Sparassis crispa is edible mushroom recently cultivable in Japan. It contains significantly high content (approximately 40%) of 6-branched 1,3-beta-D-glucan showing antitumor activity in mice. We recently purified a beta-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 degrees 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.

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Nitric oxide (NO) is an important defense molecule in the nonspecific immune system of the body.1,2) However, NO also acts as a mediator of tissue damage in inflammatory diseases.3) NO is a highly reactive molecule that is normally produced from L-arginine by NO synthase (NOS). There are three isoforms of NOS: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are constitutively expressed, whereas iNOS expression is induced by stimuli such as lipopolysaccharide (LPS) and cytokines. 4,5) Large amounts of NO produced by iNOS have been shown to play a major role in sepsis and endotoxin shock. 6)Septic shock is characterized by a systemic inflammatory response syndrome. Sepsis associated with multiple organ failure and dysfunction, leads to hypotension. 7) NO is a strong mediator of vasodilation. 8)During the last decade, NO has been demonstrated to have a role in septic shock in both humans and experimental animals.9-11) NOS inhibitors have been used to treat septic shock. Indeed, it was reported that a NOS inhibitor restored hypotension in sepsis, but also had a detrimental effect. [12][13][14] Thus, the role of NO in septic shock has not been clarified. The apparent complexity of NO inhibition may be due in part to the different models of sepsis used. Generally, LPS is used to induce septic shock, however, it can not reproduce both gram-positive and -negative sepsis. We developed an animal model of sepsis in mice using two drugs, indomethacin (IND) and b-glucan, sonifilan (SPG). This model uses endogenous LPS which is contained in enteric bacterial flora.We have previously examined the activities of various bglucans [15][16][17][18][19] and found that pretreatment with b-glucan significantly increased mortality on the subsequent administration of IND in mice. [20][21][22][23] The lethality would be closely related to the translocation of enterobacterial flora to the peritoneal cavity. Since the concentrations of IFN-g, IL-6, and CSF in sera of b-glucan/IND-treated mice were significantly elevated, the mortality was likely due to a mal-adjustment of the cytokine network. A precise characterization of this model is needed to clarify the mechanism of septic shock. In this study, we examined the effect of NO on this model by inhibiting the synthesis of NO with a NOS inhibitor. MATERIALS AND METHODS AnimalsMale ICR mice between 5 and 8 weeks of age were purchased from Japan SLC, Inc., Shizuoka and maintained under specific pathogen-free conditions. The breeding and handling of all animals in this experiment were approved by the Committee on Animal Experiments of the School of Pharmacy, Tokyo University of Pharmacy and Life Science.Reagents Sonifilan (SPG) was purchased from Kaken Pharmaceutical Co. (Tokyo, Japan). N G -nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and indomethacin (IND) were obtained from Wako Pure Chemical Ind. IND was suspended with 2% polyoxyethylene (20) sorbitan monooleate in 0.5% sodium carboxymetyl cellulose (CMC).Administration of SPG/Indomethaci...

We have recently developed a protocol to obtain a soluble Candida spp. b1, 3-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (DMSO) extraction. CSBG is composed mainly of b-1,3 and b-1,6-glucosidic linkages with a small amount of branching. Antibody to b-glucan is generally difficult to produce. In the present study, the specificity of the human sera to CSBG was examined by ELISA. Using CSBG-coated plates, sera from normal human volunteers showed high reactivity that was neutralized by adding soluble CSBG as a competitor. However, the reactivity could not be neutralized by a b-1,6 branched b-1,3-glucan, grifolan. Similar specificity of the immunoglobulin (Ig) was detected in commercially available g-globulin prepared from pooled human sera. Comparing various standard glucans, a major epitope was present on b-1,6 glucan segment. Drug Dev.

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