Motohiro Arao

Long term effects of phlebotomy on biochemical and histological parameters of chronic hepatitis C

OBJECTIVE:There is considerable evidence that iron is a risk factor for liver injury in chronic hepatitis C. Known as iron reduction therapy, phlebotomy reduces serum ALT activity. This effect might continue with maintenance phlebotomy and result in slower progression of liver fibrosis.METHODS:We examined the biochemical parameters and liver histology of patients with chronic hepatitis C treated by maintenance phlebotomy. For biochemical evaluation, 25 patients were treated by initial phlebotomy to reduce serum ferritin levels to 10 ng/ml or less and then observed for 5 yr with maintenance phlebotomy to maintain the iron-deficient state. For histological evaluation, liver biopsies were performed before and after the study period in 13 of the patients. Thirteen patients who were virological nonresponders to interferon alone and had undergone second liver biopsies after more than 3 yr served as histological controls.RESULTS:Serum aminotransferase levels were decreased significantly by initial phlebotomy and remained at the same levels during the study period (p < 0.05). The grading scores were improved significantly in the study group (p < 0.05) and unchanged in the controls. The staging scores remained unchanged in the study group but were increased in the controls (p < 0.005). Disease progression was significantly different between the two groups (p < 0.05).CONCLUSIONS:These results suggest that phlebotomy with maintenance lowers serum aminotransferase levels, improves liver inflammation, and suppresses the progression of liver fibrosis in chronic hepatitis C.

Prevalence of diabetes mellitus in Japanese patients infected chronically with hepatitis C virus.

Background: To examine the relationship between hepatitis C virus (HCV) infection and diabetes mellitus (DM) in Japanese populations, a retrospective study was done in 866 patients with chronic viral disease. Methods: The present study included 707 HCV-infected and 159 hepatitis B virus (HBV)-infected patients. The prevalences of HBV- and HCV-related cirrhosis were 32% and 33%, respectively. A case-control study was also conducted to determine the seroprevalence of HCV infection in a cohort of 459 diabetics. Results: The prevalence of DM was higher in HCV-infected patients (20.9%; P < 0.02) than in HBV-infected subjects (11.9%). In the cirrhotic patients, DM was observed in 30.8% of the subjects with HCV compared with 11.8% of those with HBV (P < 0.01). Multivariate analysis revealed that the major independent variables associated with type II DM were male sex (odds ratio, 1.54; p = 0.020) and cirrhosis (odds ratio, 1.97; P = 0.0007). The relative odds of the development of DM were calculated to be 3.2 times higher in HCV-infected cirrhotic patients than in HBV-infected ones. In the case-control study of the diabetic cohort, 10.5% of patients were infected with HCV compared with 1.1% with HBV (P < 0.0001). The results indicate that HCV infection is closely associated with DM, compared with HBV infection. Cirrhosis was an independent risk factor for DM. Conclusions: Taken together, the findings indicate that cirrhosis appears to be a more important predictor of glucose intolerance than HCV infection, and the combination of both factors increases the risk of DM in our populations.

Production of tumor necrosis factor, interleukin 1, and interferon-γ by peripheral blood mononuclear cells from patients with primary biliary cirrhosis

Since patients with primary biliary cirrhosis (PBC) have evidence of abnormal function of the immune system, we evaluated production of various cytokines by peripheral blood mononuclear cells (PBMCs) and monocytes from patients with this disease, using an enzyme-linked immunosorbent assay. The mean amounts of production of tumor necrosis factor alpha(TNF alpha), interleukin 1 beta (IL1 beta), and interferon-gamma (IFN-gamma) by PBMCs from patients with PBC tended to be increased in cultures in the presence of stimulating agents in comparison with controls, but there was no significant difference because of a wide scatter of results. Monocytes from PBC patients also tended to produce higher amounts of TNF alpha and IL1 beta than control monocytes did, although the percentage of monocytes in PBMCs was similar in PBC and controls. A significant correlation was found between TNF alpha production and IL1 beta production in PBC patients. The number of TNF alpha or IFN-gamma positive infiltrating mononuclear cells detected by immunohistochemical staining in liver biopsy sections correlated with the production of these cytokines by PBMCs in vitro. However, cytokine production did not correlate with serum biochemical or hepatic histologic findings, except for serum alkaline phosphatase values. In patients with type B chronic active hepatitis, IL1 beta and IFN-gamma production was similar to controls, while TNF alpha production tended to be enhanced. Thus the cytokines studied here may play some role in the pathogenesis of PBC.

Effects of various cytokines on collagen synthesis by normal rat hepatocytes in primary cultures and fibroblasts.

Collagen formation is an important function of liver parenchymal and nonparenchymal cells that may be relevant to the pathogenesis of hepatic fibrosis. In the present study, the effect of various kinds of cytokines and prednisolone on collagen synthesis by hepatocytes obtained from perfused rat livers and confluent human skin fibroblasts were investigated. The cells were cultured in serum-free medium for 24 h with [3H]proline and various additives. The amounts of collagen and noncollagen protein synthesis were calculated from the incorporation of [3H]proline into collagen-sensitive and collagen-resistant proteins. The additions of interleukin 1 alpha, interleukin 1 beta and transforming growth factor alpha enhanced both collagen and noncollagen protein synthesis by hepatocytes as well as by fibroblasts. Transforming growth factor beta also increased both collagen and noncollagen protein synthesis by fibroblasts while it selectively inhibited collagen synthesis by hepatocytes. Interferon preparations and prednisolone showed a selective suppression of collagen synthesis by both cells, indicating that these additives did not affect either cell viability or proliferation during the observed period. These results suggest that collagen synthesis by liver parenchymal cells is either positively or negatively modulated by various kinds of cytokines and hormone.

Randomized controlled trial of twice-a-day administration of natural interferon β for chronic hepatitis C

Abstract We conducted a randomized controlled trial to assess the efficacy of twice-a-day administration of natural interferon β (IFNβ) as an induction of IFN therapy for chronic hepatitis C. Seventy-one patients with chronic hepatitis C were enrolled into the trial and randomly assigned into three treatment groups. Six million units (MU) of IFNβ were administered once-a-day for the first 4 weeks, and then thrice weekly for 12 weeks in 20 patients (once-a-day group). Three milion units of IFNβ were administered twice-a-day for the first 2 weeks, 6 MU once-a-day for the next 2 weeks, and then thrice weekly for 12 weeks in 23 patients (twice-a-day+β group), or 6 MU of lymphoblastoid IFNα were administered thrice weekly for the last 12 weeks instead of IFNβ in 28 patients (twice-a-day+α group). Four patients in once-a-day group (20%), 9 in twice-a-day+β group (39%), and 12 in twice-a-day+α group (43%) obtained sustained response. Sustained response rate in twice-a-day groups was higher than in once-a-day group, although there was no statistical significance. The present study suggested the possible superiority of twice-a-day administration of IFNβ as an induction therapy to once-a-day administration, but further studies are needed to confirm this regimen.

Randomized controlled trial of lymphoblastoid interferon α for chronic hepatitis C (comparison of 9-MU and 6-MU doses)

Abstract Objective: We conducted a randomized controlled trial to compare the efficacy of two different dosages of lymphoblastoid interferon α (IFN) for the treatment of chronic hepatitis C. Methods: Eighty-four patients with chronic hepatitis C were enrolled and randomly assigned into the two groups; group A was treated with 6 million units (MU) and group B with 9 MU daily for the first 2 wk, and then thrice weekly for an additional 14 or 22 wk. Results: Eighty patients were evaluated (39 patients in group A and 41 in group B); 14 patients in group A (35.9%) and 15 in group B (36.6%) obtained sustained response. The percentages of patients who became negative for HCV RNA at the end of the second wk differed slightly between the groups, without statistical significance (56.4% and 68.3%). When assessed in detail, patients with genotype 1 and

Interferon α and γ positive mononuclear cells and HLA antigen expression on hepatocyte membrane in the liver in chronic type B and non-A, non-B hepatitis

SummaryTo evaluate the role of local interferon on pathogenesis in chronic type B and non-A, non-B chronic hepatitis, we examined the cellular localization of interferon α and γ, and the expression of HLA antigens in cryostat liver sections from 33 chronic carriers of HBs antigen and 10 patients with non-A, non-B chronic hepatitis, using an immunoperoxidase procedure with monoclonal antibodies. Infiltrating mononuclear cells were the main cell element staining positive for interferon α or γ. Among type B chronic liver disease, the percentages of both interferon α- and γ-positive mononuclear cells were highest in patients with chronic active hepatitis, and their levels were higher in patients who had serum HBe antigen, indicating that local expression of interferon closely correlates with activity of disease and virus replication. On the other hand, both the frequency of interferon α or γ positive cells, and the intensity of HLA class I antigens on hepatocytes were much lower in patients with non-A, non-B, chronic active hepatitis in comparison with those with type B chronic active hepatitis. These findings suggested that locally produced interferons have role of varying degrees on the host defence mechanism in chronic hepatitis, reflecting disease activity and the etiology of the disease.

Recombinant Human Interleukin 1α is Cytotoxic for and Increases Surface Expression of HLA-A,B,C Antigens of a Human Hepatoma Cell Line, PLC/PRF/5

Our study was undertaken to determine whether human recombinant interleukin 1 alpha (rIL 1 alpha) has any effect on the proliferation and expression of HLA-A,B,C antigens of human liver cell lines. The addition of rIL 1 alpha reduced the cell number of the human hepatoma cell line, PLC/PRF/5. This effect was determined to be cytotoxic, but not growth inhibitory, rIL 1 alpha did not change the number of Chang cell or SK-Hep-1 at a concentration as, high as 25,000 U/ml. rIL 1 alpha enhanced the expression of HLA-A,B,C antigens on PLC/PRF/5, but had no effect on Change cell or SK-Hep-1. Receptor binding studies showed that 125I-rIL 1 alpha bound to PLC/PRF/5 in a specific and saturable manner, but did not bind to Chang cell or SK-Hep-1. Scatchard plot analysis of the binding to PLC/PRF/5 revealed a single type of high affinity binding site with an apparent dissociation constant of approximately 5 x 10(-5) M and the presence of approximately 150 binding sites per cell. These findings suggest that IL 1 alpha may play a role in host defense against some hepatomas as cytotoxic factor and may be an enhancer of expression of HLA-A,B,C antigens on tumor cells.

Interferon γ and bile duct damage in primary biliary cirrhosis

Aberrant expression of HLA DR antigens on bile duct epithelium in prima{y biliary cirrhosis (PBC) could be important in presenting self antigens • Expression of HLA antigens can be induced by interleukin 2 (IL2) and interferon T (IFNT).2 To explore the relationship between localization of IL2 and IFN 7, and display of HLA antigens on bile ducts, we have examined with monoclonal antibodies to IL2, IFN Zand HLA in cryostat liver biopsy sections from 4 patients with PBC using immunoperoxidase procedure. Five patients with HBeAg-positive chronic active hepatitits (B-CAH) and 5 patients with non-A, non-B CAH (NANB-CAH) were studied as controls. The number of IL2 or IFN T positive infiltrating mononuclear cells (MNC) and the total number of MNC at portal tracts were counted. Bile ducts in controls did not express HLA-DR whereas in 2 PBC patients with early histological stages there were DR staining in interlobular bile duct epithelium. In these patients, HLAA,B,C expression on biliary epithelium was also increased. IL2 or IFN T positive MNC were noted in 2 PBC cases with the early stages although the percentages of both cytokines positive MNC were highest in B-CAH (Table). In 2 PBC patients with the late stages there was no change in HLA antigens on bile duct or no cytokine positive MNC. Present study indicated that abnormal HLA-DR expression and increased A,B,C expression on bile duct were associated with IL2 and IFN T positive infiltrating MNC at portal tracts in the early stages of PBC. However, it is not clear why the same does not happen in CAH. Thus it is more likely that DR expression in bile duct epithelium is a primary event in PBC. Table. Display of HLA antigens and cytokine positive infiltrating mononuclear cells in the liver tissue HLA display on bile duct Diagnosis (no.) A,B,C % of cytokine positive cells DR IL2 IFN T PBC (4) +~% -~q+ 3.8 + 4.5 6.0 + 9.5 B-CAH (5) + 9.6 + 7.1 8.0 + 8.1 NANB-CAH (5) + ~q+ 1.2 + 2.6 0.8 + i.i Results were expressed as mean + SD. References i. Balardini G, Mirakian R, Bianchi FB, et al: Lancet 1984; 2: 1009-1013 2. Farrar WL, Johnson HM, Farrar JJ: J Immunol 1981; 126: 11201125