Hepatitis C virus (HCV) is the major etiological agent of acute and chronic non-A, non-B hepatitis with genetic information located in a positive single-strand RNA molecule of about 9.4 kb in length. 1 HCV replication in liver has been demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) 2 ; however, RT-PCR is unable to provide information about the cellular localization and intracellular distribution of HCV RNA in the tissue. HCV has been detected by in situ hybridization in liver biopsies predominantly in the cytoplasm of hepatocytes 3-11 ; however, neither the relative viral load of the infected cells nor its relation with serum viremia levels has been established. In the present study we present a simple method based on image analysis of in situ hybridization signals that allows the following: 1) the mapping of the distribution of HCV infection in large areas of the liver biopsy; 2) the estimation of the relative number of HCV genome copies per infected cell; and 3) the analysis of the relationship between the relative amount of HCV in the biopsies and the serum viremia levels.
PATIENTS AND METHODS PatientsLiver sections from ten patients with a histologically proven chronic active hepatitis were analyzed in this study. All patients had abnormal alanine aminotransferase levels and were antibody to HCV and serum HCV RNA positive for at least 1 year before obtaining the liver biopsies. All patients were hepatitis B surface antigen-, antihepatitis B core-, and anti-human immunodeficiency virusnegatives and did not have markers of autoimmune hepatitis.Liver specimens were obtained by percutaneous liver biopsy using Tru-Cut needles (Baxter Healthcare Corporation, Deerfield, IL). A portion of the liver tissue was snap-frozen in liquid nitrogen until required for analysis and the remainder was fixed in formaldehyde and embedded in paraffin wax for histological diagnosis and the detection of HCV RNA by in situ hybridization.
We describe a simple procedure that allows the use of fluorescence in situ hybridization (FISH) for in situ detection of DNA strand breaks in single cells (DBD-FISH: DNA Breakage Detection-FISH). After trapping within an agarose microgel, cells are incubated in an unwinding alkaline solution, deproteinized and dehydrated. Areas of single-stranded DNA are generated by the alkaline solution in proportion to the degree of DNA strand breakage. These then act as targets for FISH of whole genomic or region-specific probes (telomeric, human chromosome 8 painting, human alphoid DXZ1 locus, and human c-erbB-2 cosmid probes). Measurement of the amount and surface of FISH signals provides information on the breakage level in probed areas, permitting the assessment of possible intragenomic differences in sensitivity as well as intercellular heterogeneity in DNA damage induction or repair.
(TTAGGG)n sequence repeats in human telomeres and in Chinese hamster interstitial centromeric areas were digested in situ with exonuclease III (ExoIII) and exonuclease Bal 31. Incubation with AluI was performed beforehand to increase DNA breaks near telomere sequence areas. DNA removal at these specific regions was quantified by digital image analysis of the fluorescence in situ hybridization signal produced by a telomeric probe. Exonuclease III was 2.6 times more active in interstitial than in terminal telomeric sequence areas. Exonuclease Bal 31 was 2.3 times more effective in terminal than in interstitial telomeric sequence regions. These results support the hypothesis that chromatin is differentially organized in both telomeric sequence areas, despite their similar DNA composition.
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