Yoshihide Sakagami

Effects of an anti-tumor polysaccharide, schizophyllan, on interferon-γ and interleukin 2 production by peripheral blood mononuclear cells

We examined the effect of schizophyllan, a neutral glucan isolated from the culture filtrate of Schizophyllum commune Fries, on the production of interferon-gamma (IFN-gamma) and interleukin 2 (IL 2) from the mitogen-stimulated human peripheral blood mononuclear cells (PBMC). When the levels of IFN-gamma and IL 2 in the culture medium of phytohemagglutin (PHA)- or concanavalin A (Con A)-stimulated PBMC were measured by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) respectively, a significant increase in the production of both cytokines by schizophyllan was demonstrated. These results suggest that the increased production of IFN-gamma and IL 2 may be responsible for the anti-tumor activity of this glucan.

Release of peptide leukotrienes from rat kupffer cells

Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, and the levels of peptide leukotrienes (LTC4, LTD4, and LTE4) contained in the culture supernatant were determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. In response to A23187, Kupffer cells released LTC4, LTD4, and LTE4. After 10 min-preincubation of Kupffer cells with AA861, a 5-lipoxygenase inhibitor, the generation of LTC4, LTD4, and LTE4 from A23187-stimulated Kupffer cells was significantly suppressed. Platelet activating factor (PAF), a phospholipid mediator, significantly enhanced the release of LTC4, LTD4, and LTE4 from Kupffer cells stimulated with A23187. These results suggested that Kupffer cells may participate in inflammatory and immunologic events in the liver tissue by the release of peptide leukotrienes.

Changes in leukotrienes and prostaglandins in the liver tissue of rats in the experimental massive hepatic cell necrosis model

When heat-killed Propionibacterium acnes is intravenously injected into rats followed by an intravenous injection of a small amount of Gram-negative lipopolysaccharide (LPS) 7 days later, massive hepatic cell necrosis is induced and most of the rats die within 24 hours of LPS injection. Using this experimental model, we studied the changes in the levels of leukotrienes (LTs) and prostaglandins (PGs) in the liver tissue and bile of rats with experimentally-induced massive hepatic cell necrosis. Both the levels of LTs and PGs in the liver tissue and LTs in the bile increased before the microscopic appearance of hepatic cell necrosis. These results suggest that arachidonic acid metabolites may play an important role in the induction of liver cell injury.

Protective effects of a leukotriene inhibitor in an experimental massive hepatic cell necrosis model

SummaryWhen heat-killedPropionibacterium acnes was intravenously injected into mice and seven days later, a small amount of gram-negative lipopolysaccharide was also intravenously injected, most of them died of massive hepatic cell necrosis. However, when azelastine hydrochloride, a leukotriene antagonist chemically known as l(2H)-phthalazinone, 4-[(4-chlorophenyl)methyl]-2-(hexahydro-l-methyl-1H-azepine-4-yl)-, monohydrochloride, or AA861, 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-trimethy l-1, 4-benzoquinone, a specific inhibitor of 5-lipoxygenase, was administered during this experimental induction of massive hepatic cell necrosis, the survival rate of the mice increased and the histological changes of the liver improved remarkably. These results suggested that leukotriene may be important for the induction of massive hepatic cell necrosis in our experimental model.

Release of leukotriene B4 from rat Kupffer cells

In order to examine the production of leukotriene B4 (LTB4) from Kupffer cells, Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, opsonized zymosan, or platelet activating factor (PAF), and the amount of LTB4 in the culture supernatant was determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. As a result, when activated in vitro with calcium ionophore A23187, Kupffer cells generated LTB4. When Kupffer cells were stimulated with calcium ionophore after 10-min preincubation with AA861, a selective 5-lipoxygenase inhibitor, the release of LTB4 from Kupffer cells was markedly suppressed. PAF, which is a phospholipid mediator having a wide spectrum of biological activities, significantly enhanced the release of LTB4 from Kupffer cells stimulated with calcium ionophore or opsonized zymosan. Even when the Kupffer cell were not stimulated with calcium ionophore or opsonized zymosan, LTB4 production was significantly increased by PAF. Thus, our studies indicate that Kupffer cells could generate LTB4 as well as polymorphonuclear leukocytes and macrophages. In addition, it is suggested that Kupffer cells may be able to modify inflammatory and immunological events in the liver tissue by the release of LTB4.

Effects of the polysaccharide chain of lipopolysaccharide in an experimental massive hepatic cell necrosis model

When a small amount (1 μg) of lipopolysaccharide (LPS) purified from Salmonella minnesota wild, Salmonella minnesota R60, and Salmonella minnesota R345 was intravenously injected into mice 7 days after heat-killed Propionibacterium acnes was intravenously injected, massive hepatic cell necrosis was induced and most of the mice died within 24 hours. However, when LPS from Salmonella minnesota R5 and Salmonella minnesota R7 and lipid A from Salmonella minnesota R595 were administered, the survival rate was much higher and no histological changes in the liver such as necrosis could be seen in any of the mice. In each of the LPS used in this study, the structure of the polysaccharide chain was different, and it decreased in the following order: Salmonella minnesota wild → Salmonella minnesota R60 → Salmonella minnesota R345 → Salmonella minnesota R5 → Salmonella minnesota R7 → Salmonella minnesota R595. This suggested that the polysaccharide chain of LPS played an important role in the induction of massive hepatic cell necrosis in this experimental model.

Experimental immunological intrahepatic cholestasis model

SummaryHeat-killedPropionibacterium acnes (P. acnes) was intravenously injected into tuberculin-sensitized guinea pigs, and 7 days later, purified protein derivative (PPD) was also injected intravenously resulting in marked reductions in bile flow and bile acid excretion. Serum levels of bile acids and cholesterol were increased, and the activities of alkaline phosphatase (ALK-p) and leucine aminopeptidase (LAP) were also elevated. Histological examination revealed dilatation of bile canaliculi and diminution of microvilli. These results indicated that intrahepatic cholestasis was induced. These pathological changes were not detected in guinea pigs in which PPD was not injected. In this experimental model, the tuberculin-sensitized lymphocytes were infiltrated into the liver byP. acnes injection and the cholestatic factor was produced by PPD injection, resulting in the induction of intrahepatic cholestasis.

Effects of bile acids on liver cell injury by cultured supernatant of activated liver adherents cells

SummaryWhen heat-killedPropionibacterium acnes (P. acnes) is intravenously injected into mice followed by an intravenous injection of a small amount of lipopolysaccharide (LPS) 7 days later, most of the mice die of massive hepatic cell necrosis within 24 hours of LPS injection. In addition, when the liver adherent cells including Kupffer cells are separated from the mice 7 days after P.acnes injection and incubatedin vitro with LPS, remarkable activity of the cytotoxic factor is found in the culture supernatant. This cytotoxic factor is thought to cause liver injury. Using this experimental model, the effects of various bile acids on liver cell injury were studied. As a result, ursodeoxycholic acid and dehydrocholic acid suppressed liver cell injury induced by the cytotoxic factor. However, cholic acid, deoxycholic acid and chenodeoxycholic acid did not have any hepatocytoprotective effects.

Estradiol receptors in the cytosol of peripheral blood mononuclear cells in hepatitis B virus carriers treated with interferon-alpha.

SummaryEstradiol receptors in the cytosol of peripheral blood mononuclear cells and the effects of interferon-α (IFN-α) on estradiol receptors were studied in asymptomatic hepatitis B virus (HBV) carriers, patients with chronic hepatitis B and normal controls. The level of estradiol receptors in the cytosol of mononuclear cells was significantly lower in asymptomatic HBV carriers and patients with chronic hepatitis B, compared to normal controls. This low level of cytosol estradiol receptors in patients with chronic hepatitis B was increased by the administration of IFN-α. In addition, when peripheral blood mononuclear cells from patients with chronic hepatitis B were incubated with IFN-αvitro, the level of cytosol estradiol receptors also increased by increasing the concentration of IFN-α. We previously reported that the response of mononuclear cells to estrogen is impaired in HBV carriers, and our present results suggested that this may be due to the low level of estradiol receptors in the cytosol of mononuclear cells.

The protective effect of cyclosporin a on experimentally-induced acute hepatic injury in mice

SummaryWhen heat-killedPropionibacterium acnes (P. acnes) and a small amount of endotoxin lipopolysaccharide (LPS) were intravenously injected into mice at a week’s interval, most of them died of massive hepatic cell necrosis. This experimentally-induced acute liver injury was significantly inhibited by cyclosporin A (CsA), resulting in a remarkable improvement of the survival rate. This protective effect of CsA on acute liver injury was also histopathologically confirmed. To study the mechanism by which CsA protected the mice from fatal hepatic injury, adherent cells prepared from the murine liver 7 days afterP. acnes injection were incubated with LPS in the presence of CsA, and the effect of CsA on the production of the cytotoxic factor from the adherent cells was estimated. As a result, CsA inhibited the activation of liver adherent cells and suppressed the release of the cytotoxic factor.