Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.     

Desmoplasia and regression of lung adenocarcinoma after intratumor injection of OK-432: report of a case.

Intratumor injections of an immune modulator, OK-432, were administered to a 61-year-old man with inoperable lung adenocarcinoma. He received intratumor injections of OK-432, 20 K.E., twice weekly, under chest ultrasound guidance. A total dose of 240 K.E. was given in a six-week period. The tumor size decreased during a six-month follow-up period after the OK-432 injections. The immunologic profile of the patient showed neutrophilia, a decrease in the lymphocyte count in the peripheral blood and an increase in immunoglobulins after the OK-432 injections. The peripheral T-lymphocyte subsets showed a significant reduction in cytotoxic T cells and a decrease in the OKT4/OKT8 ratio. Histologic examination of the tumor after OK-432 injections showed extensive desmoplastic fibrosis. There was no evidence of lymphocyte infiltration. Intratumor injections of OK-432 may be an alternative for local control of inoperable lung cancer.     

Study on enhancement of antitumor immunity in cervical cancer--with reference to administration of immunopotentiators

The immunopotentiator OK-432 was injected intracutaneously, locally, or both, to patients with cervical cancer. The results were as follows: Histological changes: There was a marked increase in the infiltration of T3, Ia1, M1, Leu7 into the interstices surrounding the cancer and the regional lymphnodes. An analysis of the mononuclear cells in the regional lymphnodes by flow cytometry showed an increase in the percentage of M1 and Leu7. Cytotoxicity: There were significant increases in NK activity, LAK activity and killer activity in the mononuclear cells around the cancer and of the regional lymphnodes. These changes in the intracutaneous administration concomitantly with local administration were more marked than in the intracutaneous or local administration.     

Clinical studies of long-term administration of OK-432 in patients with gynecologic cancer

The clinical value of OK-432 treatments, was evaluated in 215 patients with various types of gynecologic cancer. Five KE OK-432 was administrated by intramuscular injection every other day for up to 5 years, unless severe side-effects were observed. The 5-year survival rate was determined in 51 patients in the OK-432 group and 93 patients in the control group. The survival rate in the OK-432 group was significantly higher in the cervical cancer patients in stages III and IV than in the control group, at from 20 to 29 months and from 37 to 42 months. These results indicated that long period administration of OK-432 might improve the survival rate in patients with gynecologic cancer, especially in those with advanced cervical cancer.     

Immunotherapy using the streptococcal preparation OK-432 for the treatment of uterine cervical cancer. Cervical Cancer Immunotherapy Study Group

The efficacy of immunotherapy using a streptococcal preparation, OK-432, was evaluated in each clinical stage of uterine cervical cancer. The 382 eligible patients were stratified by clinical stage and presence/absence of surgery. Within each stratum, patient's were randomly allocated to OK-432 treatment or to control treatment. OK-432 significantly inhibited recurrence in patients with stage II cervical cancer; the recurrence-free interval and survival time were remarkably prolonged in patients with stage II disease who underwent surgery. However, OK-432 did not significantly prolong these parameters in patients with stage III disease. Retrospective analyses revealed that in patients with or without lymph node metastases who underwent surgery, the recurrence-free interval and survival time were significantly prolonged by OK-432 treatment. These results indicate that OK-432 is an effective and useful postoperative immunotherapeutic agent for uterine cervical cancer.     

透明质酸在宫颈上皮及宫颈癌组织中的表达

目的 :探讨透明质酸 (HA)在正常宫颈上皮及宫颈癌中的表达及其与肿瘤发生发展的关系。方法 :用免疫组化法检测 5 9例宫颈浸润癌、35例宫颈上皮内瘤样病变 (CIN)及11例正常宫颈组织中HA和CD4 4v6的表达。结果 :正常宫颈上皮不表达HA。慢性炎症者上皮底层细胞以及细胞间质表达HA ,随病变加重及CIN发生 ,HA表达增强。癌旁组织的上皮细胞HA呈强阳性表达 ,伴有基质HA强阳性染色。癌细胞HA阳性率为 6 7.2 7% ,与病理类型有关 ,角化癌高表达 ,而腺癌不表达 ,在低分化鳞癌中多见不规则灶性HA阴性区。宫颈鳞癌的肿瘤基质HA表达普遍增强。肿瘤基质的HA表达与CD4 4v6呈正相关。结论 :正常宫颈上皮不表达HA ,但炎症与致瘤因素可促使HA表达 ,HA的代谢失衡可能与宫颈癌的发生及浸润行为有关     

The effect of intraperitoneal administration of OK-432 with recombinant interleukin-2 to patients with peritonitis carcinomatosa of recurrent gynecological cancer and changes in ascitic lymphocyte subsets

The effect of intraperitoneal administration of OK-432 followed by intraperitoneal instillation of recombinant interleukin-2(rIL-2) was examined in tumor bearing animals and in four recurrent gynecological cancer patients with peritonitis carcinomatosa which had been resistant to chemotherapeutic drugs. Seven days after intraperitoneal inoculation of tumor cells (10(6) cells/body of MH134 hepatoma into C3H/He mice and 10(5) cells/body of Meth-A fibrosarcoma into BALB/C mice, respectively), OK-432 was administered intraperitoneally. Two days later, a 14 day course of daily intraperitoneal instillation of rIL-2 followed. The survival time for animals treated with OK-432 combined with rIL-2 was significantly prolonged. Three of the 8 C3H/He mice and one of the 8 BALB/C mice in this group survived more than 150 days without forming ascites. However, the group treated by rIL-2 alone did not survive for more than 40 days. The group treated by OK-432 alone as well as the untreated group did not survive for more than 20 days. Ascitic fluid disappeared clinically in two of four cases and decreased in the rest. Ascitic cancer cells disappeared in one case and decreased in three cases. The serum CA125 level declined significantly in all cases. The surface markers of ascitic lymphocytes were analyzed by flow cytometry on day 8. The CD4+ subset accounted for 70-90% whereas the CD8+ subset accounted for only 7-17%. In three cases in which two color analysis was performed, the CD4+, CD29+ helper inducer T cell was dominant. We could conclude that LAK cells were not the main effector cells.     

Effect of immunotherapy using the non-specific immunopotentiator OK-432 in malignant ovarian tumors--selecting appropriate cases for immunotherapy

Immunotherapy for malignant ovarian tumors has been discussed in a number of recent studies. However, the effects of this therapy are produced by an indirect mechanism, and are, therefore, difficult to evaluated. In order to select appropriate cases for immunotherapy using OK-432 against malignant ovarian tumors, clinical and experimental studies were carried out. Clinically patients with ovarian cancer were divided into two groups; OK-432 treated group and no immunotherapy groups. As a parameter of immunoresponse to OK-432, PHA and Su-PS skin tests were performed periodically. Natural killer (NK) cell activity was also measured before and after administration of OK-432. Peripheral blood lymphocyte subsets were analysed by using monoclonal antibody and a laser flow cytometer. Finally tumor growth velocity was evaluated in tumor transplanted nude mice. The survival rate was used as an index for evaluation. The survival rate of the OK-432 treated groups was better than the control groups in advanced cases, but a statistically significant difference was found between the two groups. Among fundamental studies which were carried out to select appropriate cases, Su-PS skin test, the NK response of OK-432 seemed to be actually utilized.     

子宫颈上皮内病变及子宫颈癌患者外周血及局部免疫功能分析#br#

Objective?To investigate the distribution of T cell subsets in peripheral blood of different cervical lesions and the level of cytokines secreted, and it’s difference in the location and density of immune cell distribution in the epithelium and stroma of cervical lesion tissues. Methods?We used CD series cell detection slide quality control kit to detect the distribution of T cell subsets in peripheral blood of 55 patients with different cervical lesions, analyzed the changes of peripheral blood cytokines, and analyzed the localization and density distribution of T lymphocytes (CD4+T, CD8 +T) in the tissue microenvironment of 64 patients with different cervical lesions by immunohistochemistry. Results?The level of CD4+T and CD8+T in peripheral blood of patients with low grade lesion (LSIL) were lower, with 479.13±229.65 unit and 378.00±231.74 unit respectively. The level of CD4+T and CD8+T in high grade cervical squamous intraepithelial lesion(HSIL)was 816.30±284.65 unit and 668.75±268.92 unit respectively, and the level of CD4+T and CD8+T in carcinoma was 824.80±330.65 unit and 564.00±58.31 unit. Meanwhile the concentration of IL-17 in the serum of LSIL patients was higher than that of HSIL (P<0.05). CD8+T cells in epithelial and stroma tissues of cervical cancer were higher than those in control, LSIL and HSIL tissues, with statistical significance (P<0.05). However, CD4+T cells in the stroma of cancer tissues were higher than those of control, LSIL and HSIL tissues, with statistical significance (P<0.001). Conclusion?The patients with cervical lesion have abnormal immune function in the systemic immunity and local microenvironment of the lesion, both CD8+T cells and CD4+T cells play a key role in eliminating HPV-infected cervical epithelial cells. The development of cervical lesions is related to the cellular inflammatory factors.     

Effect of malignant ascitic fluids and OK-432 on the induction of LAK activity in peripheral blood mononuclear cells of gynecological cancer patients]

Recently, successful treatment of peritonitis carcinomatosa by intraperitoneal administration of lymphokine-activated killer (LAK) cells followed by intraperitoneal recombinant interleukin 2 (rIL-2) has been reported by several authors, in spite of the well documented results of the immunosuppressive activity of malignant ascitic fluid. We investigated the effect of malignant ascitic fluid on in vitro induction of LAK cells obtained from patients' peripheral blood mononuclear cells. We also examined whether Streptococcal preparation OK-432, which has been instilled into the peritoneal cavity to treat malignant ascites, is able to synergize with rIL-2 to induce LAK activity, and the following results were obtained; 1. A little augmentation of LAK activity was observed at a lower concentration of malignant ascitic fluid, while the results were diversified at a concentration higher than 10%. In two cases out of six, a severe suppressive effect was observed at a concentration higher than 40%. At the higher concentration, fewer cells were recovered after a 5 day culture in all cases. 2. No augmentation of LAK activity following the combination of OK-432 with rIL-2 was observed. At a lower concentration of OK-432 (ranging from 0.01-0.04KE/ml), the number of cells increased in comparison with rIL-2 alone. These results suggest that the potential of adoptively transferred LAK cells followed by rIL-2 was not effectively suppressed by malignant ascitic fluid in vivo and that the administration of OK-432 followed by rIL-2 could induce a larger number of various killer cells than rIL-2 alone.     

Studies on immunotherapy of uterine cervical cancer by administration of schizophyllan (SPG) (author's transl)

This study was designed to investigate effects of immunopotentiator on cervical cancer. We administered schizophyllan (SPG) as immunopotentiator to patients of cervical cancer therapy with irradiation, and examined successively immunological parameters such as blastformation of lymphocytes, skin reaction of PPD, lymphocyte counts, concentration of serum immunoglobulins, and other clinical inspections. Moreover we examined ability of interferon induction of SPG. We also calculated and compared 3 year relative survival rate of the cases administered SPG and historical controls therapy with only irradiation. In patients administered SPG the results revealed: 1) an enhancement of stimulation index of lymphocytes blastformation with PHA, 2) an enlargement of skin reaction to PPD, 3) no effects to number of lymphocytes, serum immunoglobulins, liver function and renal function, 4) an interferon activity on patients' sera of cervical cancer after administration of SPG, and 5) patients administrated SPG had significantly high relative survival rate in comparison with the historical control of cervical cancer cases treated solely by irradiation.     

Studies of natural killer activity and augmentation by OK-432 in patients with gynecological malignancies

The natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities were determined in peripheral blood lymphocytes of patients with gynecologic malignancy. The effects of a preparation of Streptococcus pyogenes (OK-432) and of interferon were determined. Patients with advanced cancer exhibited lower natural cytotoxicity than normal women. NK and ADCC activities were decreased following surgery and NK activity was decreased following chemotherapy. Radiation reduced the percentage of Leu-7-positive cells in the circulation of the patients. OK-432 augmented the NK activity of both healthy women and patients with early malignancy. Interferon was less effective than OK-432 in augmenting NK activity.     

Efficacy and safety of OK-432 sclerotherapy for giant cystic hygroma in a newborn

BACKGROUND: OK-432, a lyophilised incubation mixture of group A Streptococcus pyogenes of human origin, was used as a sclerosant for the involution of a giant cervical cystic hygroma in a newborn. RESULTS: There were no systemic side effects. Blood tests and double immune diffusion tests showed no systemic infection or generalised inflammatory response, or antibody production. Cellular and cytokine-induced localised inflammatory reaction within the cystic hygroma, was observed on analysis of the intracystic fluid. CONCLUSIONS: The leucocytosis induced and activated by OK-432 probably increases the endothelial permeability of the lymphatics. This probably accelerated lymph drainage leading to involution of the cystic hygroma. Intralesional injection of OK-432 was safe and effective therapy for cystic hygroma in this newborn as its inflammatory reaction was localised.     

Significance of peritoneal macrophages on fertility in mice.

OBJECTIVE: To elucidate the role of peritoneal macrophages in infertility, we investigated how the presence of peritoneal macrophages would affect fecundity in mice. Moreover, we also studied the effects of interleukin-1 on embryonic development. STUDY DESIGN: Mice were administered OK-432 intraperitoneally to induce macrophage infiltration of the peritoneal cavity; ovulation was then induced and animals were mated. On day 13 of gestation, fetuses were counted. After injection of OK-432 or interleukin-1, the mice were mated. Three days later, embryos were collected and the stage of embryo development was determined. RESULTS: In mice given OK-432 (n = 33), four (12%) became pregnant and the mean litter number was 6.0 +/- 3.6, whereas in the control group 23 of 30 mice (77%; p < 0.01) became pregnant and the litter number was 14.1 +/- 5.3 (p < 0.01). OK-432 and interleukin-1 administered intraperitoneally significantly suppressed embryo development (p < 0.01). CONCLUSION: Increased numbers of peritoneal macrophages negatively affect fecundity, probably by suppressing embryonic development.     

Macrophages and not granulocytes are involved in cervical ripening

To clarify the role of leucocytes in human cervical ripening and dilatation, cervical biopsies were obtained from six non-pregnant women, eight women undergoing early termination of pregnancy, 18 pregnant women undergoing elective Caesarean section at term (both with and without a ripe cervix as determined by Bishop score) and 11 women after term vaginal delivery. Leucocytes were localised by immunohistochemistry labelling and quantified in subepithelial and deep stromal areas. CD45+ leucocytes were more numerous in the subepithelial area of the cervix than in the deep stroma in all groups (P<0.01). CD14+ macrophages and CD15+ granulocytes were increased in both the subepithelial and deep stromal areas only in the vaginal delivery group (P<0.01). The number of macrophages in the ripening cervix (Bishop score above 4) was higher than in the unripe cervix (Bishop score 4 or less; P<0.05) with no differences in other leucocyte populations. CD3+ CD8+ T cells in the subepithelial area were reduced in late pregnancy and after vaginal delivery (P<0.01), but showed no relationship to Bishop score. Macrophages and granulocytes may be involved in the process of cervical dilatation, but macrophage infiltration into the ripening cervix before labour suggests their role in the ripening process. Reduced numbers of CD3+ CD8+ T-lymphocytes in late pregnancy and after vaginal delivery suggests that local immunity is down-regulated in the late pregnancy period. Regional differences in leucocyte subpopulations in the cervix indicate that leucocyte infiltration is likely to be regulated by local factors.     

Treatment with biologic response modifiers in patients with ovarian cancer.

The therapeutic and immunomodulating potential of biological response modifiers (BRM) such as OK-432 (a streptococcal preparation) and recombinant interferon gamma (rIFN-gamma) has been evaluated in 15 patients with advanced chemotherapy resistant ovarian cancer, presenting malignant ascites and/or pleural effusions. OK-432 was injected intracavitary in 10 patients in increasing doses from 0.2 up to 7.5 mg weekly. Five women were treated intracavitary with rIFN-gamma twice a week. The initial dose was 0.1 mg/m2 which was raised up to 12 mg/m2 over 6 weeks. With OK-432 a complete response was achieved for 14.1 + 8.9 months in 4 patients, a partial response for 1.7 + 0.3 months in 3 patients. The survival time of the 4 responders was significantly longer (21.1 + 8.3 months) than the survival time of the patients with partial or no response (4.9 + 2.7,4.1 + 2.3 months, respectively). In the rIFN-gamma therapy group, we found a partial response in one and no response in 4 patients. Toxicity observed under OK-432 and rIFN-gamma was minimal in all patients, suggesting a lack of systemic effect of intracavitary-applied BRM. With both agents, augmentation of certain immune responses, especially in the peritoneal cavity and to a lesser extent in the peripheral blood, has been documented. In 5 patients treated with OK-432, we found an overall augmentation of the effusion macrophage killer activity. rIFN-gamma augmented natural killer activity in 2 of 3 patients.     

Apoptosis and apoptosis-related proteins (Fas, Fas ligand, Blc-2, p53) in lymphoid elements of human ovarian tumors

Different types of lymphocytes have different roles in tumor suppression. Thus, their expression of apoptosis-related proteins (ARP - Fas and Fas ligand, bcl-2, p53) in lymphocytes and their apoptosis were analyzed immunohistochemically in ovarian tumors of different grades. Ovaries without oncologic disorders had few lymphocytes, mainly T cells, and no ARP. Benign cysts presented features of weak immune reaction: small lymphoid infiltration and few lymphocytes. The ARP were present in 13.7% to 23.5% of the lymphocytes, and apoptosis was rare. In borderline tumors, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a tenfold rise in total lymphocytes, reflecting intensification of the immune response. Most lymphocytes were T cells (92%) predominated by CD8+ cells that were in direct contact with tumor epithelial cells. ARP species were found in 47% to 65% of the lymphocytes, and apoptosis in 2.2%. In carcinomas with ligh lymphoid infiltration, lymphocytes were 2.5 times more abundant, and the apoptotic index as well as the number of CD20+ and CD25+ lymphocytes rose sharply, whereas bcl-2 positive lymphocytes decreased to 8% of their number in borderline tumors. In carcinomas with low lymphoid infiltration, the total lymphocyte count decreased eightfold compared to carcinomas with high lymphoid infiltration, reflecting the deep subcompensation of the lymphoid system. Few p53-positive lymphocytes were found in the carcinomas. In conclusion, we found a positive correlation between apoptosis and the numbers of CD4+ or CD8+ lymphocytes in epithelial ovarian tumors. This correlation could reflect the antitumor activity of T cells. However, the high expression of ARP studied by immune cells at the vicinity of the tumor ARP reveals the lymphoid vulnerability to apoptosis, resulting in devastation of the lymphoid tissue, and consequently in tumor progression.     

Increased cyclooxygenase-2 expression is correlated with suppressed antitumor immunity in cervical adenocarcinomas

Cyclooxygenase-2 (COX-2) inhibition suppressed the growth of various tumors. The augmentation of antitumor immunity by increasing cytotoxic lymphocytes may be an important mechanism for COX-2 inhibition. Among cervical cancers, adenocarcinomas present more aggressive behavior and overexpressed COX-2. The expression of COX-2 and the CD8+ lymphocyte infiltrations were evaluated in this study by immunohistochemistry. We studied COX-2 expression and CD8+ lymphocyte infiltration in 55 women with cervical adenocarcinomas. COX-2 expression and tumor stromal CD8+ lymphocytes were evaluated by semiquantified methods. Tumor intraepithelial lymphocytes were counted under microscopic field of x200. Correlations between these data and other clinicopathologic features were investigated. Thirty-seven out of 55 (67.3%) cervical adenocarcinomas significantly expressed COX-2. Patients who died within 5 years showed higher percentage of COX-2 expression than survivors (100% vs 58.1%, P < 0.05). Victims also showed lesser intraepithelial CD8+ lymphocyte counts than survived patients (3.4 vs 26.4, P < 0.05). COX-2 expression and tumor intraepithelial lymphocyte count were reversely correlated with each other (correlation index: -0.38, P < 0.01). Up-regulated COX-2 expression and lesser tumor intraepithelial CD8+ lymphocyte count are poor prognostic indicators for cervical adenocarcinoma patients. COX-2 may play an important role in the suppression of host antitumor immunity in cervical adenocarcinomas.     

Pseudomyxoma peritonei: effect of chronic continuous immunotherapy with a streptococcal preparation, OK-432 after surgery

Three cases of pseudomyxoma peritonei with ovarian neoplasma were treated by adjunctive immunotherapy with OK-432 after surgery. In 2 of the 3 cases, the periods of intramuscular injection of OK-432 were 11 and 16 months, respectively, and the 2 patients remain clinically free from evidence of disease, 7 and 6 years after surgery. The third patient is still undergoing treatment, with no evidence of recurrence at 4 months after surgery. These results, although for a small number of patients, clearly suggest that adjunctive immunotherapy with OK-432 may be suitable for treating pseudomyxoma peritonei of ovarian tumor origin.     

Reduction of pleural effusion by OK-432 in a fetus complicated with congenital hydrothorax

A 29-year-old, primiparous woman was referred to our hospital at 21 weeks of gestation because of right pleural effusion in the fetus shown by routine ultrasonographic examination. Cytology revealed abundant lymphocytes, suggesting chylothorax. We removed the pleural effusion and injected OK-432 into the chest cavity at 24 and 25 weeks of gestation. Pleural effusion declined and an adhesion between the lung surface and the pleural membrane seemed to form. At 33 weeks of gestation, a female infant was born by cesarean section (1,090 g and Apgar score 6/8). Although she demonstrated slight retraction and tachypnea, management could be achieved by administration of oxygen alone without mechanical ventilation. Later, the baby was diagnosed as suffering from the Cornelia de Lange syndrome with characteristic features. OK-432 injections could thus prevent complications of chylothorax and hypoplastic lungs, without injury to either the baby or the mother.