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.

Vacuolar hepatocyte degeneration induced by infusion of 20% glucose solution with insulin after hepatopancreatectomy in rats.

To investigate the causes of hepatic dysfunction after extensive resection of the liver together with pancreatectomy, rats were subjected to sham operation, to 68% hepatectomy alone, to 90% pancreatectomy alone, or to 68% hepatectomy combined with 90% pancreatectomy (hepatopancreatectomy). Solutions of 5% or 20% glucose were infused post-operatively for 48 h at a constant rate (250 ml/kg body weight/day) under fasting conditions. To improve the survival rates of pancreatectomized and hepatopancreatectomized rats given 20% glucose, it was necessary to use insulin. In hepatopancreatectomized rats, infusion of 20% glucose with insulin (1 U/5 g glucose) induced prominent hepatocyte vacuolar degeneration and mitochondrial swelling, associated with reduced hepatic protein content. The severity of histological changes was proportional to the insulin dose and the activity of hepatic glucokinase, a key glycolytic enzyme. were observed in These histological changes pancreatectomized rats albeit in a milder form, but not in sham-operated or hepatectomized rats given 20% glucose nor in any rats given 5% glucose. Our results suggest that hepatopancreatectomy followed post-operatively by a high glucose load and exogenously administered insulin enhances the development of hepatocyte swelling.

Extensive cell death in thymocytes in colon 26-induced cachectic mice.

Extensive atrophy has been reported to occur in the thymus in a cancer-burden state but the mechanisms of this atrophy have not been fully elucidated. We investigated changes in the thymus in tumour-bearing mice inoculated with two subclones of the murine colon 26 adenocarcinoma cell line: clone 5 (non-cachectic) and clone 20 (cachectic). In clone 20 mice, body weights and thymocyte numbers decreased significantly compared with controls. Flow cytometric analysis of the thymocytes demonstrated that the frequency of single positive cells (CD4+ CD8- and CD4- CD8+) was significantly increased and that of double positive cells (CD4+ CD8+) was significantly decreased in clone 20 mice and, to a lesser extent, in clone 5 mice compared with controls. Serum levels of interleukin 6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly elevated. These results suggested that thymocyte apoptosis was accelerated in the cancer-cachectic state, and increased GM-CSF might be partly responsible for thymic atrophy.

Autologous serum deprivation restored IL-1 receptor antagonist production by peripheral blood mononuclear cells in patients with gastric cancer.

It has been established that cancer patients have immunosuppressive substances in their sera that depress cellular immunity. Although plasma exchanges have been attempted to remove these substances and to improve immunity to cancer, little is known about its mechanism from the viewpoint of cytokine pattern. The levels of the cytokines, tumour necrosis factor-alpha, interleukin 1beta, interleukin 6, interferon-gamma and interleukin-1 receptor antagonist (IL-1ra) by peripheral blood mononuclear cells (PBMC) were determined simultaneously by the whole-blood assay and the PBMC assay in 20 patients with gastric cancer and in 10 healthy volunteers. In both assays the cytokine levels were lower in patients with cancer compared with healthy controls, with the exception of IL-1ra. In the PBMC assay, the IL-1ra level in cancer patients was significantly higher than that in controls. No statistical correlation between the cytokine levels determined by the two assays was found. We suggest that autologous serum deprivation restored and enhanced IL-1ra production, and normalized the cytokine cascade in immune response, in patients with gastric cancer.

Effect of streptococcal lyzate OK-432 on peripheral blood mononuclear cells in gastric cancer patients.

OK-432, a killed preparation of Streptococcus pyogenes, as well as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum are all known biological response modifiers. To examine the immunomodulatory effects of OK-432, natural killer cell activity and cytokine production by peripheral blood mononuclear cells (PBMCs) were assessed in 32 patients with gastric cancer. Skin tests for Streptococcus pyogenes A-3Su (Su-PS) and BCG were performed in all patients. Other nutritional and immunological parameters were also determined. OK-432-treated PBMCs showed a significant increase of cytotoxicity against K562 cells (p < 0.01). Increased levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) were found in the supernatants of cultures treated with OK-432 in 29 (90.6%), 20 (62.5%), and 8 (25.0%) out of 32 patients, respectively. Natural killer cell activity, IFN-gamma production, and the Su-PS skin test were positively correlated (p < 0.01). In contrast, the BCG test and other markers were not correlated with natural killer cell activity and IFN-gamma production. These results suggest that the Su-PS skin test could predict OK-432-induced natural killer cell activity and IFN-gamma production in patients with gastric cancer, and was therefore useful to determine whether patients were responders to OK-432.

Soluble intercellular adhesion molecule-1 and natural killer cell activity in gastric cancer patients.

Intercellular adhesion molecule-1 (ICAM-1), a molecule bound to the cell surface, is a ligand for leukocyte function antigen-1 (LFA-1), and the ICAM-1/LFA-1 system mediates various cell-cell interactions involved in immunity. Soluble ICAM-1 (sICAM-1) is a circulating substance and binds with LFA-1 of leukocytes, thus, making leukocytes less available for binding with cell surface ICAM-1 on target cells. The serum level of soluble ICAM-1 (sICAM-1) was found to be significantly elevated (p<0.01) in patients with early and advanced gastric cancer compared with healthy controls. Natural killer activity (NK activity) was assessed by measuring the cytotoxicity of peripheral blood mononuclear cells (PBMCs) for K562 cells. There was no significant difference in NK activity between gastric cancer patients and healthy controls when heat-inactivated fetal calf serum was used in assays. However, addition of patient serum significantly decreased (p<0.05) NK activity when the serum was from patients with advanced gastric cancer compared with healthy volunteers. Addition of anti-ICAM-1 monoclonal antibody 0 to 5.0 microg/ml caused little change in NK activity in healthy controls, but its addition at 10 microg/ml remarkably decreased NK-activity in gastric cancer patients, probably through antibody binding with ICAM-1 on target cells. In other experiments, liver metastasis was induced in mice by inoculation of colon 26 murine colon cancer cells. In vitro pretreatment of colon 26 cells with the anti-ICAM-1 monoclonal antibody significantly increased the number of metastatic nodules. These results suggest that both sICAM-1 and anti-ICAM-1 monoclonal antibody act as immunosuppressive factors by inhibiting the ICAM-1/LFA-1 system.

Intratumoral injection of a streptococcal preparation, OK-432, before surgery for gastric cancer. A randomized trial. Cooperative Study Group of Preoperative Intratumoral Immunotherapy for Cancer.

BACKGROUND: The preoperative intratumoral injection with OK-432 (Picibanil, Chugai Pharmaceutical Co., Tokyo, Japan), an immunomodulatory agent prepared from an attenuated strain of streptococcus pyogenes, activates the regional immune system and causes degeneration of cancer tissue in carcinoma of the stomach. METHODS: A multi-institutional randomized trial of OK-432 to determine its clinical usefulness was conducted. Three hundred and ninety-five patients with gastric cancer were assigned randomly either to receive or not to receive a preoperative intratumoral injection of OK-432. Among them, 277 patients with advanced cancer were treated by common postoperative chemoimmunotherapy consisting of mitomycin C, tegafur, and OK-432. All patients were followed for at least 5 years. RESULTS: The adverse effects of OK-432 injected intratumorally predominantly were mild fever, anorexia, and abdominal pain, however, no treatment was required for these symptoms. Overall, there were no differences in outcome between the OK-432 and control groups. However, analysis based on stage showed that a preoperative intratumoral injection of OK-432 significantly improved the 5-year survival rate of patients with Stage III cancer (P = 0.0229), at 47.7% for the OK-432 group and 27.5% for the control group. In subset analysis, when the 5-year survival of patients with and without tumor infiltrating lymphocytes (TIL) was compared, OK-432 injected intratumorally had a significant positive effect on the group showing a moderate to marked number of TIL (P = 0.0438). CONCLUSION: These results showed that the intratumoral injection of OK-432 may improve survival of patients with Stage III gastric cancer. Cancer 1994;3097-3103.

The enhancement of cell surface ICAM-I and HLA class I antigens in human gastric cancer cell lines by IFN-gamma.

Cytotoxic lymphocytes, including natural killer cells, lymphokine-activated killer cells, and cytotoxic T lymphocytes, adhere to and lyse cancer cells by recognizing cell surface antigens. Among the cell surface antigens, intercellular adhesion molecule-1 (ICAM-1) and HLA class I antigen are important for the cytotoxic activity of lymphocytes. The ICAM-1 and HLA class I antigen were examined in gastric cancer cell lines MKN-28 and MKN-45 by flow cytometry to determine whether their expression on the cell surface is enhanced by interferon gamma (IFN-gamma). The cell expression rate [stained cells/10(4) cells x 100(%)] was only 10% in ICAM-1 and about 20% in HLA class I antigen without IFN-gamma, but reached 70% in ICAM-1 and up to 60% in HLA class I antigen after incubation with IFN-gamma for 24-96 h. This enhanced expression of cell surface ICAM-1 and HLA class I antigen by IFN-gamma might increase sensitivity for cytotoxic lymphocytes.

[The expression of neural cell adhesion molecule (NCAM), neural invasion and recurrence patterns in rectal cancer--a study using anti-NACM (neural cell adhesion molecule) antibody].

Neural invasion may be one of the main causes of local recurrence, but its mechanism has not been sufficiently clarified. We previously reported that the expression of NCAM on cancer cells was correlated with neural invasion in pancreatic cancer. In this study, we examined the neural affinity of rectal cancer cells and the relationship between neural invasion and recurrence patterns of rectal cancers. A total of 64 rectal adenocarcinoma were examined. Specimens from 17 patients (27%) revealed perineural invasion. The incidence of neural invasion increased with the frequency of venous invasion and the degree of lymph node metastasis, but not significantly. The incidence of the expression of NCAM in rectal cancer cell was 45.3 percent. Neural invasion of rectal carcinoma was significantly related to the expression of NCAM (p < 0.05). NCAM immunocytolocalization was classified into the focal type, and the diffuse type. Concerning the recurrence type, 8 of 9 cases that developed local recurrence showed either presence of neural invasion or expression of NCAM. Liver metastasis was associated with absence of neural invasion or NCAM expression. The expression of NCAM may contribute to local recurrence, whereas the absence of NCAM may predict liver metastasis.

Augmentation in cytotoxicity of lymphnode lymphocytes by OK-432.

It has been demonstrated that natural killer (NK) cell activity of lymphnode lymphocytes (LNL) is very low and hardly augmented by interferon. In this study, OK-432 was injected into the gastric cancer mass through endoscopy one week before the operation, through which NK activity of LNL was significantly increased. A single cell assay could divide the OK-432-injected patients into two groups; responder and non-responder. In responders, the increased activity induced by OK-432 was found also in the distal lymphnodes.

Coeliac axis-oriented approach in surgery for advanced gastric cancer.

To achieve the systemic curative lymph node dissection in gastrectomy for advanced gastric cancer, coeliac axis-oriented approach was proposed. Mobilization of the spleen as well as the tail and body of the pancreas from the left retroperitoneum and Kocher's maneuver lifting up the hepatoduodenal ligament and head of pancreas from the right retroperitoneum permits the observation of the coeliac axis under direct vision and enables the para-aortic node dissection with ease. This approach was applied to 131 cases with advanced gastric cancer with neither mortality nor notable complication.

[Effects of gabexate mesilate (FOY) on the gallbladder, sphincter of Oddi and duodenum of the normal and gastrectomized dogs].

FOY induced a dose-dependent inhibitory response on the gallbladder, sphincter of Oddi and duodenum of normal and gastrectomized dogs, although it induced an excitatory response in some dogs. The inhibitory response was not reduced or terminated by pretreatment with atropine, guanethidine, hexamethonium or/and proglumide. The FOY-induced inhibitory response reversed to the excitatory response in the sphincter of Oddi and duodenum by pretreatment with tetrodotoxin, but not in the gallbladder. These results suggested that the FOY-induced inhibitory response of the sphincter of Oddi and duodenum was caused by stimulation of nonadrenergic noncholinergic inhibitory neurons, not by FOY-induced cholecystokinin secretion. The excitatory response was induced by direct stimulation of their smooth muscles. The inhibitory response of the gallbladder to FOY was induced by direct stimulation of the smooth muscles.

Primary malignant lymphoma of the breast mimicking breast carcinoma: a case report.

A case of primary malignant lymphoma of the breast in a 52 year old woman is described herein. She presented with a painless tumor of the right breast, which was elastic-hard and measured 2 X 1 cm, although there was no lymphadenopathy noted in the axilla or any other regions. Both the biopsy carried out prior to surgery and frozen sections revealed evidence of malignancy, however, histologically, it was difficult to differentiate between malignant lymphoma and carcinoma until staining with the monoclonal antibody MB1 was performed. This allowed a diagnosis of B cell malignant lymphoma of the diffuse large-cell type to be made. The patient remains alive and well 24 months after her mastectomy.

Augmentation of cytotoxicity of tumor-infiltrating lymphocytes by biological response modifiers.

Lymphocyte infiltration into a tumor has been regarded as an expression of host immunity against cancer, but tumor-infiltrating lymphocytes (TIL) have little or no cytotoxicity. This study examined two different approaches to augment this low cytotoxicity. Firstly, biological response modifiers (OK-432, PSK) were injected into gastric cancer intralesionally. Intralesional injection of OK-432 or PSK significantly augmented the cytotoxicity of TIL. By the injection of OK-432, the ratio of OKT8-, Leu7-positive cells were increased in the TIL subset. In the second approach, TIL of gastric or pulmonary cancer patients were cultured with interleukin-2 (IL-2) in vitro. Co-culturing with IL-2 augmented the low cytotoxicity of TIL, and broad-reactive lymphokine-activated killer (LAK) cells were generated from TIL.

A case of necrosis of hepatocellular carcinoma and tumor thrombus in the portal vein induced by transcatheter arterial lipiodol chemoembolization.

A case of hepatocellular carcinoma is reported in which the main tumor, intrahepatic metastases and a tumor thrombus in the portal vein were necrotized completely after Lipiodol chemoembolization. In this case, the tumor thrombus seemed to act as a portal embolus. This phenomenon is interesting because Lipiodol chemoembolization alone usually can not necrotize intra- or extra-capsular invasion, intrahepatic metastasis or tumor thrombus in the portal vein. This case is considered to be suggestive of a possible therapy for hepatocellular carcinoma.

The effect and distribution of a protein-bound polysaccharide preparation, PSK (Krestin), intratumorally injected prior to surgery into gastric cancer patients.

In order to improve the postoperative prognosis of gastric cancer patients we have performed preoperative endoscopic intratumoral administration of various biological response modifiers. In the present study we have investigated the kinetics and the immune response augmenting effect of intratumorally injected PSK, a protein-bound polysaccharide preparation, by immunohistochemical methods using anti-PSK antibody and various other antibodies. PSK-containing cells were located in the tumor tissues and follicular marginal zones of regional lymph nodes. Intratumorally administered PSK appeared to be phagocytized by the histiocytes and to cause them to become antigen-presenting cells. These cells may play a major role in augmenting immune responses in gastric cancer patients.

[Favorable response of advanced hepatocellular carcinoma to proper hepatic arterial administration of cytokines and the significance of the administration].

The objectives in administering anti-cancer therapeutics to the feeding artery of the tumor are to allow the agent to come in direct contact with the tumor cells, to lower the concentration of the agent in body circulation, to lessen the severity of side effects, and to augment efficacy of the agent. A remarkable partial regression was observed in two patients with advanced hepatocellular carcinoma, both at the stage in which surgical excision was diagnosed impossible; one was given successive and daily bolus administration of OH-1, a anti-tumor agent consisting of natural human tumor necrosis factor-alpha (nHuTNF-alpha) and natural human interferon (nHulFN-alpha), and the other a successive and daily combined bolus administration of OH-1 and 5-FU. On investigating the role of the anti-cancer activity of OH-1 by analyzing the NK activity of rat liver large granular lymphocytes, we found that the NK activity was suppressed dose-dependently by nHuTNF-alpha, but not significantly. Thus, an increase in TNF dose in hepatic artery therapy seems undesirable from the standpoint of NK activity. The authors are presently carrying out investigations to elucidate the effector mechanism of the anticancer activity of OH-1.

Natural killer activity of peripheral blood lymphocytes and its relation to histopathological factors of lung cancer.

This investigation was intended to determine whether the natural killer (NK) activity of peripheral blood lymphocytes (PBL) correlated with the histopathological factor, which is thought to be a result of a balance between tumor aggression and host resistance. The NK activity of PBL from 60 patients with lung cancer was measured by the lysis of 51Cr-labelled K562 target cells. The activity was significantly decreased with advancing stages of the disease, and inversely correlated with increased immunosuppressive substance levels of the serum. Histopathological factors, such as low grade pleural invasion of the tumor and abundant lymphoid cell infiltration around the tumor, were significantly associated with the high NK activity of PBL. These results show that a decrease in NK activity may play a role in identifying those individuals with a greater risk of cancer development.