Keiji Arima

Frequency and significance of antibodies to P450IID6 protein in Japanese patients with chronic hepatitis C.

BACKGROUND/AIMS The aims of the current study were to assess the frequency and the significance of antibodies to cytochrome P450IID6 protein (anti-P450IID6) in various diseases among Japanese patients. METHODS Sera from 541 patients were tested by indirect immunofluorescence, and the specificity of anti-P450IID6 was ascertained by either enzyme immunoassay (ELISA) or Western blot using recombinant antigen or rat liver microsomes. RESULTS Anti-P450IID6 was found in only 6 of 235 patients (2.6%) with chronic active hepatitis (CAH) positive for hepatitis C virus (HCV) antibody and quantitative HCV-RNA with genotypes II and IV. The predominant epitopes on immunoblots were 66 and 50KD, a 10KD band being the newly underfined microsomal antigen. Even in the patients negative for autoantibodies to nuclear antigens (ANA) by routine indirect immunofluorescence test, various ANA were detected by the newly developed recombinant ELISA. These patients were younger, with lower gamma-globulin and IgG levels than patients with autoimmune hepatitis. Three of five patients with anti-P450IID6 responded well to interferon therapy and one received prednisone when interferon was ineffective. Interestingly, only this patient was diagnosed as definite autoimmune hepatitis according to the criteria proposed by the International Autoimmune Hepatitis Group (IAHG). The other five patients who did not satisfy the IAHG criteria might be considered as CAH-C with autoimmune features. No autoimmune hepatitis patients positive for anti-P450IID6 were identified in the current study, indicating that the variant is very rare in Japan. CONCLUSIONS Anti-P450IID6 in CAH-C patients in Japan is not as rare as expected. Anti-P450IID6 among Japanese patients has uncertain significance and precludes further characterization of CAH-C with autoimmune features, which might require interferon therapy.

Characterization of anti-histone antibodies in patients with type 1 autoimmune hepatitis

We have recently found that antibodies to total histones are common in a group of American patients with type 1 autoimmune hepatitis (AIH). In an attempt to determine the profile and clinical association of anti‐histone antibody (AHA), 45 Japanese AIH patients were studied for serum isotypic reactivity with individual histones (H1, H2A, H2B, H3, H4) by enzyme‐linked immunosorbent assay and western blotting. The results revealed that 40% of sera had reactivities with at least one of individual histones and that the antibodies were detected in all three classes of immunoglobulins (IgG, IgM, IgA). Immunoglobulin G type anti‐H3 showed the dominant reactivity and it characterized 72% of sera with AHA. The titre of anti‐H3 decreased significantly (P < 0.0075) after steroid therapy and the index of decrease for anti‐H3 was correlated in individuals with that for serum aminotransferase. In general, patients with AHA showed higher serum level of alanine aminotransferase (P < 0.05), immunoglobulin G (P < 0.025), and higher frequency of A2‐DR4 haplotype (53 vs 17%) than their seronegative counterparts. However, the titre of AHA was low in this disease condition and histone class‐specific antibodies did not distinguish patients with distinctive clinical features, although patients with anti‐H3 tended to be younger than those without AHA.

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection.

BACKGROUND/AIMS It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.

Assessment of the presence and severity of esophagogastric varices by splenic index in patients with liver cirrhosis

Purpose To determine whether spleen size is related to the severity of esophageal varices or associated gastric varices and liver functions in patients with cirrhosis. Method The authors retrospectively studied spleen size on CT (splenic index [SI] = length × width × height of the spleen), liver functions, and the results of esophagogastric endoscopy in 110 patients with cirrhosis. They also analyzed SI in 112 controls. Results In controls, body weight, height, and age affected the SI. The SI in patients with uncompensated cirrhosis was greater compared with the SI in those with well-compensated disease (p = 0.0363). The SI in patients with esophageal varices was greater than in patients without esophageal varices (p < 0.0001), but patients with and without gastric varices had similar SI values. The SI in patients with the red color signs (red wale marking, cherry red spot, and hematocystic spot) on esophageal varices or with risky varices (enlarged tortuous varices with beady, nodular, or tumor shape associated with red color signs) was greater than in patients without these signs (p = 0.0029 and p = 0.0030, respectively). Conclusion The SI is a good indicator of the severity of esophageal varices and hepatic functional reserve in patients with cirrhosis.

Interferon therapy in LKM-1 positive patients with chronic hepatitis C: follow-up by a quantitative radioligand assay for CYP2D6 antibody detection.

BACKGROUND/AIMS Liver/kidney microsomal type 1 (LKM-1) antibodies described by indirect immunofluorescence using frozen sections of kidney, stomach and rat liver define a group of patients with type 2 autoimmune hepatitis. Sera react with a non-glycosylated 50-kD protein of the endoplasmic reticulum, which was recently identified as cytochrome P4502D6 (CYP2D6). LKM-1 antibodies may also be associated with hepatitis C virus infection (HCV+/LKM-1+). For this subset of patients, the choice of steroids or interferon alpha therapy may be difficult because of the association of hepatitis C virus infection and autoimmune manifestations. Recently we developed a quantitative immunoprecipitation radioligand assay using 35S-methionine-labeled CYP2D6 protein produced by in vitro transcription and translation reaction. This method detects antibodies against linear and conformational epitopes in both AIH-2 and HCV+/LKM-1+ patients. The aim of this study was to analyze the time-course of HCV+/LKM-1+ patients, applying our radioligand assay over a long follow-up. METHODS We studied five patients who were positive for CYP2D6 antibodies from among 235 chronic hepatitis C virus hepatitis patients (2.1%) treated with interferon alpha for a minimal follow-up of 2 years. We analyzed LKM-1 antibody titer sequentially by radioligand assay, HCV RNA titer and alanine aminotransferase activity in these patients. RESULTS We found no aggravation of liver disease in this group of patients. Three of these patients showed a sustained biochemical and virological response after interferon. Two others responded partially to interferon therapy. Alanine aminotransferase levels and HCV-RNA decreased during interferon therapy in responder patients. CYP2D6 antibodies did not change in three responder patients during follow-up. One responder patient decreased CYP2D6 antibody level by radioligand assay, but indirect immunofluorescence titers showed a similar pattern. One partial responder patient decreased CYP2D6 antibody level but was negative by indirect immunofluorescence. CONCLUSIONS Our results show that patients with hepatitis C virus who are positive for CYP2D6 antibodies may be treated with interferon, and respond in the same way as CYP2D6 antibody negative patients. Radioligand assay could be helpful for monitoring HCV+/LKM-1+ patients receiving interferon therapy.

CD28-negative CD8-positive cytotoxic T lymphocytes mediate hepatocellular damage in hepatitis C virus infection.

The pathogenic mechanism for hepatocellular damage in hepatitis C virus (HCV) infection has not been clearly understood. Analysis of costimulatory molecules on lymphocytes may give us insight into the pathogenic mechanism of hepatocellular damage in HCV infection. Peripheral blood mononuclear cells (PBMCs) and liver infiltrating mononuclear cells (LIMCs) isolated from the HCV-infected patients were analyzed with antibodies directed against a variety of costimulatory molecules by flow cytometry. Blocking experiment against HLA-A24-restricted HCV-specific CTLs and immunohistochemical analysis were also performed. PBMCs expressing CD8, CD28, CD80, or CD154 were significantly reduced in HCV-infected patients compared with the healthy controls. CD28(+)CD8(+) PBMCs in the patients inversely correlated with ALT levels. Conversely, levels of CD28(−)CD8(+) LIMCs correlated with ALT levels. HCV-specific CTL activity was blocked by the treatment with anti-CD8 antibody, but not with anti-CD4 or anti-CD28 antibody. Immunohistochemical analysis revealed the accumulation of CD28(+) cells around the portal area in the liver of a patient with chronic active hepatitis C. These results suggest that CD28(+)CD8(+) T cells leave the circulation, move to the livers, and are activated in the portal area in proportion to the extent of liver diseases. CD28(−)CD8(+) T cells may finally function as effector T cells causing the hepatocellular damage in HCV infection.

Histopathologic comparison of anti-mitochondrial antibody-positive primary biliary cirrhosis and autoimmune cholangiopathy

The present study aimed to investigate whether antimitochondrial antibody (AMA)-positive primary biliary cirrhosis (PBC) and AMA-negative PBC with autoantibodies differ histologically, especially with respect to infiltrating cells in portal tracts involved by chronic non-suppurative destructive cholangitis. Liver specimens were stained from 15 primary biliary cirrhosis with AMA (group 1), nine patients consistently negative for AMA but positive for antinuclear antibodies (ANA) (group 2). Group 2 showed overlapping features of PBC and autoimmune hepatitis type 1, in a pattern recently termed autoimmune cholangiopathy (AIC). We analyzed the cell population, including lymphocytes, plasma cells, large histiocytes, eosinophils and neutrophils, which had infiltrated portal tracts involved by destructive lesions. Although serum immunoglobulin M levels were higher in group 1 compared to those in group 2 (P=0.0282), patients of both groups were broadly comparable with respect to clinical features and laboratory data. Histologically, the number of plasma cell and its percentage among inflammatory infiltrating cells in the portal tract were higher in group 2 than in group 1 (P=0.0015, P=0.0070, respectively). The percentage of lymphocyte infiltration among inflammatory infiltrating cells in the portal tract were higher in group 1 than in group 2 (P=0.0052). The percentage of plasma cell infiltration among inflammatory infiltrating cells in the portal tract was correlated to immunoglobulin G levels in group 2 (r=0.949, P=0.0016). In conclusion, AMA-positive PBC and AIC showed differences in inflammatory cell population in involved portal tracts in this preliminary study.

Analysis of CD28 and bcl-2 Expression on Peripheral Blood and Liver-Infiltrating Mononuclear Cells in Patients with Autoimmune Hepatitis

Because the underlying mechanism of hepatocellular damages in autoimmune hepatitis (AIH) still remains unclear, analysis of CD28 and bcl-2 molecules, which are critical for T cell activation and survival, was performed in patients with AIH. The number of CD28(+)CD4(+) peripheral blood mononuclear cells (PBMC) in corticosteroid (CS)-treated patients was comparable to normal control individuals but decreased in untreated AIH patients. In contrast, the number of CD28(+)CD8(+) PBMC was decreased in both CS-treated and untreated AIH patients. Analysis of liver-infiltrating mononuclear cells (LIMC) showed that the number of CD28(+)CD4(+) and CD28(−)CD8(+) LIMC were positively correlated with the histology activity index score. Bcl-2(+)CD4(+) LIMC were observed in the portal area of the liver and the numbers fluctuated with disease activity during the time course after CS administration. By contrast, CD8(+) LIMC were shown not to express bcl-2. Taken collectively, these results suggest that bcl-2(+)CD28(+)CD4(+) and bcl-2(−)CD28(−)CD8(+) cells may play critical and distinct roles in hepatocellular damage in AIH.

Electron Microscopic Studies of Peripheral Blood Mononuclear Cells in Chronic Type C Hepatitis Treated with Interferon-α

Electron microscopic studies of peripheral blood mononuclear cells (PBMC) in chronic type C hepatitis revealed several interesting structures after interferon-α (IFN-α) therapy. Fifteen of 20 patients were treated with IFN-α. Tubuloreticular inclusions (TRI) were observed in only one patient who did not receive therapy. In contrast, TRI were observed in 10 of 15 (66.7%) patients who underwent the therapy for 2 weeks to 6 months. Cylindric confronting cisternae (CCC) were identified in the cytoplasm of PBMC in 4 of 16 (25%) patients who underwent the therapy. CCC were found only after IFN-α therapy. The appearance of both TRI and CCC in the PBMC was significantly correlated to IFN-α therapy. Although there is little evidence about the morphogenesis of TRI and CCC, these structures may be a host response to IFN-α induced by hepatitis C virus infection.

Involvement of Annexins I an II in human liver cirrhosis and hepatocellular carcinoma

Annexin (AX) is the name of a new family of Ca2+-dependent membrane binding proteins of which 13 members have been reported to date. Among these, AXI and AXII have been reported to possess many biological functions in vitro. Their actual roles in vivo, however, are as yet unknown. There have been no reports previously demonstrating the direct involvement of AXI and AXII in chronic hepatitis (CH), liver cirrhosis (LC) or hepatocellular carcinoma (HCC). The involvement of AXI and AXII in the etiological processes of CH, LC and HCC was investigated by Western blot and/or immunohistochemistry using anti-AXI and AXII antibodies. AXI and AXII were rarely detected in CH liver tissues, while they were found to be expressed at high levels in LC and HCC. AXI and AXII were present in the hepatocytes of LC and HCC and their subcellular localization was mainly cytoplasmic. These results indicate that AXI and AXII may act together in the occurrence and development of LC and HCC.