In situ simultaneous detection of hepatitis C virus RNA and hepatitis C virus-related antigens in hepatocellular carcinoma

BACKGROUND: The overwhelming evidence that chronic infection with the hepatitis C virus (HCV) is an important cause of hepatocellular carcinoma (HCC) is based on epidemiologic, case-control, and cohort studies as well as laboratory investigations. To address better the pathogenesis of HCV infection at the single-cell level, the authors developed a specific reproducible method for the simultaneous detection of HCV specific sequences and antigens in liver tissue, using a combination of nonradioactive in situ hybridization and immunohistochemistry. METHODS: After immunohistochemical staining of the liver sections for E2/NS-1, C22-3, C33c, C100-3, and NS-5 antigens with immunogold-silver technique, in situ hybridization was performed on the same sections using digoxigenin-labeled HCV 5' NonCoding specific probes. The hybridization signal was detected by an antidigoxigenin, Fab fragment-alkaline phosphatase conjugate. This simultaneous detection permitted the subcellular localization of HCV RNA and antigens with excellent preservation of tissue morphology and absence of background staining. In addition, the types and percentages of cells harboring HCV in tissue could be determined. RESULTS: The in situ detection of HCV showed positive signals in both cancerous and noncancerous areas of liver tissue in six of six HCV-infected patients with HCC and in none of four controls, including three HCV negative HCC patients and one patient with epithelioid hemangioendothelioma. Two classes of infected cells were distinguished throughout the liver: (1) cells containing large amounts of negative-stranded HCV RNA, which were probably undergoing active viral replication; and (2) cells displaying positive-stranded HCV RNA only, with unpredictable levels of viral replication. Both types expressed core, envelope, and NS-3, -4, and -5 proteins. HCV RNA and antigens were exclusively cytoplasmic. Detection of viral proteins was highly predictive of the presence of large amounts of HCV RNA in the same cell. Fewer HCV positive cells were consistently demonstrated in the cancerous area. CONCLUSIONS: These findings support the contention that HCV infects hepatocytes and replicates in them, even after their malignant transformation

CIRCULATING LEVELS AND LIVER-TISSUE DISTRIBUTION OF INTERCELLULAR-ADHESION MOLECULE-1 DURING INTERFERON-BETA THERAPY OF HEPATITIS-C VIRUS-ASSOCIATED CHRONIC ACTIVE LIVER-DISEASE

Intercellular adhesion molecule-1, an immunoglobulin supergene family member, is known to account for important steps in cell activation and the immune response. By a non-isotopic slot-dot immunoblotting assay, we measured circulating levels of intercellular adhesion molecule-1 in 26 patients with hepatitis C virus-associated chronic active liver disease before and after beta-interferon therapy, in 6 patients with non-A, non-B acute self-limiting hepatitis and in 13 healthy subjects. Circulating intercellular adhesion molecule-1 was found in 10 of 13 (77%) normal controls at low concentrations which were not statistically different from those measured in patients with hepatitis C virus-associated chronic active liver disease responsive to beta-interferon, whereas significantly higher levels were found in unresponsive patients. Higher serum intercellular adhesion molecule-1 levels were found in 4 of 10 (40%) beta-interferon-responsive patients compared with 13 of 16 (18%) unresponsive patients. Intercellular adhesion molecule-1 levels persisted after discontinuation of beta-interferon treatment and did not correlate with hepatocytolysis (as indicated by alanine aminotransferase serum activity) either in chronic active liver disease or acute hepatitis. However, a good correlation was found between intercellular adhesion molecule-1 and its expression on liver cells, thus emphasizing that induced circulating levels may reflect the state of activation at the sites of the inflammatory process. These data strongly support the view that intercellular adhesion molecule-1 plays an important role in liver cell damage in hepatitis C virus-associated acute and chronic liver disease, and that its circulating levels may be a good prognostic parameter of responsiveness to beta-interferon therapy

DETECTION OF HEPATITIS C VIRUS (HCV) PROTEINS BY IMMUNOFLUORESCENCE AND HCV RNA GENOMIC SEQUENCES BY NON-ISOTOPIC IN SITU HYBRIDIZATION IN BONE MARROW AND PERIPHERAL BLOOD MONONUCLEAR CELLS OF CHRONICALLY HCV-INFECTED PATIENTS

Immunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36.4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. IF signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers