Toll-like receptor (TLR) family proteins are well elucidated to play a role in recognizing bacterial components and inducing interleukin (IL)-12 from macrophages. Above all, peptidoglycan seems to be the agonist of TLR2 rather than the obverse. In our present study, the role of TLR2 for the recognition of OK-432 by macrophages and the effects of OK-432 are examined on murine allergic rhinitis model. Interestingly, results show IL-12 production by macrophages derived from TLR2 knock-out (ko) mice was significantly decreased, in comparison with that of macrophages derived from wild-type mice. Moreover, in TLR2 ko mice, no regulatory effect of OK-432 was observed on an allergic rhinitis model. These data indicate that TLR2 signaling is involved in regulating OK-432-induced anti-T helper 2 (Th2) immunity, and may offer a new prophylactic and therapeutic approach using OK-432 to downregulate allergic disorders, such as allergic rhinitis.

Microwave ablation combined with OK-432 induces Th1-type response and specific antitumor immunity in a murine model of breast cancer

BackgroundMinimally invasive therapies, such as microwave ablation (MWA), are widely used for the treatment of solid tumors. Previous studies suggest that MWA is feasible for the treatment of small breast cancer, and thermal ablation may induce adaptive antitumor immunity. However, the induced immune responses are mostly weak, and the immunomodulation effects of MWA in breast cancer are unclear. Immunostimulant OK-432 can induce tumor-specific T-cell responses and may augment the immunity induced by MWA.MethodsWe treated 4T1 breast cancer bearing BALB/c mice with MWA, OK-432, MWA plus OK-432, or left without treatment. Survival time was evaluated with the Kaplan–Meyer method comparing survival curves by log-rank test. On day 25 after ablation, surviving mice received tumor rechallenge, and the rechallenged tumor volumes were calculated every 5 days. Immunohistochemistry and flow cytometry were used to evaluate the T-cell immune responses in ablated tissues and spleens. The tumor-specific immunity was assessed by enzyme-linked immunospot assays. Besides, the cytokine patterns were identified from enzyme-linked immunosorbent assay.ResultsMicrowave ablation plus OK-432 resulted in longer survival than single treatment and protect most surviving mice from tumor rechallenge. Both local and systemic T-cell responses were induced by MWA and were further enhanced by subsequent administration of OK-432. Moreover, the combination of MWA and OK-432 induced stronger tumor-specific immune responses than MWA alone. In addition, OK-432 and MWA synergistically promoted the production of Th1-type but not Th2-type cytokines, and polarized T-cell responses to Th1-dominant state.ConclusionsThe T-cell immune responses were activated by MWA in breast cancer. Furthermore, the combination of MWA and OK-432 induced Th1-type response and elicited specific antitumor immunity.

OK-432 (Sapylin) Reduces Seroma Formation After Axillary Lymphadenectomy in Breast Cancer

ABSTRACTPurpose/aim: Modified radical mastectomy is the standard surgery for breast cancer in developing countries. However, seroma formation regarded as the most frequent postoperative complication limits the therapeutic benefit of mastectomy and axillary surgery. The purpose of this study was to evaluate the efficacy of OK-432 in reducing seroma formation after axillary dissection. Methods: This prospective cohort study included 80 patients with advanced breast cancer who underwent modified radical mastectomy. Patients were randomized into two groups, which differed with the OK-432 administration. N = 40 patients per group were treated with either OK-432 plus closed suction drainage or drainage-only. Result: In comparison with the drainage-only group, we found that patients in the OK-432 group had a lower drainage volume (p = .030) and a shorter duration of axillary drainage (p < .01). Besides, the use of OK-432 could reduce the incidence of seroma formation (p < .01) and the volume of seroma (p = .040). There were also significant differences in reducing the chance of evacuative punctures (p = .036) and the healing time (p < .01) between control and OK-432 group. Conclusion: OK-432 not only shortened the suction drainage duration, but also significantly reduced seroma formation as well as the needs for aspiration punctures after modified radical mastectomy.

Gorham’s disease of the proximal tibia successfully treated with local administration of OK-432, followed by reconstruction with distraction osteogenesis

Gorham's disease (GD) is a rare and intractable disease characterized by marked progression of osteolysis associated with lymphangioma and/or hemangioma. Here, we describe a case of GD of the proximal tibia occurring in a 10-year-old boy. Although we could not correctly diagnose it at first, we finally diagnosed him as having GD. Progression of osteolysis of the tibia stopped 3 months after the local administration of OK-432. Thereafter, the huge bone defect with varus and extension deformity was reconstructed successfully by distraction osteogenesis using the Ilizarov method. The present case suggests that local administration of OK-432, followed by distraction osteogenesis is a treatment option for GD.

Effect of soluble factors derived from oral cancer cells on the production of interferon-γ from peripheral blood mononuclear cells following stimulation with OK-432

The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an invitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-β and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432.

Overcoming regulatory T‐cell suppression by a lyophilized preparation of Streptococcus pyogenes

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4(+) CD25(+) regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK-432), which stimulates Toll-like receptors, could overcome Treg-cell suppression of CD4(+) T-cell responses in vitro and in vivo. OK-432 significantly enhanced in vitro proliferation of CD4(+) effector T cells by blocking Treg-cell suppression and this blocking effect depended on IL-12 derived from antigen-presenting cells. Direct administration of OK-432 into tumor-associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4(+) CD25(+) Foxp3(+) Treg cells. Furthermore, when OK-432 was used as an adjuvant of vaccination with HER2 and NY-ESO-1 for esophageal cancer patients, NY-ESO-1-specific CD4(+) T-cell precursors were activated, and NY-ESO-1-specific CD4(+) T cells were detected within the effector/memory T-cell population. CD4(+) T-cell clones from these patients had high-affinity TCRs and recognized naturally processed NY-ESO-1 protein presented by dendritic cells. OK-432 therefore inhibits Treg-cell function and contributes to the activation of high-avidity tumor antigen-specific naive T-cell precursors.

Intrathoracic Administration of OK-432 Elevates the Serum Procalcitonin Levels

The intrathoracic administration of OK-432, a lyophilized preparation of the heat- and penicillin-treated Su-strain of type 3, group A Streptococcus pyogenes, is performed in Japan for pleurodesis of malignant pleural effusion or pneumothorax. Persistent fever is often observed after pleurodesis. To elucidate whether procalcitonin (PCT) is useful for distinguishing between the side effects of OK-432 and infection, we measured the serum PCT levels before and after pleurodesis. We performed a prospective study of 12 patients with refractory pleural effusion or pneumothorax who required pleurodesis using OK-432 between August 2011 and February 2012. The serum PCT and C-reactive protein (CRP) levels were measured on days 1 and 3. Of the 12 patients, five had pneumothorax and seven had uncontrolled pleural effusion with carcinomatous pleurisy. The median serum levels of PCT and CRP increased from 0.055 to 1.59 ng/mL (p=0.0022) and from 1.52 to 16.82 mg/dL (p=0.0022), respectively. The fevers subsided without antibiotic administration. The serum PCT level may not be useful for distinguishing fever caused by side effects of OK-432 from that caused by bacterial infection. The intrathoracic administration of OK-432 increased the serum levels of both PCT and CRP in the absence of any bacterial infection.

Transbronchial Occlusion of a Malignant Bronchopleural Fistula With Cyanoacrylate Glue

A 73-year-old man who had undergone surgery for lingual cancer, followed by radiation therapy and chemotherapy, presented with severe dyspnea. A chest radiograph revealed a left tension pneumothorax. An immediate tube thoracostomy alleviated his dyspnea. A chest computed tomography scan showed multiple large cystic tumors in both lungs. Squamous cell carcinoma was cytologically proven in the pleural effusion. A malignant bronchopleural fistula due to a metastatic pulmonary tumor was diagnosed. Surgical interventions were tried twice. However, air leakage occurred when the rapidly recurring tumor caused bilateral pneumothoraces. Thus, bronchoscopic closure was conducted. A flexible video bronchoscope and a thin catheter were inserted into the targeted left bronchiolus. The lumen of the catheter was filled with water. Cyanoacrylate glue and lipiodol were mixed in 2 syringes, and the mixture was slowly injected and then flushed with water. As soon as the lipiodol accumulation was visualized by radiograph fluoroscopy, the air leakage stopped. Pleurodesis with the intrathoracic administration of OK-432 was added to both pleural cavities, and the left and right chest tubes were removed on the third and fifth days, respectively. The patient was discharged the following day. However, he died of the disease a month later.

The neural and vascular effects of killed Su-Streptococcus pyogenes (OK-432) in preterm fetal sheep.

Fetal exposure to inflammatory mediators is associated with a greater risk of brain injury and may cause endothelial dysfunction; however, nearly all the evidence is derived from gram-negative bacteria. Intrapleural injections of OK-432, a killed Su-strain of Streptococcus pyogenes, has been used to treat fetal chylothorax. In this study, we evaluated the neural and cardiovascular effects of OK-432 in preterm fetal sheep (104 +/- 1 days, term 147 days). OK-432 (0.1 mg, n = 6) or saline vehicle (n = 7) was infused in the fetal pleura, and fetuses were monitored for 7 days. Blood samples were taken routinely for plasma nitrite measurement. Fetal brains were taken for histological assessment at the end of the experiment. Between 3 and 7 h postinjection, OK-432 administration was associated with transient suppression of fetal body and breathing movements and electtroencephalogram activity (P < 0.05), increased carotid and femoral vascular resistance (P < 0.05), but no change in blood pressure. Brain activity and behavior then returned to normal except in one fetus that developed seizures. OK-432 fetuses showed progressive, sustained vasodilatation (P < 0.05), with lower blood pressure after 4 days (P < 0.05), but normal heart rate. There were no changes in plasma nitrite levels. Histological studies showed bilateral infarction in the dorsal limb of the hippocampus of the fetus that developed seizures, but no injury in other fetuses. We conclude that a single low-dose injection of OK-432 can be associated with risk of focal cerebral injury in the preterm fetus and chronic central and peripheral vasodilatation that does not appear to be mediated by nitric oxide.

Abstract 5325: Inhibition of CD4+CD25+ regulatory T cell suppression to anti-tumor responses by OK-432 (lyophilized Streptococcus pyogenes)

Abstract It is important to control the suppressive activity to anti-tumor immune responses mediated by CD4+CD25+ regulatory T cell (Treg) for effective immunological cancer treatment. Recently, a number of studies have shed new light on recognition of pathogen-associated molecular patterns through toll like receptors (TLRs) to break the suppressive environment present at the tumor local site. TLR signals are not only able to block the suppressive activity of CD4+CD25+ Tregs but can also break CD8+ tolerance even in the presence of CD4+CD25+ Tregs. Recently, we reported that while high-avidity NY-ESO-1-specific CD4+ T cell precursors are indeed present in naïve T cell populations, their activation following peptide vaccination is kept under the strict control of Tregs, resulting in a selective activation/expansion of low-avidity NY-ESO-1 peptide-specific CD4+ T cells. Additionally, following DNA vaccination, high-avidity NY-ESO-1-specific CD4+ T cells can only be observed transiently and become rapidly suppressed by Tregs. Here, we focused on OK-432 (lyophilized preparation of Streptococcus pyogenes) that can stimulate TLRs and explored 1) whether OK-432 administrated into tumor-associated exudate fluids (chest or abdominal cavity) inhibits local Treg function and accumulation, and 2) whether OK-432 used as an adjuvant of cancer vaccine can break Treg suppression and induce the activation of high-avidity tumor antigen-specific T cell precursors. Levels of Tregs in cells collected from pleural effusion or ascites of cancer patients treated with OK-432 were assessed by staining with antibodies for CD4, CD25 and Foxp3. The frequency of Foxp3+ Tregs significantly decreased after OK-432 administration. Furthermore, suppressive function of Tregs from patients also decreased after OK-432 treatment. PBMCs were collected from patients receiving vaccination with tumor antigens (HER2 and NY-ESO-1), formulated in choresteryl hydrophobized pullulan (CHP) together with OK-432 and the presence of high-avidity NY-ESO-1-specific CD4+ T cells in effector/memory (CD25-CD45RO+) T cell population was examined. NY-ESO-1-specific CD4+ T cells were detected within CD45RO+ memory T cell population after vaccination. In contast to peptide vaccination, CD4+ T cell clones induced from these NY-ESO-1-protein vaccinated patients in the presence of OK-432 had high-affinity T cell receptors and recognized naturally-processed NY-ESO-1 protein presented by dendritic cells. Taken together, OK-432 appears to inhibit Treg function and contribute to the durable differentiation of high-avidity tumor antigen-specific naive T cell precursors into effector/memory T cells with relative resistance to Tregs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5325.

Acute Behavioral Effects of Intrapleural OK-432 (Picibanil) Administration in Preterm Fetal Sheep

Objective: To develop a model to study the fetal effects of intrapleural infusion of OK-432 (Picibanil), a pleurodesis agent derived from killed Gram-positive streptococci. Methods: OK-432 (0.1 mg, n = 5), or normal saline (n = 5) were infused over 20 min into the pleural space of chronically instrumented preterm fetal sheep at 0.7 gestation. Fetal physiological parameters, including breathing and nuchal activity were monitored in utero from 6 h before infusion until 12 h afterward, and fetuses were killed after 7 days recovery. Results: OK-432 was associated with transient suppression of fetal EEG activity, breathing and body movements from 3–6 h after infusion. Hypotension and hypoxia did not occur. At postmortem, local pleural adhesions were seen around the site of OK-432 infusion but not in saline treated fetuses. Conclusions: Intrapleural administration of OK-432 is associated with marked but transient fetal behavioral effects. This model will enable preclinical investigation of the neural and cardiovascular safety of OK-432 at a clinical relevant stage of development.

Adjuvant 5-fluorouracil and leucovorin with or without OK-432 in colorectal cancer: A single-institutional study of long-term follow-up

15128 Background: This study was to investigate whether adding a streptococcal preparation, OK-432, to chemotherapy of 5- fluorouracil (5-FU) and leucovorin (LV) could improve overall survival in patients who had undergone curative resection for Dukes’ B2 and C colorectal cancer. Patients and Methods: A total of 269 patients with a performance status of 0 to 2 received 5-FU, 400mg/m2, i.v. and LV, 20mg/m2, i.v. (FL), which was given daily for 5 days in every 4 weeks for a maximum of 12 cycles, ± OK-432 (2 KE s.c. weekly during chemotherapy) for colon cancer, and FL and radiotherapy (RTx) (5040 - 5400 cGy) ± OK-432 for rectal cancer. Results: From January 1991 to December 1996, 69 among 139 patients with colon cancer and 62 among 130 patients with rectal cancer were treated with the addition of OK-432 to FL adjuvant therapy. The median follow-up duration was 11.5 years. Regardless of addition of OK-432, there was no statistically significant difference in overall survival between in colon (FL, 75.6%; FL+OK-432, 82.2%, P=0.7449) and rectal (FL + RTx, 60.3%; FL+RTx+OK-432, 76.6%, P=0.139) cancer patients. However, overall survival (FL + RTx, 57.9%; FL+RTx+OK-432, 75.6%, P=0.0478) was different significantly in subgroup with Dukes’ C rectal cancer with addition of OK-432. Toxicities following OK-432 administration were not different, except for the flu-like symptom and rash. Conclusion: Chemoimmunotherapy with a regimen of FL and OK-432 does not improve survival in patients with curatively resected colorectal cancer, but further trials for patients with Dukes’ C rectal cancer are required to study whether chemoimmunotherapy is effective or not. No significant financial relationships to disclose.

Experimental pseudocyst model resembling human ganglion

There is no animal model of ganglion. We describe a simple and reproducible animal model of pseudocystic diseases. First, we experimented to establish a pseudocystic model. We used cylindrical glass implants (6 mm diameter, 30 mm long) to create fibrous capsules in rats. The implants were inserted in the subcutaneous tissue in the dorsum of rats. Sixty implants were carried out (two implants per rat). Twelve weeks after implantation, the glass implants were removed and 0.5 mL sodium hyaluronate solution was injected into each cavity. Next, we tested the model by histological examination after OK-432 administration. Microscopic examination revealed that the wall was composed of a layer of collagenous fibers similar to those noted in ganglia; the lumen was retained for 3 weeks. Histopathological changes after OK-432 administration showed nonspecific inflammatory response induced by OK-432, resulting in in vivo activation of many inflammatory cells and then fast and reliable closure of cavities. No harmful reactions to OK-432 were observed histopathologically. These data suggest that our experimental cyst is a suitable model for studying pseudocystic diseases. This model can be used for research evaluating safe drug doses, conducting therapeutic comparison of several agents, and histopathological time course studies of the affected tissues. OK-432 administration on this model showed the potential of one of the ideal agents to treat pseudocystic lesions like ganglion.

Intralesional injection of OK-432 for vision-threatening orbital lymphangioma

Surgical excision of orbital lymphangiomas is difficult, and almost always incomplete due to the diffusely infiltrative pattern of these tumors. The present report describes the successful use of intralesional OK-432 administration to treat two patients with intractable hemorrhagic proptosis due to orbital lymphangiomas. A 3-year-old girl (case 1) presented with aggressive proptosis and ptosis, and a 1-year-old boy (case 2) presented with massive proptosis and exposure keratopathy, associated with recurrent intracystic bleeding of an orbital lymphangioma. In case 1, 0.02 mg OK-432 was intracystically injected in a volume of 2 ml of physiologic saline. Due to a lack of therapeutic response, a second injection of 0.05 mg OK-432 in 1 ml was administered. In case 2, a single dose of 0.05 mg OK-432 in 1 ml was administered. In both cases, intracystic administration of 0.05 mg of OK-432 in a 1-ml volume resulted in a successful outcome. The adverse effects were minor (mild transient fever and lid swelling), and rebleeding and intraocular pressure elevation did not occur. Proptosis and eyelid swelling gradually improved over 1 month, and completely resolved within 3 months of treatment. Intralesional administration of 0.05 mg/ml OK-432 (delivered in 1 ml) resulted in the successful treatment of two cases of orbital lymphangioma. Although this drug concentration is higher than in previous reports, there were no major adverse effects.

Involvement of nitric oxide in anti-tumor effects of OK-432, a streptococcal anti-tumor immunotherapeutic agent

We examined the role of nitric oxide (NO) induced by OK-432, a streptococcal immunotherapeutic agent, in anti-tumor effects of the OK-432 by in vitro and in vivo experiments using an NO synthase inhibitor, N-monomethyl- l-arginine acetate (NMA). The in vitro treatment of mouse splenocytes with OK-432 increased the expression of inducible NO synthase (iNOS) gene and NO production in a dose-dependent manner. Although it is well known that OK-432 induces cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, both of which are known to be potent NO inducers, we observed only a partial reduction of OK-432-induced NO production with the addition of anti-IFN-γ and/or anti-TNF-α neutralizing antibodies. The cytotoxicity of the splenocytes increased by in vitro OK-432 stimulation was almost completely inhibited by the treatment with NMA. OK-432 administration resulted in a marked prolongation of survival and a significant inhibition of tumor growth in syngeneic tumor-bearing mice, whereas NMA significantly inhibited the anti-tumor effects of OK-432. Although the increased cytotoxicity of adherent splenocytes derived from OK-432-treated tumor-bearing mice was almost completely inhibited by NMA, only partial inhibition by NMA was observed in the cytotoxicity of the nonadherent splenocytes. These findings strongly suggest that the iNOS/NO induced by OK-432 is intimately involved in the anti-tumor effects of OK-432.

Effect of Intraperitoneal Neutrophils Induced by OK432 on Malignant Ascites

To evaluate the efficacy of a streptococcal preparation, OK432, on malignant ascites in mice. PC-C203U (PC203) is a preparation of another strain of the streptococcal family, with the lowest antineoplastic action. To examine the survival curves of mice after the inoculation of BAMC-1 tumor cells, we gave intraperitoneal OK432, PC203, or saline as a control. Intraperitonal neutrophils were counted by cytospin, and interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and macrophage inflammatory protein (MIP)-1alpha were measured by enzyme-linked immunosorbent assay, and 8 h after the administration of OK432, PC203, and saline. Using electron microscopy, we examined the greater omental milky spots, where the in situ proliferation of neutrophils or macrophages takes place. The OK432 group had the best survival and the control group the worst. The ratio of intraperitoneal neutrophils to BAMC-1 was highest in the OK432 group and lowest in the control group. Quantitative IL-1beta, IL-6 and MIP-1alpha levels were correlated closely with survival. Electron microscopic examination of the milky spots revealed massive proliferation of neutrophils in the OK432 group, but not in the PC203 or control groups. OK432 effectively activated intraperitoneal neutrophils and a series of immunological chain reactions through an increase in IL-1beta, IL-6, and MIP-1alpha levels. Milky spots could have important antitumor effects in terms of the spread of neutrophils.

Stable long-term induction of perforin-positive CD8+ T cells in gut by oral administration of streptococcal preparation OK-432

Perforin is known as a pore-forming cytotoxic granule released from cytotoxic T cells. Previous experiments in vitro revealed the presence of precursor cells that are capable of producing perforin in the immune system cells. The present study was undertaken to examine whether perforin-positive cells could be induced in the digestive tract and to characterize their precursor cells. Expression of perforin-positive cells in the intestine of Balb/c mice induced by OK-432 was analyzed by immunohistochemical staining and RT-PCR. Oral treatment of Balb/c mice with OK-432 resulted in the occurrence of perforin-positive cells in the inferior segment of small intestine, the superior segment of large intestine, mesenteric lymph nodes and spleen. In the small intestine, perforin-positive cells were found in the lamina propria mucosa. The presence of perforin-positive cells was also noted following long-term OK-432 treatment. Similar results were obtained following treatment with biological response modifiers such as lipopolysaccharide. In mice with GVHD (graft-versus-host disease), the presence of perforin-positive cells was noted in the small intestine and spleen. When the serial sections of the small intestinal mucosa from OK-432-treated mice were immunostained with anti-perforin, anti-CD8 and anti-asialo-GM1 antibodies, the perforin-positive cells were found to be CD8-positive. The results suggest that CD8(+) cells in lamina propria mucosa play a significant role as effectors in the mucosal immune system which is activated by various stimuli.