Biochemical modulation of 5-fluorouracil with a streptococcal preparation, OK-432, against murine colon-26 carcinoma

Conventional therapy for colorectal carcinoma using 5-fluorouracil (5-FU) has shown limited antitumor action. The purpose of our study was to investigate synergistic antitumor effects of the streptococcal preparation of OK-432 and 5-FU, and to elucidate the mechanisms of interaction between the 2 agents in mice. Biochemical modulation of OK-432 and 5-FU were determined in vivo against colon-26 carcinoma. The concentration of 5-FU and its metabolites, and the activity of thymidylate synthase and thymidine kinase, respectively, were measured using cytosolic extracts of the tumors. Combination treatment with OK-432 produced a significant increase in intratumor 5-FU and 5-FU in RNA (F-RNA) concentrations, increased the thymidylate synthetase inhibition rate, and decreased thymidine kinase activity, as compared with the results observed in the control mice. These additive antitumor effects are obtained by use of the 2 agents; the mechanism of action is considered to be the suppression of both the de novo and the salvage pathway for DNA synthesis, along with the suppression of RNA synthesis.     

Combined effects of intraperitoneal administration of recombinant interleukin-2 and streptococcal preparation OK-432 in murine tumors

The combined effects of rIL-2 and OK-432 were investigated against a Meth-A tumor, a syngeneic tumor of inbred BALB/c mice. An analysis of the effector cells was also performed. The treatment resulted in an inhibition in vivo of tumor growth and increased survival of the Meth-A tumor-bearing mice. Splenic cells obtained from Meth-A inoculated mice which received combination therapy were not only NK-sensitive YAC-1 and LAK-sensitive EL-4 cells, but also NK-resistant Meth-A cells, as shown in a 4-h 51Cr-release assay. Syngeneic killer cell activity against Meth-A cells was abolished almost completely with anti-Thy 1.2 treatment and about 70% of the activity was abolished with anti-asialo GM1 treatment in a complement-dependent cytotoxic assay. It was not changed by the removal of macrophages and B cells from the splenic cells. Mice which survived for 60 days after the start of therapy rejected Meth-A inoculation when rechallenged, suggesting the establishment of a specific immunity. Combination therapy appeared to be beneficial against Meth-A cells and T-cells appeared to play a determining role in the treated Meth-A bearing mice. It was suggested that more than two populations of killer cells exist in the spleen treated with the combined therapy and they may have the same characteristics as activated T and NK cells with or without specific killer T-cells.     

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.     

Percutaneous treatment of renal cysts with OK-432 sclerosis

PURPOSE: The aim of this study was to demonstrate OK- 432 sclerotherapy efficacy for treatment of simple renal cysts. MATERIALS AND METHODS: Twenty patients with 25 symptomatic or large simple cysts were treated by ultrasonography (US)-guided percutaneous aspiration and injection of OK-432 (8 men and 12 women, mean age 63.6 years, SD 9.5). Six patients presented with flank pain, 14 presented with renal mass; renal cyst location was right, left, or bilateral sided in 9, 8, and 8 kidneys, respectively. Patients were evaluated by clinical assessment, US, or CT scan 3 months following the procedure. Complete and partial success was defined as symptom resolution with either total cyst ablation or greater than 70% reduction, respectively. Failure was defined as 30% of cyst size recurrence and/or persistent symptoms. RESULTS: Average reduction was 93.0%. Complete and partial resolution occurred in 11 (44.0%) and 13 (52.0%) cysts, respectively. One case was defined as failure, with a 64.2% size reduction from 10.9cm to 3.9cm (volume reduction rate 95.4%). Renal pain improved in all patients, regardless of complete or partial resolution. Minor complications occurred in 3 patients, 2 developed leukocytosis and 1 had mild fever (< 38.5 degrees C) following aspiration and sclerotherapy. Successful treatment was achieved with conservative measures and NSAID therapy. CONCLUSION: Percutaneous treatment of simple renal cysts with OK-432 sclerotherapy was found to be a safe, effective and minimally invasive procedure.     

Immunohistochemical studies on local antitumor effects of streptococcal immunopotentiator, OK-432, in human solid malignant tumors

Immunocytochemical techniques were used to clarify the local inhibitory effects of a streptococcal immunopotentiator, OK-432, against solid malignant tumor growth. Natural killer (NK) cells and fibronectin were chosen as immunostaining markers to demonstrate the antitumor effects. Immunocytochemical staining was performed by the avidin-biotin-peroxidase complex method. These investigations demonstrated that (1) local administration of OK-432 seems to promote a marked induction of NK cells and fibroblasts around or entering into the cancerous lesions and (2) the cancer cell-killing effect of NK cells and the fibronectin-enriched stromal reaction augmented by the injection of OK-432 suggest at least the possibility of protection against neoplastic growth with invasion and the spread of distant or nodal metastases of solid carcinomas.     

OK-432增强脐血树突状细胞瘤苗抗胃癌作用的实验研究

目的： 用脐血浆代替胎牛血清，观察OK-432对脐血来源的树突状细胞（DCs）功能的影响，探讨制备高效抗肿瘤DCs疫苗的优化方案。方法: 分离脐血单个核细胞并在含10%同源脐血浆、GM-CSF和IL-4的培养体系中诱导培养，部分加入肿瘤冻融抗原和/或OK-432冲击,MTT法检测DCs体外刺激同种混合淋巴细胞增殖的免疫活性，LDH法检测DCs诱导的CTL对不同靶细胞的细胞毒作用。结果: 收集第9 d的各组DCs，肿瘤冻融抗原冲击后的DCs呈成熟形态，能呈递胃癌相关抗原给淋巴细胞刺激其增殖并诱导对胃癌细胞株特异的CTL毒性。OK-432联合肿瘤冻融抗原冲击能增强其抗原呈递功能，诱导更强的混合淋巴细胞增殖反应及针对胃癌细胞株的强烈CTL杀伤效应。结论: 该DCs培养方案具有培养时间短、细胞因子消耗少、试剂简单等优点,可用于制备DCs肿瘤疫苗,有临床应用价值。     

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.     

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.     

沙培林联合肿瘤冻融抗原刺激的小鼠骨髓源性树突状细胞瘤苗的抗胃癌效应

目的:观察沙培林(OK-432)对小鼠骨髓源性树突状细胞(DCs)功能的影响,及由此产生的DCs对荷胃癌小鼠的抗癌效应.方法:OK-432联合肿瘤冻融抗原诱导小鼠骨髓源性DCs,ELISA法检测DCs分泌白介素12(IL-12)水平.建立荷胃癌小鼠模型,尾静脉注射DCs,观察其对肿瘤生长的抑制作用,RT-PCR法检测移植瘤IL-12mRNA表达情况,ELISA法检测移植瘤IL-12蛋白表达情况,免疫组织化学法检测移植瘤血管内皮生长因子(VEGF)蛋白表达情况.结果:OK-432联合肿瘤冻融抗原促进DCs分泌IL-12.OK-432联合肿瘤冻融抗原刺激的DCs抑制移植瘤生长,促进移植瘤IL-12mRNA和IL-12蛋白表达,抑制移植瘤VEGF蛋白表达.结论:OK-432联合肿瘤冻融抗原刺激能有效增强DCs瘤苗的抑瘤效应,其机制可能与纠正肿瘤组织免疫缺陷,促进肿瘤组织表达IL-12和抑制肿瘤组织表达VEGF有关.     

Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.     

Production of interleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.     

Increased plasma IL-6, IL-8, TNF-alpha,and G-CSF in Japanese narcolepsy

Narcolepsy is a chronic hypersomnia involving excessive daytime sleepiness and cataplexy. Some susceptibility genes and environmental factors suggest that post-infectious immunological alterations underlie its pathophysiology. To investigate the immunological alterations in narcolepsy patients, we examined cytokines.     

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.     

Activation and augmentation of guinea pig macrophages with streptococcal preparation OK-432 and stimulated spleen cell products

A mechanism of macrophage activation by a streptococcal preparation (OK-432) was studied. Peritoneal exudate macrophages from normal guinea pigs treated in vitro with OK-432 were activated, manifesting increased glucose consumption, increased spreading, and morphological alterations under scanning electron microscopy. Macrophages showed extensive spreading within 1 hr after OK-432 treatment; they then became rounded with characteristic ruffles in their surfaces after 6 hr of treatment. Highly purified macrophages were activated as effectively as a crude macrophage preparation, suggesting that the macrophage activation resulted from a direct interaction between macrophages and OK-432. However, when spleen cells were treated with OK-432, a factor(s) capable of activating macrophages was produced in the culture supernatant. Spleen macrophage-rich preparation was found to release the factor(s) upon stimulation with OK-432, but this did not occur with the lymphocyte-or granulocyte-rich preparations. These results indicate that OK-432 not only activates macrophages by direct interaction without lymphokine participation, but also augments the activation by affecting spleen cells, probably macrophages, in such a way as to produce monokines.     

Correlation between the presence of intracellular OK-432 and antitumor activity of peritoneal macrophages

The mechanism of the generation of tumoricidal macrophages by intraperitoneal administration of OK-432 was investigated. An association between the presence of intracellular FITC-conjugated OK-432 and the increase of macrophage cytostatic activity was observed after i.p. administration of OK-432. When peritoneal exudate cells were fractionated on the basis of cell size by velocity sedimentation, a higher proportion of macrophages and a strong cytostatic activity were shown in the large cell fractions. Furthermore, in these fractions, increased numbers of macrophages with intracellular FITC conjugated and 3H-acetate labelled OK-432 were observed. Thus, the more cytocidal macrophages can be identified by the presence of intracellular microorganisms. A role for OK-432 in the direct activation of macrophages is suggested.     

肿瘤抗原冲击致敏的IL-2基因修饰的巨噬细胞治疗肾癌的实验研究

目的 :观察体外肿瘤抗原冲击致敏的白细胞介素 2 (IL 2 )基因修饰的巨噬细胞对肾癌小鼠的治疗效果并探讨其相关的免疫机理。方法 :通过重组腺病毒的介导 ,将IL 2基因转入新鲜分离的小鼠腹腔巨噬细胞 ,经肿瘤抗原冲击致敏后回输治疗原位肾癌小鼠 ,采用 4h5 1 Cr释放法检测脾脏NK和CTL活性。结果 :IL 2基因修饰的巨噬细胞经肿瘤抗原冲击后体内回输可使肾癌小鼠肺转移结节明显减少 ,存活期明显延长 ,40 %肾癌小鼠达到长期存活。治疗后荷瘤小鼠脾脏NK和CTL活性显著提高。结论 :IL 2基因修饰的巨噬细胞经肿瘤抗原冲击后自体回输是治疗肾癌的有效方法。     

Combination tumor immunotherapy with radiotherapy and Th1 cell therapy against murine lung carcinoma

Mice bearing established Lewis lung carcinoma (LLC) expressing model tumor antigen, ovalbumin (OVA) (LLC-OVA) marginally responded to local radiotherapy, but none of the mice was cured. In contrast, treatment of the tumor-bearing mice with intratumoral injection of tumor-specific T helper type 1 (Th1) cells and tumor antigen (OVA) after radiotherapy dramatically prolonged the survival days and induced complete cure of the mice at high frequency (80%). Radiation therapy combined with Th1 cells or OVA alone showed no significant therapeutic activity against LLC-OVA. Such a strong therapeutic activity was not induced by intratumoral injection of Th1 cells plus OVA. Compared with other treatment, radiation therapy combined with Th1 cells and OVA was superior to induce the generation of OVA/H-2^b tetramer⁺ tumor-specific cytotoxic T lymphocyte (CTL) with a strong cytotoxicity against LLC-OVA in draining lymph node (DLN). Moreover, the combined therapy is demonstrated to inhibit the growth of tumor mass, which grew at contralateral side. These results indicated that radiotherapy combined with Th1 cell/vaccine therapy induced a systemic antitumor immunity. These findings suggested that combination therapy with radiotherapy and Th1 cell/vaccine therapy may become a practical strategy for cancer treatment. Hiroshi Yokouchi and Kenji Chamoto are equally contributed.