Effect of PSK on Th1/Th2 balance in tumor-bearing mice

We investigated the effect of PSK on Th1/Th2 balance in tumor-bearing mice. PSK was intraperitoneally administered to Meth A-bearing BALB/c mice, and PSK caused regression of the Meth A tumor. The results of Winn assay suggested that the effect of PSK was dependent on CD4+T cells. Furthermore, spleen cells were cultured with mitomycin C-treated Meth A, after which the cytokine concentration was measured by ELISA. IFN-gamma production was increased and IL-4 showed almost no change in PSK-administered mice. In another experiment, PSK was orally administered to colon 26-bearing mice in which tumors were inoculated into the subserosal space of the cecum. Mesenteric lymph nodes cells were cultured with mitomycin C-treated colon 26 cells. IFN-gamma production was increased, but not so much as to be statistically significant, and IL-4 was significantly decreased in PSK-administered mice. PSK increased IFN-gamma and IL-12 p70 production and decreased IL-4 production when spleen cells were stimulated with Con A together with PSK in vitro. As suggested from these results, PSK might induce cytokine production that works for Th1 differentiation, and suppress cytokine production that works for Th2 differentiation, and shift the Th1/Th2 balance toward Th1 dominance in tumor-bearing mice.     

A suppressive effect of PSK, a protein-bound polysaccharide preparation, on tumor growth: a new effect of PSK on cell motility

We found that PSK has ambidextrous effects on cell motility. PSK enhances macrophage motility and inhibits tumor cell motility, when the capillary tube method was used in vitro. Macrophage motility was enhanced with increasing concentration of PSK. PSK inhibited tumor cell motility, i.e. Ehrlich tumor cells, EL-4 lymphoma cells and human leukemic cells, in dose dependent fashion. Macrophages and tumor cells were incubated with medium containing PSK for varying times at 37 degrees C and then washed with medium to eliminate PSK thoroughly. The motility of macrophages and tumor cells was enhanced and inhibited, respectively, with increasing pre-incubation time. When PSK-treated tumor cells were injected into the abdominal wall of C57BL/6 mice, PSK-treated tumor cells were less invasive than non-treated ones in mice. These phenomena are concern neither with the change of cellular viability nor that of cell proliferation.     

Induction of immunopotentiation activity by a protein-bound polysaccharide, PSK (review)

A protein-bound polysaccharide, PSK, extracted from the mycelium of Coriolus versicolor (Fr.) Quel, has been recognized for its host-mediated induction of antitumor and antimicrobial activities in mice. Intravenous administration of PSK, in association with OK-432 (Picibanil), transiently induced endogenous production of a cytotoxic factor (CF) (possibly tumor necrosis factor, TNF) in normal mice. The ability to produce CF depended greatly on both dose and interval between administration of the PSK and OK-432. Although PSK has been reported to contain several active ingredients, unfractionated PSK has been used in almost all experiments performed so far. We recently reported that, of the four subfractions separated by successive filtration through membrane filters, only the highest molecular weight fraction F4 (MW greater than 200 kD) induced significant antimicrobial activity in mice. PSK stimulated the NBT-reducing activity of mouse peritoneal macrophages and the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). Among the subfractions of PSK, the highest molecular weight fraction F4, and the fraction precipitated at pH 4.0-4.5 (Fr. 4), stimulated macrophage NBT-reducing activity and PMN iodination most. In contrast, natural and chemically modified glucans had little or no stimulating activity. PSK, F4 or Fr. 4 additively or synergistically stimulated TNF-induced cytotoxicity against L-929 cells, differentiation of human myelogenous leukemia cell lines toward monocytes/macrophages, and iodination of human peripheral blood PMN. The active PSK subfractions significantly reduced the down regulation of specific 125I-TNF or 125I-IFN-gamma binding to cellular receptors. These data suggest that (i) immunopotentiation activity of PSK might be ascribed, at least in part, to stimulation of cytokine action and production, and (ii) PSK might have some unique structural features.     

Effectiveness of immunochemotherapy with PSK, a protein-bound polysaccharide, in colorectal cancer and changes of tumor marker

In the present study, curatively resected patients of colorectal cancer at pTNM stages II and III were selected. Patients receiving postoperative combined PSK, a protein-bound polysaccharide, and fluoropyrimidine therapy (PSK + chemotherapy group) were compared with patients receiving postoperative chemotherapy alone (chemotherapy group) during the same period of study. Three-year disease-free survival rates were evaluated and the postoperative changes of serum type IV collagen level were investigated. The results confirmed a significant improvement of the three-year disease-free survival rate in the PSK + chemotherapy group compared to the chemotherapy group, suggesting that PSK is useful as postoperative prognosis control including relapse prevention for colorectal cancers at pTNM stage II and III. Analysis of the postoperative changes of serum type IV collagen level showed significantly higher levels in the chemotherapy group than in the PSK + chemotherapy group, and this tendency was sustained for 12 months after surgery. This observation is speculated to be caused by inhibition of vascular basement membrane destruction by PSK, leading to inhibition of release of type IV collagen into the blood. These results indicated a possibility that combined PSK and chemotherapy inhibited metastasis, thereby reducing the risk of relapse and leading to improvement of the three-year disease-free survival rate.     

PSK,a protein-bound polysaccharide,overcomes defective maturation of dendritic cells exposed to tumor-derived factors in vitro

Dendritic cells (DC) are the most potent antigen-presenting cells that induce specific anti-tumor immunity. To obtain potent efficacy of immunotherapy using infusion of activated DC, it is necessary to overcome defective function of DC in tumor-bearing patients. We examined whether the treatment with PSK, a biological response modifier derived from Basidiomycetes, could allow DC to avoid inhibition of functional maturation by tumor-derived factors in vitro. CD14+ monocyte-derived DC were generated by stimulating with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 in the presence or absence of PSK (100 microg/ml), by exposure to a tumor culture supernatant (TSN) of MKN-45P human gastric cancer cells. TSN-exposed DC were not effective in inducing cytotoxic T lymphocyte-mediated growth inhibition of target HT29 human colon cancer cells. In contrast, the presence of PSK significantly resuscitated the defective cytotoxicity. This beneficial outcome was accompanied by an increase in phagocytic activity as measured by fluorescein isothiocyanate-conjugated dextran, expression of CD83 (maturation-specific phenotype), overexpression of a CD86 co-stimulatory molecule, preserved production of IL-12 that plays a key role in the induction of Th1-type immune regulations, and protection against TSN-induced apoptosis of DC. These results demonstrated that PSK overcomes defective maturation of DC exposed to tumor-derived factors in vitro, and suggest the efficacy of PSK in DC-based immunotherapy in cancer patients.     

Influence of PSK (Krestin) on resistance to infection ofPseudomonas aeruginosa in tumor-bearing mice

Summary C3H/He mice were inoculated withPseudomonas aeruginosa by various routes 1 day after X5563 transplantation or 4 days after cyclophosphamide (CY) administration. Administration of PSK (Krestin) i.p. or p.o. to the tumor-bearing mice or CY-treated tumor-bearing mice resulted in an increase in survival rates. ViableP. aeruginosa were inoculated i.v. on day 0 into mice inoculated with tumor cells on day -12 and vaccinated with killedP. aerouginosa on day -10, or into mice inoculated with tumor cell on day -15, treated with CY on day -14 and vaccinated on day -10. Resistance to infection, which is enhanced by vaccination, was depressed by tumor burden or treatment with CY, but such depression was prevented by PSK administration.Abbreviations used CY cyclophosphamide - i.p. intraperitoneally - s.c. subcutaneously - p.o. orally - i.v. intravenously     

Antitumor effector mechanism at a distant site in the double grafted tumor system of PSK, a protein-bound polysaccharide preparation

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.     

Restoration of impaired T cell functions in tumor-bearing mice by the administration of interleukin 1

The effect of in vivo administration of recombinant human interleukin 1 (IL-1) on T cell functions in tumor-bearing mice was studied using an in vitro assay system. The in vitro induction of trinitrophenyl (TNP)-specific cytotoxic T cell and proliferative T cells responses from spleen cells was impaired in X5563 plasmacytoma-bearing C3H/He mice. However, the administration of IL-1 alpha or IL-1 beta to tumor-bearing mice restored T cell functions in a dose-dependent manner. Antigen-presenting activities of spleen cells in tumor-bearing mice for T cell activation were not restored by the administration of IL-1. The activities of cytotoxic T cells and cytostatic T cells specific for X5563 cells were also enhanced by the administration of IL-1. Furthermore, in IL-1-treated mice, NK cell activity of spleen cells detected in terms of the killing of Yac-1 cells was also restored. In accordance with these results, the growth of X5563 cells was significantly inhibited and the lymphocytes from IL-1-treated mice specifically inhibited the growth of tumor cells. These results suggest that the in vivo administration of IL-1 restored the impaired T cell and NK cell functions in tumor-bearing mice and activated protective immunity against tumor cells. Thus, recombinant IL-1 can be applied for tumor immunotherapy.     

Neonatal inoculation with the protein-bound polysaccharide PSK increases resistance of adult animals to challenge with syngeneic tumor cells and reduces azoxymethane-induced precancerous lesions in the colon.

We have investigated the results of neonatal inoculation with a protein-bound polysaccharide, PSK, as it affects the defense mechanism of animals against cancer. Male BALB/c mice received a single i.p. injection of 10 mg/kg PSK within 48 h of birth. When the mice were 8 weeks of age, colon adenocarcinoma 26 (C26 tumor) cells were transplanted s.c. Injection of PSK increased the number of tumor-rejecting mice from 10 to 50% compared with the control mice transplanted with 5 x 10(3) tumor cells and prolonged the median survival period to 174% of control mice with tumors. When the number of transplanted tumor cells was increased to 1 x 10(6), PSK injection significantly prolonged the survival period, although tumors grew in all mice. The survival period was also significantly prolonged in male C57BL/6 mice that received an injection neonatally with PSK and were given a s.c. transplant of Lewis lung carcinoma or B16 melanoma at 8 weeks of age. The effect on survival was dependent on the PSK dose and the number of transplanted tumor cells. PSK was as effective for male mice 30 weeks of age as for mice 8 weeks of age treated with PSK during the neonatal period. However, prolongation of the survival period of tumor-bearing mice was not observed in the offspring (F1). Neonatal injection of PSK also significantly reduced the number of metastatic foci in the liver of mice inoculated with 1 x 10(5) C26 tumor cells in the splenic vein after 8 weeks of age. In addition, neonatal injection of PSK significantly reduced the number of aberrant crypts and aberrant crypt foci, the precancerous lesions in the colon of F344 rats that received injections s.c. with azoxymethane after 7 weeks of age, to 47% of that of rats that received an injection with saline at the same age. The effect on precancerous lesions was dependent on the timing of PSK injection and the dose. Regarding the mechanism, when animals thymectomized during the neonatal period or when congenitally athymic animals were used instead of healthy animals, the effect on survival or precancerous lesions did not appear. Neonatal injection of PSK significantly reduced the number of CD4+ CD8+ T cells and significantly increased the number of CD4+ CD8- and CD4- CD8+ T cells in the thymus of healthy mice 10 weeks of age and C26 tumor-bearing mice. Furthermore, neonatal injection of PSK significantly elevated the T-cell differentiation induced by a mouse thymus extract 10 weeks of age. These findings suggest that neonatal injection of PSK induces resistance in adult mice to challenge by syngeneic tumor cells and reduces the azoxymethane-induced precancerous lesions in the colon of adult rats via the thymus functions.     

Comparative virulence of Candida albicans strains in CFW1 mice and Sprague-Dawley rats

Summary. To verify host-species specificities of virulence of Candida albicans in experimental systemic mycoses, 10 ATCC strains of Candida albicans were compared for their virulence in CFW1 mice and Sprague-Dawley rats. Virulence was parallel in mice and rats, four strains were avirulent (ATCC 10231, 18804, 38245, 44831), one strain had an intermediate virulence (ATCC 32354), and five strains (ATCC 10261, 44373, 44505, 62342, 90028) were highly virulent in both host species. Infection doses of 2 times 10⁶CFU per mouse and 5 times 10⁶ CFU per rat were comparable with respect to mortality of animals within 10 days; this represents a 4:1 ratio on the basis of body weight. In Sprague-Dawley rats haemorrhage occurred in infections with all virulent strains which was not observed in CFW1 mice.
Zusammenfassung. Zur Bewertung Wirtsspezies-spezifischer Virulenzunterschiede von Candida albicans bei tierexperimentellen systemischen Mykosen, führten wir für 10 ATCC-Stämme von Candida albicans Virulenzvergleiche für CFW1-Mäuse und Sprague-Dawley-Ratten durch. Die Virulenz der Stämme war parallel in Maus und Ratte. Vier Stämme waren apathogen (ATCC 10231, 18804, 38245, 44831), ein Stamm zeigte eine mitlere Virulenz (ATCC 32354), fünf Stämme (ATCC 10261, 44373, 44505, 62342, 90028) waren hochgradig virulent für beide Wirtsspezies. Infektionsdosen von 2 times 10⁶ KBE pro Maus und 5 times 10⁶ KBE pro Ratte waren für die Letalität über 10 Tage vergleichbar, auf das Körpergewicht bezogen wird bei CFW1-Mäusen eine vierfach höhere Infektionsdosis benötigt als bei Sprague-Dawley-Ratten. Bei Sprague-Dawley-Ratten wurden Blutungen bei der Infektion mit allen virulenten Stämmen beobachtet, was bei CFW1-Mäusen nicht auftrat.     

Immunoregulatory effects of PSK on the Th1/Th2 balance and regulatory T-cells in patients with colorectal cancer

It is very important for immunotherapy to release Th2-dominated and Treg-dominated immunological conditions in patients with malignant diseases. In the present study, we assessed the population of CD4+IL-10+T-cells and CD4+Foxp3+T-cells in peripheral blood in patients with colorectal cancer using a flow cytometric analysis, and we investigated whether Th2-dominated and Treg-dominated condition could be modulated by PSK. Peripheral blood samples were collected preoperatively from 40 patients with colorectal cancer before and after oral administration of PSK (3 g/day × 1 week). After PSK treatment, CD4+IL-10+T-cell percentages decreased in 63% of patients and CD4+Foxp3+T-cells percentages decreased in 63% of patients. However, no correlation was found between the ratio of CD4+IL-10+T-cell percentages and that of CD4+Foxp3+T-cell percentages after/before PSK treatment. These results suggest that PSK could release Th2-dominated and Treg-dominated immunological condition in patients with colorectal cancer. Further examinations are needed to investigate the after/before percentage ratio of CD4+Foxp3+T-cells can be useful predicting parameters for the selection of responders of PSK.     

枸杞多糖对酒精性肝损伤小鼠肾脏的保护作用

目的:研究枸杞多糖对酒精性肝损伤小鼠肾脏的保护作用。方法:将60只小鼠随机分为正常对照组,模型组和枸杞多糖低、中、高剂量组(分别为75、150、300 mg/kg)。枸杞多糖组动物连续灌胃受试物16 d,第10天开始,给予枸杞多糖组和模型组50%酒精连续灌胃7 d,建立酒精性肝损伤模型。最后一次灌胃16 h后处死小鼠,取血清和肾脏。用自动生化分析仪检测血清中葡萄糖(GLU)、尿素氮(BUN)和肌酐(CREA)浓度,用生化试剂盒测定肾脏中超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和丙二醛(MDA)浓度,用ELISA方法测定肾脏中肿瘤杀伤因子-α(TNF-α)、白细胞介素-1β(IL-1β)浓度。结果:与对照组相比,模型组小鼠体质量下降,肾脏指数均升高,血清GLU、BUN、CREA浓度升高,肾脏SOD活性、GSH浓度下降,而MDA上升,肾脏炎性因子TNF-α和IL-1β升高(P均<0.01),表明模型组小鼠在大量酒精刺激后,机体健康受到影响,肾脏受到损伤。与模型组相比,枸杞多糖中剂量组小鼠体质量增加,各剂量组肾脏指数均降低,中、高剂量组小鼠血清GLU浓度(7.63、7.82 mmol/L)降低,高剂量组BUN浓度和CREA浓度降低,而SOD活力升高,各剂量组GSH浓度升高,MDA浓度下降,各剂量组TNF-α浓度降低(P<0.05),高剂量组IL-β浓度与模型组相比降低(P均<0.05)。结论:枸杞多糖对于乙醇诱导的小鼠酒精性肾脏损伤具有一定的保护作用。     

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.     

Selective suppression of t-cell activity in tumor-bearing mice and its improvement by lentinan,a potent anti-tumor polysaccharide

The cellular site of immunosuppression in Ehrlich tumor-bearing mice was analysed with particular reference to the T- and B-cell activities. The B-cell activity as measured by the anti-dinitrophenyl (DNP) antibody responses to DNP-thymus-independent carriers (TID) was not impaired in tumor-bearing mice as compared with normal mice, whereas the anti-DNP antibody responses to DNP-thymus-dependent carriers (TD) and the development of helper T-cell activity to TD were markedly suppressed in tumor-bearing animals or mice pretreated with cell-free cancerous ascitic fluid. The selective suppression of T-cell response was not mediated by the generation of suppressor cell activity toward TD, which may depress the manifestation of developed helper T-cell activity. A marked suppression of T-cell response was observed when the animals were inoculated with tumor cells or injected with cancerous ascitic fluid prior to antigenic stimulation, but not when the animals were rendered tumor-bearing by such treatments after the immunization. The suppression of T-cell activity in both sarcoma 180 tumor-bearing mice and cell-free Ehrlich cancerous ascitic fluid-treated mice was prevented by treatment with lentinan, a potent anti-tumor polysaccharide. The applicability of this experimental system to the search for immunopotentiators relevant to tumor immunotherapy is discussed in the light of the preventive effect of lentinan on the suppression of T-cell response in tumor-bearing animals.     

Comparative virulence of Candida auris with Candida haemulonii,Candida glabrata and Candida albicans in a murine model

The incidence of invasive fungal infections (IFIs) caused by uncommon Candida species with diverse virulence and susceptibility profiles has increased in recent years. Due to scarce clinical and experimental data on the pathogenicity of Candida auris, the aim of this study was to evaluate and compare the virulence of two rare clinically relevant species, C. auris and Candida haemulonii with Candida glabrata and Candida albicans in an immunocompetent murine model of disseminated infection. Immunocompetent ICR female mice were infected with three inoculum sizes (1 × 10⁵, 1 × 10⁶ and 1 × 10⁷ CFU/mouse) of two C. auris strains and one isolate of C. haemulonii, C. glabrata and C. albicans. Tissue burden on days 5 and 10 postchallenge and mortality rate were used as virulence markers. A high virulence was found for C. albicans, followed by C. auris, C. glabrata and C. haemulonii, respectively. Candida albicans showed high virulence with a medium survival time of 9.5 days for mice infected with 1 × 10⁷ CFU/mouse. For inocula at 1 × 10⁶ and 1 × 10⁷ CFU/mouse, there were significant differences in fungal burden at day 10 between C. albicans, C. auris and C. glabrata isolates compared with C. haemulonii (P < .0001). Overall, no significant differences between C. albicans with C. auris and C. glabrata were observed in mice infected with three different inocula (P > .05). In general, the highest fungal load of all isolates was detected in kidney followed by spleen, liver and lung tested with three different inocula on the two different experimental days. Histopathological examination revealed the abundant presence of yeast cells with pseudohyphae for C. albicans and only yeast cells for C. auris, C. glabrata and C. haemulonii, in all the kidney tissue samples. In conclusion, C. albicans is a highly virulent opportunistic fungus, as the clinical and experimental data demonstrate, and also our results demonstrate a low virulence of C. haemulonii in immunocompetent animals. Altogether, this study highlights the pathogenic potential of C. auris.     

Adherence in tissues of immunocompromised mice of a non-mycelium producing strain of Candida albicans

Summary. A non-mycelial strain of wild-type Candida albicans strain 3153 A was produced by repeated subculturing on Sabouraud glucose agar, and maintained on yeast extract-peptone-glucose medium resulting in hydrophobic cells at 26  °C and hydrophilic cells at 37  °C. The behaviour of cells of this strain was studied in male BALB/c mice, immunocompromised by treatment with cyclophosphamide and cortisone acetate. An ex-vivo assay of cell adherence to tissue sections of liver, spleen, kidney and lymph-nodes was used. The adherence of yeast cells at 26 and 37  °C was predominantly produced by hydrophobic cells and was significantly greater in spleen and liver of immunosuppressed mice compared with the organs of control animals. Adhesion was observed in the white and red pulp as well as in the marginal zone of the spleen.