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For the induction of antigen-specific immune responses, adjuvants as well as antigens are essential. CpG-ODN is a potent agonist of toll-like receptor 9 (TLR9) and is known as an adjuvant to induce cellular immune responses. We previously developed a therapeutic oligonucleotide delivery system based on the formation of a complex between schizophyllan (SPG), a kind of β-1,3-glucan, and poly(dA), which actively delivered CpG-ODN to antigen-presenting cells (APCs) in the draining lymph nodes and induced antigen-specific immune responses. However, unfortunately, the signaling pathway of TLR9 is negatively regulated by an intracellular protein called suppressor of cytokine signaling-1 (SOCS-1), which suppresses the adjuvant effect of CpG-ODN. To solve this, we focused on microRNA-155 (miR-155), which regulates innate and autoimmune processes by targeting SOCS-1. In this study, we proposed a strategy of combining miR-155 and CpG-ODN, each complexed with SPG (denoted as SPG/miR-155 and SPG/CpG, respectively), to induce a more potent immune response. As a result, we showed that the efficient delivery of miR-155 to APCs by a complex form could induce much more potent cellular immune responses than SPG/CpG alone. Furthermore, the mice treated with the combination of SPG/miR-155 and SPG/CpG showed a long delay in tumor growth occurrence and improved survival after tumor inoculation. These results indicate the possibility of therapeutic strategies for cancer.

Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a β-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD., (© 2012 New York Academy of Sciences.)

Modern medical practice relies heavily on the use of highly purified pharmaceutical compounds whose purity can be easily assessed and whose pharmaceutical activity and toxicity show clear structure-function relationships. In contrast, many herbal medicines contain mixtures of natural compounds that have not undergone detailed chemical analyses and whose mechanism of action is not known. Traditional folk medicine and ethno-pharmacology coupled to bioprospecting have been an important source of many anticancer agents as well as other medicines. With the current decline in the number of new molecular entities from the pharmaceutical industry, novel anticancer agents are being sought from traditional medicine. As the example of medicinal mushrooms demonstrates, however, translating traditional Eastern practices into acceptable evidence-based Western therapies is difficult. Different manufacturing standards, criteria of purity, and under-powered clinical trials make assessment of efficacy and toxicity by Western standards of clinical evidence difficult. Purified bioactive compounds derived from medicinal mushrooms are a potentially important new source of anticancer agents; their assimilation into Western drug discovery programs and clinical trials also provides a framework for the study and use of other traditional medicines.

Kupffer cells (KC), resident macrophages of the liver, have been strongly implicated in lipopolysaccharide (LPS)-induced liver graft injury. However, our recent study showed that sizofiran (schizophyllan glucan) (SPG), which activates KC, did not influence cold ischemia-reperfusion liver injury of LPS-exposed rats. Here we investigated some mechanisms by which SPG does not aggravate LPS-enhanced cold ischemia-reperfusion rat liver injury. Control and SPG-treated rats were exposed to LPS for 2 h prior to hepatectomy. The livers were cold-preserved in University of Wisconsin solution followed by reperfusion with Krebs-Henseleit buffer. We found that SPG dramatically inhibited LPS-induced increases of tumor necrosis factor-alpha (TNF-alpha) in the plasma and bile in vivo. Moreover, LPS-induced TNF- release into the washout solution after cold ischemia was also abrogated by SPG pretreatment. However, SPG increased TNF- release into the perfusate after reperfusion. On the other hand, SPG completely abolished expression of c-myc protooncogene, which is known to sensitize cells to TNF-alpha cytotoxicity. In conclusion, inhibition of both TNF- release after LPS challenge and c-myc expression may explain why activation of KC with SPG does not aggravate endotoxin-enhanced cold ischemia-reperfusion liver injury.

Gynecologic Oncologists in Japan has increasingly much interest in the phase III study to define the standard treatment modality. It is very difficult, however, to conduct a phase III trial because a very few medical staff including doctors have experience and understanding of such studies and also poor understanding of patients to be entered. According to the literature and papers in the meetings, several phase III studies were conducted unexpectedly. I reviewed these data and pointed out the problems included in the studies. The results of these trials and related problems prompted Japanese Gynecologic Oncologists to conduct a high quality phase III study to clarify the standard treatment modality for gynecologic cancer.

Clinical effects of doxifluridine (group A, 600 mg/body/day; group B, 800 mg/body/day) combined with radiotherapy and immunotherapy were evaluated in patients with advanced cancer of the uterine cervix. Response rates were 84.2% (16/19 patients) in group A and 100% (18/18 patients) in group B, respectively (p=0.230). There was no significant difference in adverse reaction incidence between the methods but significantly higher grade adverse reaction were observed in group B than in group A (p=0.048). Time to progression (TTP) was longer in group B than in group A (p=0.081). The optimal 5'-DFUR dose was 800 mg/body (group B), by which higher grade adverse reactions were fully controlled and TTP was prolonged.

Thymidine phosphorylase (dThdPase) is reportedly identical to platelet-derived endothelial cell growth factor (PD-ECGF). We conducted immunohistochemical staining of dThdPase to assess correlation between its expression in cancer tissue and efficacy of a combination therapy with 5'-DFUR, radiotherapy and sizofilan (SPG) in uterine cervical cancer patients. No difference in response rates was observed between dThdPase positive and negative tumor and stromal cells. Survival curves significantly differed between stromal dThdPase positive and negative groups (p=0.032). Results showed that dThdPase immunostaining is possibly prognostic and predictive in determining success of the combination therapy.

Medicinal properties have been attributed to mushrooms for thousands of years. Mushroom extracts are widely sold as nutritional supplements and touted as beneficial for health. Yet, there has not been a critical review attempting to integrate their nutraceutical potential with basic science. Relatively few studies are available on the biologic effects of mushroom consumption, and those have been performed exclusively in murine models. In this paper, we review existing data on the mechanism of whole mushrooms and isolated mushroom compounds, in particular (1-->3)-beta-D-glucans, and the means by which they modulate the immune system and potentially exert tumor-inhibitory effects. We believe that the antitumor mechanisms of several species of whole mushrooms as well as of polysaccharides isolated from Lentinus edodes, Schizophyllum commune, Grifola frondosa, and Sclerotinia sclerotiorum are mediated largely by T cells and macrophages. Despite the structural and functional similarities of these glucans, they differ in their effectiveness against specific tumors and in their ability to elicit various cellular responses, particularly cytokine expression and production. Unfortunately, our data base on the involvement of these important mediators is still rather limited, as are studies concerning the molecular mechanisms of the interactions of glucans with their target cells. As long as it remains unclear what receptors are involved in, and what downstream events are triggered by, the binding of these glucans to their target cells, it will be difficult to make further progress in understanding not only their antitumor mechanisms but also their other biological activities.

The formylmethylated and aminoethylated derivatives of schizophyllan (SPG), a beta-(1-->6)-branched (1-->3)-beta-D-glucan from Schizophyllum commune Fries, were prepared through dimethoxyethylated SPG which was synthesized by the reaction of SPG with dimethylchloracetal under an alkaline condition. The degree of the substitution of formylmethyl groups in the formylmethylated derivative of SPG was estimated as approximately 0.19, and the locations of formylmethyl groups in the derivative were predominantly located at O-6 and/or O-4 positions in glucose residues. The molecular weights of these derivatives were similar to that of SPG, and the helical structure of the derivatives did not seem to be different. The antitumor activities of the formylmethylated and aminoethylated derivatives of SPG against subcutaneously implanted sarcoma 180 solid tumor in mice by intraperitoneal (i.p.) administration were increased more effectively than that of SPG at a dose of 10 mg/kg/d for 7 d starting from 7 d after transplantation of sarcoma 180. The activities inducing tumor regressing factor of the formylmethylated and aminoethylated derivatives were 1.5 to 2 times stronger than that of SPG at a dose of 100 mg/kg. Formylmethylated and aminoethylated derivatives of SPG as well as SPG itself retained the potentiating activity for the reticuloendothelial system. The productions of soluble cytotoxic factors secreted from murine macrophages by the administration of the formylmethylated and aminoethylated derivatives of SPG, which were probably superoxide anion and tumor necrosis factor as measured by the potency of the nitro blue tetrazolium reduction and the cytotoxic activity against L929 cells, respectively, appeared to be more efficient than that of SPG.

Objective: Experimental chemotherapy was conducted to investigate the combined effect of Schizophyllan (SPG) and Cisplatin (CDDP)., Methods: Rats with 7,12-dimethylbenz(a)anthracene (DMBA)-induced ovarian adenocarcinoma received SPG and/or CDDP, were observed for the anti-tumor effect of the drugs and were subjected to immunohistochemical study., Results: 1) Tumor reduction was enhanced by the combined use of SPG and CDDP; 2) The survival rate of rats given SPG alone was significantly higher than that of the control rats, and treatment with SPG combined with CDDP tended to improve the survival rate; 3) Immunohistochemically, an infiltrative increase in cytotoxic T-lymphocytes (CTL) was induced, although the development of helper T-cells and macrophages was immunohistochemically weak at the tumor site., Conclusion: The administration of SPG enhanced the anti-tumor effect of CDDP.

A prospective randomized trial on 312 patients with locally advanced cervical carcinoma (FIGO stages IB-IV) was carried out. The 5-year survival in 90 patients treated with radiotherapy and antitumor polysaccharide sizofiran, an extract from the culture broth of Schizophyllum commune Fries, in combination was significantly (P = 0.045) better than that in 82 patients treated with radiotherapy alone. Treatment with sizofiran and 5-fluorouracil in combination improved (P = 0.003) the 5-year survival in 60 patients treated with radiotherapy. In 244 cervical carcinoma patients, the percentage of activated CD8+ (CD8+HLA-DR+) T cells in the CD8+ T-cell subsets in peripheral lymphocytes increased significantly as the disease progressed. A similar tendency was observed in the percentage of activated CD4+ (CD4+HLA-DR+) T cells in the CD4+ T-cell subsets. These immunologic parameters were significantly increased by radiotherapy, but not by surgery. Sizofiran accelerated a recovery in the activated CD8+ T cells in the CD8+ T-cell subsets compared with that of sizofiran nontreated patients after radiotherapy. Our data show that possible immune impairment in cervical carcinoma may be caused by disturbances in cell-mediated immunity, and that sizofiran is an effective immunotherapeutic agent for cervical carcinoma because it stimulates a rapid recovery of the immunologic parameters impaired by radiotherapy.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) has been challenged to human cancer patients during these years. However, as the efficacy of the therapy alone is still limited, another approach, a combination with other therapies may be required to obtain more favorable antitumor effects. Tumor necrosis factor alpha (TNF) is worth considering for combination, because at least its various potential roles in immune response suggest that it might increase the killing activity of T cells against autologous tumor cells. We attempted to treat the cancer patients with TIL combined with TNF therapy. A patient with recurrent cervical cancer was treated with multiple biological therapies including TIL and TNF therapy, and irradiation. Biological therapies consisted of endogenous and exogenous TNF (EET) therapy, TIL, administration of rTNF-SAM2 combined with hyperthermia and several biological response modifiers such as schizophyllan and lentinan. After 50 days of therapy, the recurrent tumor had remarkably decreased in size, and cystic change of the tumor was observed by CT scanning. By further continuation of these therapies, the disease condition was much improved. As these antitumor effects cannot well be explained by irradiation therapy alone, multiple biological therapies are considered to be potentially effective against the highly malignant advanced tumors.

Sizofilan (SPG), a simple glucan produced in a culture medium by Schizophyllum commune Feries, was used as an assistant immunotherapy in 15 patients with head and neck cancer. The cumulative 5-year survival rate was 86.7% in the SPG-treated group and 73.4% in the control group. Immunological parameters showed that the SPG group quickly recovered the cellular immunity damaged by radiation, chemotherapy and surgical procedure. SPG was found to be effective as an assistant immunotherapeutic agent in the treatment of head and neck.

Correlations between infiltration of Langerhans cells (ILC) in tumor tissues and radiation curability were investigated in 449 patients with cervical cancer treated with radiation alone, including 390 squamous cell carcinomas and 59 adenocarcinomas. No significant difference in prognosis was noted in stage I, II, and IV squamous cell carcinomas between positive and negative ILC. However, in the patients with stage III squamous cell carcinoma, a significantly better survival was observed for patients with ILC than for those without, 10 year survival rates being 78% vs. 54%, P < 0.01. In adenocarcinoma, the patients with ILC also showed significantly better survival than those without ILC, the 10 years survival rates being 45% vs. 25%, P < 0.025. An analysis of failure patterns following radiation treatment demonstrated that the favorable prognosis in patients with ILC in squamous cell carcinoma was due to improvement of local control rates and somewhat lower metastatic rates, whereas in adenocarcinoma it was only due to better local control rate. The ILC was significantly associated with T-cell infiltration in tumor tissues. The immunological stimulation with Sizofiran in 20 patients led to an augmentation of ILC in tumor tissues. The present study suggests that the ILC in cancer tissues improves local response to radiation treatment partly by T-cell mediated anti-tumor activity.

The effect of immunotherapy using sizofiran (SPG) on the prognosis of patients with ovarian cancers was prospectively studied in a total of 68 patients, who were randomly assigned to either a cisplatin, adriamycin and cyclophosphamide (PAC) therapy group or a PAC plus SPG combination therapy group. The survival rate was significantly higher in patients with stage Ic, II or III cancers treated with the PAC plus SPG combination, compared with the patients treated with PAC alone. In the SPG-receiving patients with stage Ic or more advanced cancers who were treated with four cycles or more of PAC, the outcome was improved (Cox-Mantel, p = 0.074; generalized Kruskal-Wallis, p = 0.032). Similar improvement was also observed in the patients with non-serous adenocarcinomas (Cox-Mantel, p-0.076; generalized Kruskal-Wallis, p = 0.045). No side effects attributable to SPG were recorded. The present results suggest that the use of SPG in combination with long-term chemotherapy improves the postoperative prognosis in ovarian cancer patients.

The immunomodulating effect of sizofiran on regional lymph nodes (RLN) was studied in cervical cancer and benign gynecologic tumors comparatively between treated patients (sizofiran intramuscularly (1) 1 day (20 mg) before and (2) 8 days (20 mg) and 1 day (20 mg) before surgery; n = 34) and untreated patients (n = 21). The RLN (internal iliac lymph nodes) were dissected surgically, and freshly obtained mononuclear (MN) cells were studied to observe interleukin-2 (IL-2) production and lymphokine-activated killer cell and natural-killer cell activities. The infiltration of surface phenotype of MN cells into RLN was determined semiquantitatively by immunohistochemistry. Sizofiran augmented IL-2 production of RLN, which was accompanied by a marked increase in the number of cells stained with anti-Leu-3a and IL-2 receptor. This effect was found more often in Stage Ib disease than in Stage 0, and it was not observed in benign tumors. These results suggest that stimulation with some antigens (e.g., cancer antigen in the current study) is necessary to induce immunoaugmentation by sizofiran which has no antigenicity. The augmenting effect was seen even in lymph nodes involved by advanced cervical cancer. Therefore, sizofiran was found to be a potent biologic response modifier in patients with cervical cancer.

To evaluate the clinical effect of a biological response modifier (BRM), sizofiran (SPG), combined with irradiation, a randomized controlled study was performed in patients with stage II or III cervical cancer involving the collaboration of 52 institutes throughout Japan. Patients were randomly allocated to the control group (radiotherapy only) and the SPG group (radiotherapy + SPG). SPG was given intramuscularly, 40 mg once and 20 mg twice, a week concomitantly with radiotherapy. A total 315 patients were enrolled for the study but 23 were excluded from analysis. Of the remaining 292 patients, 121 were of stage II (43 controls and 78 SPG) and 171 of stage III (49 controls and 122 SPG). The results were as follows. (1) The complete response (CR) rate among stage II patients was higher in the SPG group (91.0%) than in the control group (79.1%); also the CR rate among stage III patients was significantly higher in the SPG group (77.9%) than in the control group (61.2%). (2) The SPG group showed a significantly rapid recovery from the decreased lymphocyte counts due to radiotherapy (P less than 0.05). (3) Side effects, probably associated with SPG administration, were observed in 11 cases (5.2%).

The efficacy of Sizofiran(SPG), a highly purified beta-1,3-D-glucan from the culture broth of basidiomycetes Schizophyllum commune Fries, in combination with local irradiation was investigated using squamous-cell carcinoma NR-S1 and syngeneic hosts of C3H/He mice. NR-S1 tumor was implanted sc in the thigh of C3H/He mice. When tumor grew to 4 mm in diameter, the local irradiation of 55 Gy was delivered. SPG was injected im at a dose of 5 mg/kg. When SPG was administered after irradiation, remarkable inhibition of tumor growth was observed in comparison with the radiation alone group. Furthermore, the combination effect of radiation and active immunotherapy using mitomycin C-treated NR-S1 cells as vaccine was examined. When radiotherapy and active immunotherapy were combined with SPG, suppression of tumor growth was observed from an early stage in comparison with the group which was not administered SPG. SPG also inhibited the pulmonary metastasis of NR-S1 tumor after radiotherapy.

Four patients with ovarian cancer received 20 mg of sizofiran, a beta-1,3-glucan (molecular weight: 450,000), intramuscularly one day before and 4, 7, 11, 14, 18 and 21 days after second look laparotomy and recombinant interferon-gamma (rIFN-gamma) intraperitoneally on the day of second look laparotomy and 4, 7, 11, 14, 18 and 21 days thereafter. The peritoneal cavity was washed with physiological saline and peritoneal macrophages (M phi) were isolated. The number of M phi increased 30-1600 times during the treatment period. The concentrations of interleukin-1, interferon-gamma, tumor necrosis factor and prostaglandin E2 were also found increased in the supernatant fluid of M phi cultured for 24 hours with 10 micrograms/ml of lipopolysaccharide. The present study demonstrated that the activation of peritoneal M phi could be maintained and its number was increased by repeated dosing of sizofiran and rIFN-gamma in combination every three or four days in patients with ovarian cancer. Peritoneal M phi thus activated may exert an antitumor effect on ovarian cancer.

Augmentation of anti-tumor effect of interleukin 2 (IL-2) with sizofiran (SPG) was demonstrated with in vivo and in vitro experiments. C3H/He mice with subcutaneously inoculated X5563 tumor were used as experimental models. IL-2 at a dose of 2 X 10(4) units per mouse by subcutaneous injection, and/or SPG at a dose of 0.1 g per mouse by intramuscular injection were given every other day for a total of ten times. Tumor infiltrating lymphocytes (TIL) were investigated with the avidin-biotin peroxidase complex method. Cytotoxic activity of mice spleen cells after the therapies against YAC-1, X5563 and MH134 were tested with 51Cr release assay. IL-2 or SPG alone suppressed tumor growth in vivo, although not significantly. Combination therapy with IL-2 and SPG suppressed tumor growth in vivo significantly (p = 0.04). Mice treated with the combination survived longer than the mice treated with the single drug (p less than 0.05) and the controls (p less than 0.001). Immunohistologically, more TILs were seen in the combination group than the other groups. In the cytotoxicity study, 3 days after initiation of the therapies, augmentation of natural killer (NK) activity was greater by the combination than by IL-2 or SPG alone. After incubation with IL-2, the spleen cells from mice treated with the combination showed higher cytotoxicity against X5563 or MH134 tumors than those treated with the single drug or controls. Obtained results suggested that combination therapy with IL-2 and SPG effectively induced NK cells and lymphokine-activated killer cells in vivo and may lead to greater clinical benefit in the treatment of malignancies.

Authors studied the effects of several biological response modifiers (BRMs) on bladder carcinoma. On carcinogenesis in BBN-induced bladder carcinoma rats, Schizophyllan (SPG) showed inhibition of body weight loss and thymus weight loss. Bladder weight increase was also inhibited. SPG prolonged cancer-bearing survival time, and postponed malignant changes of bladder mucosa. On thymocytes of SPG-treated cancer-bearing rats, rosette-forming thymocytes increased. In clinical study, SPG, OK-432, ubenimex and kampo medicine were used as BRMs. As a result, the number of suppressor-inducer T cells increased, and in cytotoxic T cells a slow fall was observed. On helper-inducer T cells, a slow rise and decrease were noticed in the SPG group, and the reverse tendency was seen in the ubenimex and OK-432 group.

Adjuvant immunochemotherapy using the antitumour polysaccharide sizofiran (SPG), an extract from the culture broth of Schizophyllum commune Fries, was prescribed randomly for 386 Japanese patients with resectable gastric cancer. Although the overall survival probability for 5 years did not differ between the SPG and control groups, in 264 patients with curatively resected cancer, the probability to 5 year survival and to recurrence in the sizofiran-administered patients was better than in the controls. In the multivariate analysis, four of six prognostic factors correlated with the prognosis of the 264 patients who underwent curative surgery, that is, nodal involvement (chi 2 = 21.426, P = less than 0.0001), age distribution (chi 2 = 9.262, P = 0.010), sizofiran administration (chi 2 = 6.507, P = 0.011), and primary tumour size (chi 2 = 9.345, P = 0.025). Thus, patients with a curatively resected gastric cancer had a better prognosis when sizofiran was prescribed in combination with antitumour drugs.

To investigate the immunological effect of Sizofilan (SPG) combined with radiotherapy, we evaluated the immunological parameters in 22 patients with uterine cervical cancers. Twelve cases were treated with SPG combined with radiotherapy (SPG group), and the other ten cases, with radiotherapy only (control group). As a result, 1) During radiotherapy, the numbers of lymphocyte and CD2 positive cell decreased in SPG and control groups. After radiotherapy, however, its numbers in SPG group became significantly higher than in control group (p less than 0.05). The number of CD3 positive cell also presented a tendency to increase after radiotherapy in SPG group. As for CD20 positive cell, its numbers were kept unchanged after radiotherapy in both two groups, and no significant difference was observed between them. 2) NK cell activity decreased during radiotherapy in both two groups. After radiotherapy, its activity in SPG group recovered to its pre-value and became significantly higher than that in control group (p less than 0.05). 3) SPG did not any prominent effect on CD4/CD8 ratio. 4) The adverse effect of SPG to liver or kidney function were not observed in our patients. The SCC level in SPG group decreased rapidly by radiotherapy as well as that in control group, and no significant difference was observed in SCC levels between them. So it was suggested that SPG did not suppress the cytocidal effect of radiation to cancer cells. Based on these findings, it was concluded that SPG prompted the recovery of not only lymphocyte, especially T cell, but also NK cell activity. These immunological findings presented a usefulness of clinical application of SPG to radiotherapy in patients with uterine cervical cancers.

It has been well known that Sizofiran (SPG) cultured from the Schizophyllum commune Fries activates the macrophages and induces the cytotoxic lymphocytes in some cancers. In this study, we observed electronmicroscopically the macrophages around the cancer tissue from the patients with uterine cervical cancer after the treatment with SPG. At the same time, their immune responses were also examined by analyzing lymphocyte subsets, ADCC and NK activity in peripheral blood. A considerable number of erratic macrophage with well developed Golgi apparatus, endoplasmic reticulum and mitochondria were found in the uterine cervical cancer tissue from the patients treated with SPG under radiotherapy. Simultaneously, we identified the lysosome granules with a bright filament structure which appeared to be specific for SPG. In the immune responses evaluated by analyses of peripheral blood, the number of CD 16+ cells and NK activity significantly increased in the patients treated with SPG as compared with non-treated group. The present results indicate that SPG-immunotherapy combined with radiotherapy not only induces the cytotoxic activity of macrophage but also augments NK activity in the patients with uterine cervical cancer.

The effects of radiation therapy and Sonifilan (SPG; a glucan produced by Schizophyllum commune) on the control of the primary lesion of human uterine cervix cancer and the experimental metastasis with mice were studied. SPG had excellent effects on the uterine cervix cancer compared with radiation therapy alone. Furthermore, immunohistochemical study showed that tumor infiltrated monocytes in good responders were mainly occupied with helper/induced T lymphocytes. SPG also suppressed the experimental lung metastasis especially with radiation therapy. Metastatic tumor infiltrated monocytes were many macrophages, which was the same as in inoculated local tumor.

TNF, IL-1, IFN-gamma and PGE2 secretion of peritoneal macrophage obtained by peritoneal washing at the first day after laparotomy from 19 patients with cervical cancer and 19 with ovarian cancer, who were treated with sizofiran (SPG) intramuscularly before laparotomy or with recombinant interferon gamma (rIFN) intraperitoneally at the day of laparotomy, were studied. Treatment with SPG and rIFN significantly increased the number and secretion of TNF, IL-1 and IFN-gamma (p less than 0.05) but not that of PGE2. A high correlation between secretion of TNF and IFN-gamma (r = 0.99, p less than 0.01) was observed. These data suggest that potent tumoricidal activity of peritoneal macrophage obtained from the patients with gynecological malignancies could be expected by the treatment with SPG and rIFN due to the increased secretion of TNF, IFN-gamma and IL-1 and inhibition of increased secretion of PGE2.

Female C3H mice aged 8-10 weeks with transplanted KHT sarcoma or SCCVII tumor were used to investigate the antitumor effect of SPG (Sonifilan, Schizophyllan) alone and in combination with local irradiation of pions with 4 fractions of 400 cGy (total 1600 cGy). Daily doses of 10 mg/kg of SPG were given intramuscularly to the mice bearing KHT sarcoma for 14 consecutive days from day 7, and to mice bearing SCCVII tumor for 20 consecutive days from day 7 and thereafter three times a week for another 2 weeks. The antitumor effect was evaluated by the changes in tumor volume, survival curves, and the number of pulmonary metastatic nodules on the surface of the lungs. SPG failed to exert any antitumor effect and any life-prolonging effect for the KHT sarcoma. As for SCCVII tumor, in the group treated with pions and SPG, tumor growth decreased significantly (p less than 0.01) compared with the group treated with pion only, and life prolonging effect and metastasis-suppressing effect were also observed (p less than 0.04). In conditions of minimal residual disease brought about by pion irradiation, the adjuvant effect of a Biological Response Modifier (BRM) SPG may prove to be a promising method of cancer therapy for some tumors.

Sixty-two patients with gynecological disease, who were treated with sizofiran (SPG) intramuscularly before laparotomy or with recombinant interferon gamma (IFN) intraperitoneally on the day of the laparotomy, were studied to investigate the change on the number and function of peritoneal macrophages (PM) obtained by peritoneal washing on the day and on the 1st and 4th days after the laparotomy. SPG significantly increased the number of PM by the 1st day, and IFN by the 4th day (p less than 0.05). However, IL-1 and IFN-gamma secretion by PM were not activated by SPG or IFN treatment, respectively. When patients were treated with these two agents simultaneously, the number of PM was significantly increased by both the 1st and the 4th day (p less than 0.05), and IL-1 and IFN-gamma secretion by PM were also significantly activated by the 1st day (p less than 0.05). These data suggest that SPG and IFN had a synergistic effect on increasing the number of PM and on activating the secretion of cytokines by PM, and that the priming with SPG played an important role in the activation of PM function by IFN.

Schizophyllan, a water-soluble beta-D-glucan elaborated by Schizophyllum commune Fries, was partially depolymerized by ultrasonic irradiation to a low-molecular-weight polysaccharide, designated "sonic-degraded schizophyllan". Both native and degraded polysaccharides exhibited essentially the same antitumor activities against Sarcoma-180 ascites. Both glucans are comprised solely of D-glucose residues and have a main chain of (1 leads to 3)-beta-D-glucopyranosyl residues, one out of three glucose residues being attached as single, (1 leads to 6)-beta-D-glucopyranosyl groups. Although both glucans have similar structural features, significant differences are observed in such physical properties as molecular weight and intrinsic viscosity. End-group analysis by using radioisotope-labeled glucans suggests that ultrasonic degradation occurs mainly by cleavage of glycosidic bonds of the main chain of schizophyllan. The molecular weights of the native and sonic-degraded schizophyllan were shown to be 75% of those of corresponding, original schizophyllan preparations, suggesting that there is no anomalous linkage sensitive to periodate oxidation, and ultrasonic irradiation may cause random hydrolysis of (1 leads to 3)-beta-D-glucosidic linkages in the main chain.

Effects of schizophyllan (SPG) and several antibiotics in combination against experimental Pseudomonas aeruginosa infection were examined. (1) Whereas the increase in protective effect of SPG was not observed in combination with carbenicillin or sulbenicillin, remarkable effects could be recognized in combination with dibekacin or gentamicin (GM). The combined therapy with SPG and GM proved to be most excellent. (2) The time course for changes of viable number of Ps. aeruginosa in various organs of mice revealed that the multiplication of the infected bacteria was more markedly inhibited in mice treated with SPG and GM in combination than those treated with each alone. (3) The increase in bactericidal activity of the peritoneal exudate cells was more remarkably demonstrated in the combined treatment with SPG and GM than with each alone, showing the rapid clearance of bacteria from the peritoneal cavity in the former. (4) The microscopic estimation demonstrated that the increase in both phagocytic index and intracellular digestion of bacteria by cultivated macrophages was larger in combination of SPG and GM than each alone. (5) The measurement of viable number of bacteria in cultivated macrophages by the plate-count method also showed that the ingestion and bactericidal activity within the cells were much higher in combined treatment with SPG and GM than each alone.

We reported previously that Schizophyllan (SPG) combined with MMC + 5-FU (MF regimen) or Tegafur (F regimen) had a significant life-prolonging effect over chemotherapeutic regimens in patients with inoperable and recurrent gastric cancer in a randomized controlled study. In the present paper, we have further investigated the effect of SPG on life-span followed-up for more than 2 years. A significant life-prolonging effect of SPG was reconfirmed in patients given either the MF regimen (154 patients) or the F regimen (213 patients). Thus, SPG combined with chemotherapeutics has proved to be useful for treating patients with inoperable and recurrent gastric cancer, resulting in a significant prolongation of life-span without serious side effects despite having no influence on tumor size.

The antitumor activity of schizophyllan (SPG) against syngeneic ACI/N rat tumor, AMC-60 fibrosarcoma and BC-47 bladder cancer was investigated. Intramuscular injection of SPG caused a marked suppression of AMC-60 tumor growth. Results obtained with cytotoxicity tests in vitro and lymphoblastogeneic response in vitro suggest the association of enhanced macrophage cytostasis and high reactivity of lymphoid cells with the resistance of the host to AMC-60 tumor. Moreover, SPG showed pronounced antitumor activity against BC-47 bladder cancer. The highest therapeutic effectiveness was obtained when SPG injection was started at an advanced stage of the tumor, 4 out of 15 rats treated being completely cured of cancer. Cytotoxicity tests in vitro and neutralization tests in vivo indicated the important role of cytotoxic lymphocytes as well as activated macrophages in the host defence mechanism against BC-47 cancer. This, taken together with the fact that the rats cured completely by SPG therapy acquired transplantation resistance to BC-47 cancer, shows that SPG may promote tumor immunity in the host.

Schizophyllan (SPG) was administered to 13 lung cancer patients (i.m. 20mg X 2/week) for 3 weeks without chemo or irradiation therapies, and serum proteins were analyzed by two-dimensional electrophoresis (TDE). Additionally, immunosuppressive acidic protein (IAP) was quantitatively determined by single radial immunodiffusion (SRID). By TDE analysis, human serum proteins were separated into more than 100 spots, and about 14 spots were found to show quantitative changes in cancer patients. Quantitative examination was therefore conducted on changes of 8 components among these spots, including alpha 1-acidic glycoprotein (alpha 1 AG), acidic alpha 2-macroglobulin (acidic alpha 2 M), haptoglobin (Hp) and IAP. The protein which showed the most marked decrease in cancer patients, located between transferrin and IgG on the above TDE patterns, was ascertained to have a molecular weight of about 150,000 using a gel filtration method. This protein was increased in 7 of 13 patients after SPG treatment.

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.

The chemical structure and characteristics, the antitumour activity, the antibacterial, antiviral, antifungal, antiparasitic activities, immunological properties and mode of action of immunomodulators of microbial origin, especially antitumour polysaccharide lentinan, have been discussed immunopharmacologically in comparison with pachymaran, schizophyllan, DiLuzio's yeast glucan, Coriolus preparation PS-K, Streptococcal preparation OK-432, Nocardia rubra cell wall skeleton N-CWS together with BCG and Corynebacterium parvum. Lentinan exerts its inhibitory action not only on allogeneic tumours but also on syngeneic and autologous tumours, and prevents chemical and viral carcinogenesis. The phase III randomized control study of lentinan in cancer patients with gastric and colo-rectal cancer showed the clinical efficacy of lentinan in prolonging the life span and improving host immune responses. Experimental and clinical application demonstrated the advantage of N-CWS over BCG-CWS, and a recent study on OK-432 showed similar results to those obtained with C. parvum. In future work on microbial immunomodulators, it will be essential to find a parameter of the host-tumour relationship which will indicate the protocol for application of immunomodulators, and to find a new-type of selective immunostimulant. For this purpose, exhaustive studies on lymphokines, monokines and lymphocyte tropic hormones are indispensable.

Anti-tumor activity was studied in mice injected with Schizophyllan (SPG), a glucan produced by Schizophyllum commune Fries. SPG-injected mice rejected subcutaneously inoculated sarcoma-180 and the anti-tumor activity was shown to be mediated by host spleen and lymph node cells. The anti-tumor activity lasted as long as 60 days after the SPG administration as assessed by transfer of cells into normal recipients. Cells involved in anti-tumor activity were shown to be T lymphocytes since anti-tumor activity was diminished when lymph node cells from SPG-treated mice were treated with anti-Thy-1.2 sera plus complement, whereas cells passed through a nylon wool column retained the activity. Macrophages were also shown to be involved since administration of carrageenan or trypan blue into the host decreased the inhibition ratio of tumor growth. It was concluded that anti-tumor activity in SPG-treated mice was mediated by the cooperation of T lymphocytes and macrophages, thus the impairment of either function decreased anti-tumor activity.

The immunological effect has been studied of the non-specific immunopotentiator sizofiran (SPG) on patients with advanced uterine cervical cancer treated with radiation. Ten cases (study group: SPG group) were administered weekly 40 mg of SPG throughout the course of radiation and two weekly 40 mg during 12 months after radiotherapy. Nineteen cases (control group) were treated with irradiation alone. The lymphocyte count, the PHA index and the ratio of CD4 cells/CD8 cells were significantly reduced by radiotherapy, being observed the most remarkable decrease at post-radiotherapy. But in SPG group, these decreasing values were significantly smaller than in control group and the values showed a more progressive rise during the 12 month respectively. There was no significant difference in peripheral T lymphocyte; CD3, CD4, CD8. The NK cytotoxic activity showed a progressive rise during the 12 month period following a minimal change after radiotherapy. These results suggest that the SPG have some enhancing effect on the function of the lymphocytes.

To evaluate the clinical effects of the anti-tumor polysaccharide Schizophyllan (SPG), a randomized study was done on 220 patients with Stage II or Stage III cervical cancer who had been given irradiation, concomitantly. The tumor-reducing effect of SPG was significant in patients in either stage. The time to recurrence was longer in SPG-dosed patients with Stage II cancer, compared with findings in the control group. There was no significant difference in the time to recurrence in patients with Stage III cancer between the SPG and control groups. When comparing the 48-month survival curve, the survival time of patients with Stage II cancer in the SPG group was significantly longer than in the control group. However, there was no significant difference in the survival rate of patients with Stage III cancer between the SPG-dosed and control groups.

The efficacy of schizophyllan, a beta-1, 3-D-glucan with a beta-1, 6-linked D-glucose residue isolated from Schizophyllum commune Fries, in combination with local radiation therapy was investigated using C3H mouse squamous-cell carcinoma NR-S1. Male C3H/He mice were implanted with 10(5) cells of NR-S1 tumor on day 0. On day 9 after implantation, mice received electron irradiation with a dose of 5,500 rads. Intramuscular injection of SPG (1.0 mg/kg and 5.0 mg/kg) was then started from the day following irradiation and repeated 15 times at one-day intervals. The antitumor effect was measured by the tumor size and survival time. Significant tumor growth suppression and prolongation of life-span were observed in the group given radiation and SPG (5.0 mg/kg) in comparison with the group which received radiation alone. In histopathological examination of irradiated tumor sites, stromal reactions accompanied by a much greater degree of cellular infiltration consisting of mainly lymphocytes were observed in the group given combined radiation and SPG (5.0 mg/kg).

The effect of a glucan, Schizophyllan (SPG), on pulmonary metastases in syngeneic mice bearing Lewis lung carcinoma (3LL) was examined. As a model of pulmonary metastases, 3LL cells were implanted into the footpads of C57BL/6 mice, the resulting primary tumor was removed 9-10 days later. The inhibitory effect of SPG was evaluated from the number of pulmonary surface nodules on the lungs about 3 weeks after tumor implantation. SPG was found to have antimetastatic activity, which depended on its dose and time of injection. A single injection of 100 or 200 mg/kg or daily injections of 20 or 50 mg/kg of SPG after removal of the primary tumor markedly inhibited pulmonary metastases. Combined therapy with cyclophosphamide and SPG significantly prolonged the survival of mice with pulmonary metastases. Enhancement of the in vitro cytotoxic activity of peritoneal macrophages and bronchoalveolar or whole lung cells against 3LL cells was noted in SPG-treated mice on day 7 after a single intraperitoneal injection of 100 mg/kg SPG. Intravenous transfer of peritoneal macrophages activated with SPG inhibited the development of pulmonary micrometastases.

In order to prolong the survival of patients with cervical cancer, adjuvant immunotherapy was undertaken in Japan. The result of two randomized controlled studies using biological response modifiers such as OK-432 or sizofiran are described. The three-year recurrence-free rates of 221 patients in the OK-432 group and 161 patients in the control group were 71.9% and 58.6%, respectively. The intergroup difference was statistically significant. Of all the patients with Stage II or III cancer, time to recurrence and survival rate in 99 patients in the sizofiran group were significantly longer than in 96 patients in the control, evaluated at 5 years. Based on the results of the 2 randomized controlled studies, we conclude that adjuvant immunotherapy is very useful for prolonging the survival of patients with cervical cancer.

Following a previous report we ascertained the effectiveness of sizofiran (Schizophyllan:SPG) to prolong the survival and time to recurrence of the patients with Stage II or III cervical cancer, as evaluated in a 5-year randomized controlled study conducted in 19 institutions in Japan. Of the overall patients with Stage II or III cancer, time to recurrence and survival rate in the group on SPG were significantly longer than in the control group. In the Stage II patients, there was significant difference in time to recurrence, and survival of SPG group tended to be longer than that of the control group. However, in the Stage III patients, there was no significant difference in either time to recurrence or survival rate.

Adjuvant immunochemotherapy using Schizophyllan (SPG), an extract from the culture broth of Schizophyllum commune Fries, was prescribed at random for 326 Japanese patients with resectable gastric cancer. The overall survival rates for 3 years did not differ between the SPG and control groups. In 62 patients with stage I gastric cancer and 67 with stage II, there was little difference in the 3-year survival rates. The survival rates for 100 patients with stage III were enhanced at p = 0.0811 in the SPG group, as compared to the controls. The survival rates in 97 patients with stage IV cancer were much the same. These results warrant further application of this immunopotentiating drug for treating patients with resectable gastric cancer.

Immunochemotherapy using the antitumor polysaccharide Sizofiran (SPG), an extract from the culture broth of Schizophyllum, was prescribed randomly for unresectable or resectable gastric cancer patients. In case of unresectable cases, the SPG group (treated by SPG plus tegafur--F method--or 5-FU combined with MMC--MF method--) survived longer (p = 0.009 in the MF method and p = 0.003 in the F method) than did the control group (chemotherapy only). Concerning a treatment regimen for resectable cases, all patients were given 0.4mg/kg and 0.2mg/kg of MMC on the day of surgery and the next day, respectively. Oral tegafur was initiated on the 14th postoperative day and was continued as long as possible. In the SPG group, SPG was administered intramuscularly 40mg/week concurrently with the start of oral tegafur and was continued as long as possible. Although the overall survival rates for 5 years did not differ between both groups, the 5-year survival rate for stage III in the SPG group was superior to the findings for stage III in the control (p = 0.0105).

Experimental tuberculosis in mice infected with streptomycin-resistant Mycobacterium tuberculosis SCHACHT strain was treated with streptomycin or rifampicin alone and in combination with schizophyllan. The histopathogical tests of various organs of the treated mice were carried out. 1) In the group of mice treated with streptomycin alone, the moderate focal proliferation of RE cells were seen at the beginning of infection. However, durable activation of RE cells and the prolongation of life-span could not be recognized as compared with control animals. 2) The treatment with streptomycin-schizophyllan combination appeared to be somewhat more effective than schizophyllan alone, the phagocytic capacity being more strongly stimulated. 3) In the group treated with rifampicin alone, the therapeutic effect could be exhibited by the direct antibacterial action of rifampicin, but the activation of RE cells was slight. When rifampicin was discontinued, the growth of tubercle bacilli was rapidly resumed, and the durable therapeutic effect seemed not to be expected. Degeneration of hepatic cells tended to develop. 4) In the group treated with rifampicin-schizophyllan combination, the antibacterial effect of rifampicin appeared to be potentiated by the strong activation of RE cells by schizophyllan, showing the durable therapeutic effects.