Toshiro Yadomae

真菌β-1,3-グルカンの構造と生物活性

This paper summarizes the structure, biological activities, signaling, and metabolic degradation of fungal beta-1,3-glucans. Fungal beta-glucans exist both soluble and particulate forms. Conformation of the soluble beta-glucan was classified into three groups: triple helix, single helix and random coil. Fungal beta-1,3-glucans exhibit a variety of biological and immuno-pharmacological activities, and the significance of these activities is dependent on the structure, such as solubility in water, molecular weight, degree of branching, and conformation. Many of the activities, such as nitrogen oxide synthesis of macrophage and limulus factor G activation, were dependent on the conformation, but some of the others were independent, such as interferon-gamma and colony stimulating factor syntheses. These activities are beneficial and pharmacologically useful, while some strongly related to allergic and inflammatory adverse reactions. It should be noted that the beta-glucans, once administered into the body, remain mainly in the liver and spleen for a very long period of time. The activity, at least in part, is maintained during these periods. The biochemical mechanisms of the beta-glucan, especially in soluble form, mediating biological activity was still not clearly demonstrated.

Solubilization of yeast cell-wall β-(1→3)-d-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

Immunopharmacological and immunotoxicological activities of a water-soluble (1→3)-β-d-glucan, CSBG from Candida spp.

We have established a convenient, two-step procedure to solubilize the yeast cell wall (1-->3)-beta-D-glucan using the combination of NaClO oxidation and DMSO extraction. Candida soluble beta-D-glucan (CSBG) was mainly composed of a linear beta-1,3 glucan with a linear beta-1,6-glucan moiety. In this study, we screened for several immunopharmacological activities of CSBG and found the following activities: (1) interleukin-6 synthesis of macrophages in vitro; (2) antagonistic effect for zymosan mediated-tumor necrosis factor synthesis of macrophages; (3) augmentation for lipopolysaccharide mediated tumor necrosis factor and nitrogen oxide syntheses of macrophages; (4) activation of alternative pathway of complement; (5) hematopoietic response on cyclophosphamide induced leukopenia; (6) the antitumor effect on ascites form tumor; (7) Enhanced vascular permeability; (8) priming effect on lipopolysaccharide triggered TNF-alpha synthesis; and (9) adjuvant effect on antibody production. These results strongly suggested that CSBG possessed various immunopharmacological activity.

Conformation dependency of nitric oxide synthesis of murine peritoneal macrophages by β-glucans in vitro

We have already demonstrated that various activities including NO (nitric oxide) synthesis in vivo were significantly different between triple helical (SPG) and single helical (alkaline-treated SPG, SPG-OH) beta-glucans, and that beta-glucan-mediated NO synthesis was associated with increased gene expression of IFN-gamma. In this study, we analyzed beta-glucan-mediated NO production in vitro with the concomitant use of IFN-gamma. Proteose peptone-elicited peritoneal macrophages (PM) were collected from male C3H/HeJ mice and cultured with beta-glucans in the presence or absence of IFN-gamma for 24 h. It was found that SPG-OH, but not SPG, enhanced NO synthesis in vitro, especially in the presence of IFN-gamma. Concentrations of interleukin-1 alpha, -6 and TNF-alpha in the culture supernatant of SPG-OH were significantly higher than those in that of SPG. Membrane-associated IL-1 alpha was also high with SPG-OH. Cytokine productivity of PMs, as well as NO synthesis, was elevated in the presence of IFN-gamma. These facts intensely suggest that the single helical conformer of beta-glucan (SPG-OH) is dominant in cytokine production and subsequent NO synthesis.

Relationship between Solubility of Grifolan, a Fungal 1,3-β-D-Glucan, and Production of Tumor Necrosis Factor by Macrophages in Vitro

Grifolan, GRN, is a fungal antitumor β-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150°C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.

Structure and biological activities of hypochlorite oxidized zymosan

Abstract Zymosan (ZYM), a strong complement activating yeast cell preparation, is also a potent inflammatory substance, which shows immunopharmacological activity. Major component of ZYM is β-glucan but contains other constituents, such as mannan, protein, and nucleic acid. We applied sodium hypochlorite (NaClO) treatment to ZYM to reduce impurities and compared the activity with native/parent ZYM. Oxidized ZYM (OX-ZYM) became a nitrogen-free agent. By NMR analysis of native OX-ZYM and zymolyase (endo-1,3-β-glucanase) digest, OX-ZYM was found to contain 1,3-β-linked and 1,6-β-linked glucan moieties, while the latter degraded by sodium metaperiodate treatment. OX-ZYM also contained small amounts of anionic groups, partly reducible by sodium borohydride. Degree of polymerization (DP) of 1,6-β-glucan moiety was estimated to be about DP10–DP50 by MALDI-TOF-MS analysis. In comparison with ZYM activities, OX-ZYM and derivatives showed strong antitumor activity to solid form of Sarcoma 180 in mice, and showed strong activity on alternative pathway of complement, but lost secondary response to ZYM-immune mice. These facts strongly suggested that a particulate form of β-glucan was prepared by NaClO treatment of ZYM and at least a part of ZYM-mediated biological activity was found unmediated by β-glucan moiety.

Enhanced Cytokine Synthesis of Leukocytes by a β‐Glucan Preparation, SCG, Extracted from a Medicinal Mushroom, Sparassis crispa

Abstract Sparassis crispa is edible mushroom recently cultivable in Japan. It contains significantly high content (∼40%) of 6‐branched 1,3‐β‐D‐glucan showing antitumor activity in mice. We recently purified a β‐glucan preparation designated as “SCG.” It was considered worth while to test SCG in vitro with whole blood collected from human volunteers. The present study is focusing on the cytokine productivity of SCG in an in vitro human system. The following results were observed: (i) SCG dose dependently enhanced IL‐8 synthesis of whole blood cell culture of human peripheral blood. (ii) IL‐8 synthesis was enhanced in both PBMC and PMN cultures. (iii) IL‐8 synthesis was induced in the culture with autologous plasma, but significantly reduced after 56°C treatment. (iv) The activity was also weak in heat inactivated fetal calf serum (FCS). (v) A complement fragment, C5a, was released by SCG dependently upon dose and kinetics. (vi) Anti‐SCG natural antibody was detected in human plasma. From these facts, SCG was observed to have the capacity to activate human leukocytes and related immune system.

Mechanism of Enhanced Hematopoietic Response by Soluble β-Glucan SCG in Cyclophosphamide-Treated Mice

SCG is a major 6‐branched 1,3‐β‐D‐glucan in Sparassis crispa Fr. SCG shows antitumor activity and also enhances the hematopoietic response in cyclophosphamide (CY)‐treated mice. In the present study, the molecular mechanism of the enhancement of the hematopoietic response was investigated. The levels of interferon‐ (IFN‐) γ, tumor necrosis factor‐ (TNF‐) α, granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), interleukin‐ (IL‐) 6 and IL‐12p70 were significantly increased by SCG in CY‐treated mice. GM‐CSF production in the splenocytes from the CY‐treated mice was higher than that in normal mice regardless of SCG stimulation. Neutralizing GM‐CSF significantly inhibited the induction of IFN‐γ, TNF‐α and IL‐12p70 by SCG. The level of cytokine induction by SCG was regulated by the amount of endogenous GM‐CSF produced in response to CY treatment in a dose‐dependent manner. The expression of β‐glucan receptors, such as CR3 and dectin‐1, was up‐regulated by CY treatment. Blocking dectin‐1 significantly inhibited the induction of TNF‐α and IL‐12p70 production by SCG. Taken together, these results suggest that the key factors in the cytokine induction in CY‐treated mice were the enhanced levels of both endogenous GM‐CSF production and dectin‐1 expression.

Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1→3)-β-d-glucan

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Application of Candida solubilized cell wall β-glucan in antitumor immunotherapy against P815 mastocytoma in mice

This study was designed to evaluate the antitumor activity of CSBG, purified from the cell wall of Candida albicans IFO1385. First, as an effect of CSBG on P815 mastocytoma, significant prolonged survival and suppression of the tumor growth were observed. Second, the transfer of spleen cells from CSBG-sensitized BALB/c mice to CDF1 mice led to further suppression of tumor growth as well as P815-immunized spleen cells. Third, CSBG enhanced antitumor immunity in gene therapy using B7-1-transfected P815 cells. These results strongly suggest that CSBG enhances the host defense response to tumor due in part to an adjuvant effect.