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.

Autoreactive helper T cells specific for cytokeratin 19 in patients with autoimmune hepatitis.

Recently we have demonstrated the existence of anti-cytokeratin 19 (CK19) antibodies in sera of patients with autoimmune hepatitis (AIH). In the present study, we examined the existence of T lymphocytes specific for CK19 in patients with AIH. The frequency of responders having CK19-specific T lymphocytes was significantly higher in AIH patients than in chronic hepatitis C (CH-C) patients and normal subjects. Furthermore, the stimulation index of proliferative responses of peripheral blood mononuclear cells (PBMCs) was significantly higher in AIH patients than in CH-C patients and normal subjects. The phenotype of proliferating PBMCs specific for CK19 was shown to be predominantly CD4(+) T lymphocytes and these CD4(+) T lymphocytes had a Th1 subtype. The present findings demonstrate that there is some population of CD4(+) T lymphocytes with a Th1 subtype specific for CK19 in peripheral blood of patients with AIH. The Th1-predominat pattern of cytokines may induce cytotoxic T lymphocytes (CTLs) specific for the antigenic peptides derived from the autoantigen, including CK19. In addition, these CTLs may attack the hepatocytes and this may be one of the important etiologies of AIH.

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.

Combination therapy of percutaneous ethanol injection and radiofrequency ablation against hepatocellular carcinomas difficult to treat.

Radiofrequency ablation (RFA) is an effective modality for the treatment of hepatocellular carcinoma (HCC), because it can induce large coagulated necrosis in a few sessions. We have recently reported that the combination therapy of percutaneous ethanol injection (PEI) with RFA (PEI-RFA) created enhancement of coagulated necrosis compared with RFA alone. In the present study, we adopted PEI-RFA for the treatment of HCCs located in the regions that are difficult to treat with RFA alone. Five patients with biopsy-proven HCC and liver cirrhosis underwent PEI-RFA therapy. In these patients, HCCs were located beside the gallbladder, inferior vena cava or portal vein or kidney, or immediately under the diaphragm. Prior to RFA, 99.5% ethanol was injected into the region of HCC located in the regions where RFA energy appears to be difficult to reach. In all cases, HCC was totally coagulated by PEI-RFA. Injecting ethanol prior to RFA therapy caused no major side effects. These results indicate that PEI-RFA may be effective for the treatment of HCCs located in the regions that are difficult to treat with RFA alone as well as large-sized HCCs.

Enhanced expression of Bcl-2 in lymphocytes infiltrating into the liver of patients with primary biliary cirrhosis.

The Bcl-2 family proteins are important regulators of apoptosis and have been implicated in the occurrence of autoimmune diseases. There have been reports that Bcl-2-positive lymphocytes play important roles in pathogenesis of at least some types of autoimmune diseases, including autoimmune hepatitis. In this study, we examined the role of Bcl-2-positive lymphocytes in the development of primary biliary cirrhosis (PBC). The expression of Bcl-2 in lymphocytes infiltrating into the liver was evaluated from liver biopsy specimens of 25 patients with PBC (stage I/II/III/IV, 11/3/8/3) and 24 controls with chronic hepatitis B (CH-B) or chronic hepatitis C (CH-C). Bcl-2 expression in lymphocytes infiltrating into the liver was investigated by immunohistochemistry using a monoclonal antibody to Bcl-2 with an avidin-biotin complex system. Significant overexpression of Bcl-2 in lymphocytes infiltrating into the liver was observed in the early stage of PBC, especially in areas of destructed bile ducts. Most of Bcl-2-positive cells were CD45RO-positive helper T-cells visualized by the double staining method. These results suggest that Bcl-2-positive helper T-cells may have important roles in the pathogenesis of PBC.

Clinical significance of lens culinaris agglutinin-reactive fraction of serum alpha-fetoprotein in patients with hepatocellular carcinoma.

alpha-fetoprotein (AFP) is an important marker for the diagnosis of hepatocellular carcinoma (HCC) and has been widely used in clinical settings. Recently, the importance of lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) has been indicated. However, the clinical significance of the level of AFP-L3 protein in relation to the characteristics of HCC has not been fully evaluated. In the present study, both the ratio of AFP-L3 (AFP-L3%) and the absolute value of AFP-L3 (AFP-L3-AV) were examined in 80 patients with HCC, and evaluated with respect to characteristics of HCC such as grade of differentiation, size of tumor and morphological findings. Among HCC-specific tumor markers, AFP, AFP-L3% and protein induced in vitamin K absence (PIVKA-II), AFP showed the highest positive rate in patients with HCC, while AFP-L3% showed the lowest rate. AFP-L3% and AFP-L3-AV were, however, most significantly correlated with the grade of HCC differentiation, while AFP showed the least significant correlation. Furthermore, AFP-L3% was most significantly correlated with the size of HCC in patients with solitary HCC. Conversely, neither AFP-L3-AV nor PIVKA-II showed a significant correlation with the size of HCC. In relation to morphological differences of HCC, although AFP-L3%, AFP-L3-AV and PIVKA-II were significantly higher in the diffuse type of HCC than in the nodular type of HCC, AFP was most significantly correlated with the morphological differences of HCC. These results indicate that tumor markers for HCC, such as AFP, AFP-L3%, AFP-L3-AV and PIVKA-II, may play different roles in predicting the characteristics of HCC.