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, casecontrol, and cohort studies as well as laboratory investigations. To address better the pathogenesis of HCV infection at a 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 immunoistochemical 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, -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 HCVinfects hepatocytes and replicates in them, even after their malignant transformation.
IN SITU SIMULTANEOUS DETECTION OF HEPATITIS C VIRUS RNA AND HEPATITIS C VIRUS-RELATED ANTIGENS IN HEPATOCELLULAR CARCINOMA
LAULETTA, GIANFRANCO;RUSSI, SABINO;
2010-01-01
Abstract
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, casecontrol, and cohort studies as well as laboratory investigations. To address better the pathogenesis of HCV infection at a 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 immunoistochemical 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, -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 HCVinfects hepatocytes and replicates in them, even after their malignant transformation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
