Effect of SPG/indomethacin treatment on sepsis, interleukin-6 production, and expression of hepatic cytochrome P450 isoforms in differing strains of mice.

We previously reported that a combination of beta-glucan and indomethacin (IND), a non-steroidal anti-inflammatory drug, was lethal to mice. This lethality was strongly related to translocation of enterobacterial flora to various organs and the development of a systemic inflammation. In this study, we examined expression of microsomal cytochrome P450 (CYP), a drug-metabolizing enzyme mostly found in the liver. Normal ICR mice and endotoxin-low responder C3H/HeJ mice were employed to assess effects of endotoxin on impairment of CYP. In the ICR mice, CYP3A11 expression was decreased by beta-glucan or IND. In the early stage of beta-glucan + IND-treatment, 3A11 expression decreased more significantly; when shock was induced, CYP was dramatically decreased. 3A11 expression was also decreased in C3H/HeJ mice, but the effect was milder. In contrast, in both strains, CYP2E1 expression did not vary due to beta-glucan or IND, but decreased during sepsis. To clarify the molecular mechanisms of induced sepsis in C3H/HeJ mice, the reactivity of other pathogen-associated molecular patterns (PAMPs) was assessed. Those studies showed cooperative effects between Pam(3)CSK(4) (Pam(3)) and CpG ODN (CpG-oligodeoxynucleotide) on the induction of IL-6 synthesis by C3H/HeJ spleen cells. The findings here suggest that the beta-glucan + IND combination influenced hepatic cytochrome P450 expression, particularly in the late stage of sepsis. The results also indicate that this change may be associated with not only endotoxin but other PAMPs as well, and could be affected by the integrity of a host's drug metabolism function.

SPG/IND-induced septic shock in a LPS-low responder strain, C3H/HeJ mice.

The administration of beta-glucan (sonifilan; SPG) in combination with a non-steroidal anti-inflammatory drug, indomethacin (IND), induced lethal septic shock in mice. To demonstrate the influence of bacterial lipopolysaccharide (LPS) in this lethal toxicity, LPS non-responder C3H/HeJ mice were used to compare features of sepsis and physicochemical parameters in the present study. The administration of SPG and IND induced the death of C3H/HeJ mice, lowering rectal temperature, reducing body weight, increasing serum tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) levels, shortening the gastrointestinal tract, and increasing the GOT/GPT level. Microbial translocation to various organs was also significantly increased. These results strongly suggested that LPS-non-responding strain also induced septic shock in this experimental model, and other pathogen-associated molecular patterns (PAMPs) may significantly contribute to the septic shock.

Lincomycin protects mice from septic shock in beta-glucan-indomethacin model.

We have developed a septic shock model in mice by sequential administration of beta-glucan, a biological response modifier, and indomethacin (IND), a nonsteroidal anti-inflammatory drug. Lethality was significantly related to the translocation of gut flora to various organs and mal-adjustment of the cytokine network. In the present study, we have examined the effect of antibiotics on this model to further clarify meanings of microbial flora. Schizophyllan (SPG), antitumor beta-glucan for clinical use, obtained from the culture filtrate of Schizophyllum commune, was used to induce sepsis. Lincomycin (LCM), imipenem (IPM), cilastatine (CS), and ampicillin (ABPC) were used for antibiotics treatment. The survival rate of SPG/IND-treated mice was significantly increased by administering LCM or ABPC/IPM/CS, and the effect was more significant by LCM. In in vitro spleen cell culture, LCM decreased proinflammatory cytokine production. Moreover, prednisolone, immune suppresser treatment improved survival of SPG/IND-treated mice. These findings suggest that LCM is an effective antibiotic in this endogenous septic model by modulating gut microbial flora and, at least a part, by regulating cytokine production of leukocytes.

Antibiotics protect against septic shock in mice administered beta-glucan and indomethacin.

We have developed an animal model of sepsis in mice by repeatedly administering beta-glucan, a biological response modifier, and indomethacin (IND), a nonsteroidal anti-inflammatory drug. The combination of these drugs induced bacteremia by translocation of the enterobacterial flora, resulting in increasing the number of activated leukocytes, and inducing hyper cytokinemia. In the present study, we examined the effect of antibiotics on beta-glucan and IND-induced septic shock. Treatment with antibiotics inhibited microbial translocation, inhibited contraction of the colon, reduced lipopolysaccharides (LPS)-elicited production of TNF-alpha and IL-6, and finally prolonged survival. However, the efficacy of antibiotics treatment was limited in mice administered IND orally. These findings strongly suggested that the antibiotics controlled the gut-associated action of IND and reduced various symptoms accompanying sepsis.

Effect of nitric oxide on beta-glucan/indomethacin-induced septic shock.

We have previously shown that repeated administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to mice treated with beta-glucan, a biological response modifier, induced severe lethality. The lethality would be strongly related to the translocation of enterobacterial flora to the peritoneal cavity and disruption of the cytokine network. Reports suggest that nitric oxide (NO) can have an effective or detrimental role in septic shock. In the present study, we examined the effect of NO, an inflammatory mediator, on beta-glucan/indomethacin (IND)- induced septic shock by inhibiting its synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective NO synthase (NOS) inhibitor. Nitrite concentration was used as an indicator of NO generation. Mortality in beta-glucan/IND-treated mice was increased by administering L-NAME. Numbers of bacteria in various organs of mice treated with beta-glucan/IND rose significantly within a couple of days of the administration of L-NAME. Additionally, TNF-alpha, IL-1beta, and IL-6 concentrations were enhanced in peritoneal exuded cells in culture. These results suggest a significant loss of the bactericidal activity of macrophages on the administration of a NOS inhibitor which enhanced the rate of enterobacterial invasion to the peritoneal cavity, resulting in systemic inflammatory response syndrome. The production of NO, therefore, provides a protective effect in beta-glucan/IND-induced sepsis.

Enhanced cytokine synthesis of leukocytes by a beta-glucan preparation, SCG, extracted from a medicinal mushroom, Sparassis crispa.

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.