Semi-quantitative analysis of cytokine mRNA expression induced by the herbal medicine Sho-saiko-to (TJ-9) using a Gel Doc system.

The RT-PCR method was employed to determine the cytokine mRNA expression of human peripheral lymphocytes induced by the Japanese herbal medicine Sho-saiko-to (TJ-9). The results showed that the mRNA expression of IL-12, IL-1beta, IL-10, TNF-alpha, G-CSF, and IFN-gamma increased after 6 hr in culture. This is the first reported finding that TJ-9 is an IFN-gamma inducer. Next, cytokine mRNA expression was semi-quantitatively measured using the Gel Doc system with a CCD camera and then statistically analyzed in order to determine which component of TJ-9 was the true cytokine inducer. The results showed that the scutellaria root is the main component inducing the cytokines, while the glycyrrhiza root is the secondary component. When the cytokine concentrations in the supernatants of cell cultures were measured by ELISA, the levels of IL-12, IL-1beta, IL-10, TNF-alpha, and G-CSF reflected mRNA expression levels in the cell fraction. However, the level of IFN-gamma was below the detectable limit. The effects of various reagents on many different kinds of cytokine mRNA expression could be analyzed objectively in a short time using the Gel Doc system. Many important findings could be demonstrated by this simple, easy, sensitive, and cheap method. After the clinical significance of cytokine analysis is confirmed, this method may become a useful clinical examination tool.

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 maintenance 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.

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

By using commercially available ELISA kits, serum IL-6 and TNF-alpha 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-alpha 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-gamma 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.

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-1 beta, 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-1 beta 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.

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.

In vitro effects of sho-saiko-to on production of granulocyte colony-stimulating factor by mononuclear cells from patients with chronic hepatitis C.

During the past 2 years, drug-induced interstitial pneumonia was reported in 66 Japanese patients, mainly among chronic hepatitis C patients, undergoing treatment with the Japanese herbal medicine "Sho-saiko-to" (TJ-9). As interstitial pneumonia is also induced by granulocyte colony-stimulating factor (G-CSF), we examined the effects of TJ-9 on G-CSF production in peripheral blood mononuclear cells. In patients with hepatitis B or C, G-CSF production in the absence of any stimulation was significantly lower than healthy controls (p < 0.01). G-CSF production increased along with the increase of TJ-9 levels, and this could induce excessive production of G-CSF in hepatitis C patients, and this may be a cause of interstitial pneumonia.

Effects of the Japanese herbal medicine "Sho-saiko-to" (TJ-9) on in vitro interleukin-10 production by peripheral blood mononuclear cells of patients with chronic hepatitis C.

"Sho-saiko-to" (TJ-9) consists of 7 herbal components. In Japan, it is widely prescribed to patients with chronic viral liver disease. TJ-9 is known to suppress liver cancer development and possess macrobiotic effects, but its mode of action is not fully understood. This study investigated the following: 1) cytokine production levels, mainly interleukin (IL)-10, in peripheral blood mononuclear cells of chronic active hepatitis B and C patients, and healthy volunteers; 2) effects of TJ-9 on these productions; and 3) effects of each of its herb components on cytokine production in cell fractions. Results showed that without stimulants, IL-10 production in mononuclear cells of hepatitis B and C patients was significantly lower than that of healthy subjects (P < .01). IL-10 production induced by either phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in mononuclear cells of hepatitis C patients were significantly lower than in patients with hepatitis B (P < .01) and healthy subjects (P < .05). IL-10 production induced by anti-CD3 or lipopolysaccharide (LPS) was significantly lower than in healthy subjects (P < .05). The addition of TJ-9 to the cultures strongly induced IL-10, and this induction was mainly attributable to the effects of 2 components (scutellaria root and glycyrrhiza root) on the monocyte/macrophage fraction. The production of IL-4 and IL-5 in cultures with concanavalin A (conA) was significantly higher in patients with hepatitis C than in the healthy subjects (P < .01; P < .05), but the addition of TJ-9 suppressed these increases by 25% to 33% (P < .01). Therefore, TJ-9 could adjust the decreased IL-10 production and the increased IL-4 and IL-5 production of mononuclear cells from patients with hepatitis C. Moderate regulation of the cytokine production system in patients with hepatitis C by using TJ-9 may be useful in the prevention of disease progression.

Herbal medicine 'Sho-saiko-to' induces tumour necrosis factor-alpha 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-alpha (TNF-alpha) 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-alpha and G-CSF in control cell cultures exposed to different herbal medicines were low, and this indicates the specificity of the response increases in production of these cytokines to TJ-9. TNF-alpha 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.

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-1ß (IL-1ß), 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-1ß, 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-9's 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.

Two-color analysis of peripheral lymphocyte surface antigens in inherently healthy adults.

We studied 134 "healthy persons" with no past history of any serious or latent diseases, and observed differences in their 25 lymphocyte subsets by conducting two-color analyses using 12 useful combinations of dye-labeled monoclonal antibodies. CD4+Leu8+ cells and CD25+CD3+ cells were significantly higher in males than in females, whereas CD4+Leu8- cells and CD23+CD19+ cells were significantly higher in females. In comparison of the 25 lymphocyte subsets among four age groups, CD45RA-CD4+ cells and CDw29+CD4+ cells significantly increased with age, and CD8+ CD11b- cells and CD57-CD8+ cells significantly decreased with age. In females, helper T cells significantly increased and helper inducer T cells increased, while cytotoxic T cells decreased with age. These are important findings that should be considered in studies of immune function and autoimmune disorders.

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.

Quick assay of serum HBs antibody levels using latex agglutination-integrating sphere turbidimetric assay (ISTA).

A method for determining serum HBs antibody applying the principle of integrating sphere turbidimetric assay (ISTA) by latex agglutination was developed. The minimum detectable level of HBs antibody by this method is 12.5 IU/L, indicating that this method is 3 times or more sensitive with better reproducibility and specificity than the passive hemagglutination (PHA) method. With a cut-off level of 25 IU/L, the possible highest simultaneous reading by this method was 1,000 IU/L. Serum HBs antibody can be readily measured in 10 or so minutes by this method if a fully automated EL-1000 analyzer is used. This rapid and simple method for determining serum HBs antibody will be useful not only clinically, but also in preventive medicine.

Effects of the herbal medicine "Sai-rei-to" on in vitro interferon-gamma production of peripheral blood mononuclear cells.

The herbal medicine "Sai-rei-to" has been used in the treatment of swellings and edemas for about 3000 years in China. Recently, this drug has been prescribed as an adjuvant in the treatment of rheumatoid arthritis (RA) among Japan's western medicine doctors. It is thought to possess regulatory effects on the immune system, although its mode of action is not yet fully described. In the present in vitro study, we at first induced interferon-gamma (IFN-gamma) in the cultures of peripheral blood mononuclear cells of healthy volunteers by adding pokeweed mitogen (PWM), phytohemagglutinin (PHA), recombinant interleukin-2 (IL-2), or control medium. We then added "Sai-rei-to" to these cultures and investigated the effects of this drug on IFN-gamma production levels. The results demonstrated that "Sai-rei-to" had 2 different effects: (i) it inhibited the IFN-gamma production in cultures with PWM or PHA (which induced large volumes of IFN-gamma), and (ii) it increased IFN-gamma production in the cultures with IL-2 (IL-2 induced only small volumes of IFN-gamma). These findings indicate that "Sai-rei-to" possesses regulatory effects on the IFN-gamma production. IFN-gamma is an important cytokine in the immune system, and it has also attracted attention as a factor related to the pathogeneses of RA. Therefore, concomitant administration of such a medicine which can appropriately control IFN-gamma production in vivo could be beneficial for RA patients from the immunological viewpoint. Clinical experience in the past has shown that "Sai-rei-to" has a very low incidence of side effects, and can be administered orally for long periods. We expect that concomitant administration of "Sai-rei-to" with current therapy could be clinically useful in the management of RA patients.

Effects of methylcobalamin (vitamin B12) on in vitro cytokine production of peripheral blood mononuclear cells.

Recently in Japan, one form of vitamin B12, methylcobalamin also known as methyl B12, has attracted the attention of physicians as a therapy for patients with rheumatoid arthritis. However, its immunological actions in vivo are still unknown. In this study, we induced the in vitro production of such cytokines as interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and interleukin-1 beta (IL-1 beta) by adding various mitogens (phytohemagglutinin:PHA, concanavalin A: ConA, or pokeweed mitogen:PWM) as well as recombinant interleukin-2, and we investigated the effects of methyl B12 (final concentration, 8-8,000 ng/ml) on the production of these cytokines by peripheral mononuclear cells. As compared to the controls, IL-6 production induced by PHA and ConA on Day 4 of the culture was suppressed by an average 60-70% when methyl B12 (80-8,000 ng/ml) was added to the medium. IFN-gamma production decreased dose-dependently with methyl B12, i.e., it decreased to 46% of the control when this production was induced by rIL-2, and decreased to 56-66% when it was induced by mitogens. The effect of methyl B12 on IL-1 beta production on Day I of the culture was small. These findings indicate that methyl B12 suppresses mainly the cytokine production of T lymphocytes. Such suppressive effects as shown in the in vitro situation are expected to be expressed also in vivo in patients with rheumatoid arthritis, especially at articulation lesion sites.

Efficacy of a herbal medicine "sho-saiko-to" on the improvement of impaired cytokine production of peripheral blood mononuclear cells in patients with chronic viral hepatitis.

Peripheral blood samples were collected from normal subjects and chronic viral hepatitis patients, and the in vitro capability of the peripheral blood mononuclear cells to produce various cytokine (interleukin-1 beta, interleukin-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor) were analyzed by adding pokeweed mitogen. We then investigated the effects of a herbal medicine "Sho-saiko-to" on the levels of cytokine production. The production levels of the 4 cytokines were significantly lower in the peripheral blood mononuclear cells of the patients (Patient Group) than in those of normal subjects (Control Group). The addition of Sho-saiko-to to the Patient Group resulted in improved productions of those cytokines, as well as an remarkable improvement of interleukin-1 beta production. The results demonstrated that Sho-saiko-to acts to improve such immunological abnormalities as decreased cytokine productions. Administration of Sho-saiko-to to chronic viral hepatitis patients is also expected to have immunological benefits.

Effects of the streptococcal preparation OK-432 on in vitro cytokine production of peripheral blood mononuclear cells in patients with chronic viral hepatitis.

We obtained peripheral blood mononuclear cells from 12 chronic hepatitis Type B patients, 12 Type C patients, and 15 healthy volunteers, and investigated the effects of OK-432, a streptococcal preparation, on in vitro production of 3 types of cytokines. Mononuclear cells in a concentration of 1 x 10(6) cell/ml were prepared in the culture medium. OK-432 (Chugai Pharmaceutical Co., Ltd., Tokyo) was added to this preparation and incubated for one to 4 days. Thereafter interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) levels in the culture supernatant were measured using enzyme-linked immunoassay kits. Cytokine production levels in cultures with OK-432 were significantly increased in the mononuclear cells of both patients and healthy volunteers. The largest increase was observed with IFN-gamma (p < 0.01), and then with IL-1 beta (p < 0.05). Responses of the cells from chronic hepatitis Type C patients to OK-432 were relatively good. When interferon (alpha and beta) treatment was first introduced, there were high hopes for a high efficacy. However, we now know 50-70% of patients with chronic hepatitis Type C do not respond satisfactorily to interferon. Some physicians suggest the necessity of using biological response modifier (BRM) as an adjuvant treatment for these patients. From our findings, OK-432 could be a useful BRM in patients with chronic hepatitis Type C.

Herbal medicine "sho-saiko-to" induces in vitro granulocyte colony-stimulating factor production on peripheral blood mononuclear cells.

The herbal medicine "Sho-saiko-to (Xiao-Chai-Hu-Tang)" has been used in China for about 3000 years for the treatment of pyretic diseases. This medicine is now available as one of the prescribing drugs approved by the Ministry of Health and Welfare of Japan, and has also been widely used for patients with chronic viral liver disease as one of biological response modifiers in the field of Japan's Western Medicine. However, its mode of action has not been fully described. In the present in vitro study, we added "Sho-saiko-to" (TJ-9, Tsumura, Tokyo) to the culture of peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers, and observed a dose-dependent increase in the production of granulocyte colony-stimulating factor (G-CSF). The same experiment was conducted using other herbal medicines "Dai-saiko-to" (TJ-8) and "Saiko-keishi-to" (TJ-10) which showed similar effects, or "Sho-seiryu-to" (TJ-19) which consists of very different compounds and shows different efficacy. The increases of G-CSF production were similar when "Sho-saiko-to" (TJ-9) or one of the 2 reference drugs (TJ-8 and 10) was added, whereas the increase when the control drug "Sho-seiryu-to" (TJ-19) was added, was quite small. This result shows that G-CSF induction is not a common effect of herbal medicines, but a specific effect of TJ-8, 9, and 10. Among these 3 drugs the increase produced by "Sho-saiko-to" was the largest. Based on this result, we conclude that administration of "Sho-saiko-to" may be useful not only for the treatment of chronic liver disease, but also for malignant diseases and acute infectious diseases where G-CSF is efficacious.