Masayoshi Yamashiki

Effects of the Japanese herbal medicine 'Inchinko-to' (TJ-135) on concanavalin A-induced hepatitis in mice.

Inchinko-to (TJ-135) is a herbal medicine consisting of three kinds of crude drugs, and in Japan it is administered mainly to patients with cholestasis. The present study evaluated the effects of TJ-135 on concanavalin A (con A)-induced hepatitis in mice in vivo and con A-induced cytokine production in vitro. When mice were pretreated with oral TJ-135 for 1 week before intravenous con A injection, the activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were significantly decreased 8 h after con A administration (-82%, -96% and -66% respectively). In histological investigations, sub-massive hepatic necrosis accompanying inflammatory cell infiltration was not observed in mice pretreated with TJ-135. Serum levels of interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and IL-2 were significantly lower in mice pretreated with TJ-135 compared with controls, while IL-10 levels were higher in these mice. Intrasplenic IL-12 levels were significantly lower in mice pretreated with TJ-135, while intrasplenic IL-10 levels were higher in these mice. In vitro, IL-10 production by splenocytes was increased by the addition of TJ-135 to the culture medium, whereas the production of IL-12 and IFN-gamma was inhibited. These results suggest that con A-induced hepatitis was ameliorated by pretreatment with TJ-135. With regard to the mechanism of these effects of TJ-135, we speculate that TJ-135 inhibits the production of inflammatory cytokine and enhances the production of anti-inflammatory cytokines. Therefore administration of TJ-135 may be useful in patients with severe acute hepatitis accompanying cholestasis or in those with autoimmune hepatitis.

Effects of the Japanese herbal medicine "Sho-saiko-to" (TJ-9) on interleukin-12 production in patients with HCV-positive liver cirrhosis.

Interleukin-12 (IL-12) is an important cytokine for maintainence of normal systemic defense and bioregulation. The Japanese herbal medicine Sho-saiko-to (TJ-9) has been administered to 1.5 million Japanese patients with chronic liver diseases. TJ-9 is known to significantly suppress cancer development in the liver and has macrobiotic effects. In the present study, we examined the in vitro production of IL-12 by circulating mononuclear cells from liver cirrhosis patients and the effects of TJ-9 on IL-12 production. The monocyte/macrophage fraction and the lymphocyte fraction of peripheral blood were obtained from 11 HCV-positive liver cirrhosis patients and 12 healthy subjects. Interleukin-12 levels in the supernatants were measured using ELISA kits. The levels of IL-12 produced by the patients fractions were significantly lower than those produced by healthy subjects (p < 0.01, p < 0.05). However, when TJ-9 was added to the cultures, the IL-12 production levels in both cell fractions increased approximately three fold, and the levels from the monocyte/macrophage fraction were almost the same as those from healthy subjects. This effect of TJ-9 was attributable to two of its seven herb components, that is, scutellaria root and glycyrrhiza root. One possible mechanism for the macrobiotic effects of TJ-9 on liver cirrhosis patients may be the improvement in IL-12 production.

Herbal medicine ‘Sho‐saiko‐to’ induces tumour necrosis factor‐α and granulocyte colony‐stimulating factor in vitro in peripheral blood mononuclear cells of patients with hepatocellular carcinoma

‘Sho‐saiko‐to’ (TJ‐9) is a Japanese herbal medicine that is commonly administered to patients with chronic viral liver disease in order to improve their overall physical condition and to prevent the development of liver cancer. The present in vitro study demonstrated that, by adding TJ‐9 to cell cultures, there were dose‐dependent increases in production levels of tumour necrosis factor‐α (TNF‐α) and granulocyte colony‐stimulating factor (G‐CSF) in peripheral mononuclear cells of patients with hepatocellular carcinoma accompanied by liver cirrhosis. Increases in the production of TNF‐α and G‐CSF in control cell cultures exposed to different herbal medicines were low, and this indicates the specificity of the responce increases in production of these cytokines to TJ‐9. TNF‐α and G‐CSF are known to play important roles in the biological defence mechanism. Administration of TJ‐9 may, therefore, be beneficial for patients afflicted with intractable liver diseases because it could mildly induce these cytokines.

Analysis of serum cytokine levels in primary biliary cirrhosis patients and healthy adults

By using commercially available ELISA kits, serum IL‐6 and TNF‐α levels in healthy adults, and the levels of various cytokines in patients with primary biliary cirrhosis or chronic viral liver diseases, were investigated. IL‐6 levels of healthy subjects were distributed in a wide range, and the distribution pattern was similar to those of the patients. TNF‐α levels tended to be low in females in their 30s, but there were no abnormalities in the patients. Characteristic findings, in the primary biliary cirrhosis patients, were an increase of IFN‐γ and IL‐2 levels, and a decrease of GM‐CSF levels (P<0.05). IL‐8 levels were higher in the patients than in the healthy subjects (P<0.05), and the increase was remarkable in chronic viral liver disease patients. We believe that measurement of serum cytokine levels as a clinical immunological test is highly useful. Further development of simpler, more rapid, and more sensitive analysis methods is desired. J. Clin. Lab. Anal. 12:77–82, 1998.

Effects of the Japanese herbal medicine ‘Sho-saiko-to’ as a cytokine inducer

The herbal medicine, Sho-saiko-to (TJ-9), has been widely prescribed to chronic viral liver disease patients in Japan. This study examined the inductions of such sytokines as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and granulocyte-colony stimulating factor (G-CSF), on some fractions of peripheral blood mononuclear cells (PBMC) by TJ-9 and each of its seven components. IL-1β, TNF-α, and G-CSF were highly induced by scutellaria root and glycyrrhiza root on monocytes/macrophages. By repeating the same experiments using taxol (an LPS antagonist)-treated substances, authors confirmed that these inductions were not attributable to the presence of quite low LPS in TJ-9 solution, and the cytokine inductions are the specific effect of TJ-9. Because TJ-9s macrobiotic effect in liver cirrhosis patients has been proven statistically in an etiological study, TJ-9 could be a new important therapy in chronic liver diseases.

An effective intradermal hepatitis B vaccination

Small dose intradermal (i.d.) inoculation methods of hepatitis B vaccine have been reported to be effective and economical. We determined the best method to obtain high antibody levels within a short period of time and for the long-term maintenance of these levels. A total of 173 female students were randomly allocated to seven groups: six i.d. inoculation groups, to which 6-12 micrograms was administered in three or four divided doses (Groups A-F), and a control group (Group G) which received three 10 micrograms intramuscular (i.m.) inoculations. Serum hepatitis B antibody levels were quantified in weeks 4 and 8, and months 4, 7, and 12. Positivities in all groups were not significantly different at each measurement time. In month 4, geometric mean antibody levels in the three i.d. groups (10-12 micrograms in three divided doses; 79.1-107.0 IU l-1) were significantly higher than in Group G, which had received two of three i.m. injections (17.6 IU l-1; P < 0.01, P < 0.05). In the group which received four 2 micrograms i.d. inoculations, the level was higher than in Group G in month 7, but lower in month 12. It was concluded that three i.d. inoculations, each of 4 micrograms, may be used to obtain high antibody levels within a short period of time. However, it is recommended that a 10 micrograms i.m. injection in month 6 is applied as a booster. Consequently, we could not present an economic and effective low-dose intradermal inoculation method.

FLOW CYTOMETRIC ANALYSIS OF IL-6 RECEPTORS ON PERIPHERAL LYMPHOCYTES IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS

Interleukin‐6 receptors (IL‐6R) and interleukin‐1 receptors (IL‐1R) on lymphocyte surfaces were analyzed, using flow cytometry and dye‐labeled IL‐6 and IL‐1β, to examine the clinical and immunological significance of these receptors. Incubation of peripheral blood mononuclear cells in the presence of mitogen resulted in a remarkable increase of lymphocytes expressing the IL‐6 and IL‐1β receptors on the cell surface. The increase in lymphocytes bearing these cytokine receptors may reflect an increase in stimulated lymphocytes. When peripheral blood from patients with primary biliary cirrhosis (PBC) was examined for these receptors, the percentage of IL‐6R positive cells was significantly higher in the patients than in healthy controls (P<0.01). The increase in IL‐6R positive cells was only significant for the T lymphocyte fraction (P<0.01). No significant change in IL‐1R was observed. There was a significant positive correlation between the percentage of IL‐6R positive T lymphocytes and the titer of antimitochondrial antibody in patients with PBC. These findings concerning IL‐6R may be noteworthy elucidating autoimmune etiological features of PBC. J. Clin. Lab. Anal. 12:83–87,1998.

Effects of herbal medicine "Sai-rei-to" (TJ-114) on in vitro interleukin-1 receptor antagonist production in peripheral blood mononuclear cells

Abstract Herbal medicine “Sai-rei-to” (TJ-114) has been used for the treatment of swelling and edema for about 3000 years in China. Recently, this drug has been used by Japans western medicine physicians as an adjunct therapy in the treatment of rheumatoid arthritis (RA). This drug is believed to exhibit immunoregulatory effects, but its mode of action has not yet been fully described. In this in vitro study, the Japanese herbal medicine Sai-rei-to was used. Results demonstrated that TJ-114 induced the interleukin-1 receptor antagonist (IL-1ra) dose-dependently in cultures of peripheral blood mononuclear cells (PBMC) collected from healthy persons. Another herbal medicine “Sho-saiko-to” (TJ-9), consisting of several common ingredients as in TJ-114, also showed similar, but smaller increases in production levels. However, other herbal medicines used in this study did not significantly increase IL-1ra production. Therefore, this effect was evaluated as being specific to TJ-114 and those herbal medicines containing similar ingredients. Interleukin-1 (IL-1) is regarded as the most important cytokine affecting the pathology of RA and IL-1ra inhibits the immunopathological functions of IL-1. TJ-114, which increases IL-1ra production, has a rather low incidence of adverse effects and can be administered orally for a long period. It is expected that this medicine will show a high degree of clinical usefulness for the treatment of RA.

Rapid determination of hepatitis B surface antigen (HBsAg) by latex agglutination using an integrating sphere turbidimetric assay

A method for determining levels of serum HBs antigen has been developed, applying the principles of the integrating sphere turbidimetric assay (ISTA). Using this method, the minimum detectable level of HBs antigen is 15 ng/ml, i.e., it is three times more sensitive than the reversed passive hemagglutination (RPHA) method. Reproducibility and specificity are also excellent with ISTA. With a cut-off level of 20 ng/ml, the highest reading possible with this method is 1000 ng/ml. Serum HBs antigen can readily be measured by this method if a fully automated EL-1000 analyzer is used. This rapid and simple method of measurement should be clinically useful.