Southern-blot analysis and simultaneous In Situ detection of hepatitis B virus–associated DNA and antigens in patients with end-stage liver disease

Authors

  • Kwang Hyub Han,

    1. Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030
    2. Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030
    Current affiliation:
    1. Department of Internal Medicine, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea
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  • F. Blaine Hollinger,

    1. Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030
    2. Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030
    3. Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
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  • Christine A. Noonan,

    1. Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030
    2. Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030
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  • Harvey Solomon,

    1. Department of Surgery, Baylor University Medical Center, Dallas, Texas 75246
    Current affiliation:
    1. Department of General and Transplant Surgery, St. Louis University Medical Center, 3635 Vista Ave., P.O. Box 15250, St. Louis, MO 63110
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  • Goran B. G. Klintmalm,

    1. Department of Surgery, Baylor University Medical Center, Dallas, Texas 75246
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  • Robert M. Genta,

    1. Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030
    2. Department of Pathology, Baylor College of Medicine, Houston, Texas 77030
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  • Boris Yoffe M.D.

    Corresponding author
    1. Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030
    2. Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030
    3. Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
    • Veterans Affairs Medical Center, Research Building 109, Room 139, 2002 Holcombe Blvd., Houston, TX 77030
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Abstract

To gain new insights into the pathogenesis of hepatitis B virus–induced chronic liver disease, we have used nonisotopic in situ detection methods for the simultaneous analysis of hepatitis B virus DNA and antigens at the single-cell level. Paraffin-embedded liver specimens from 23 cirrhotic patients (12 HBsAg positive and 11 HBsAg negative) who underwent liver transplantation were evaluated by in situ hybridization with a digoxigenin-labeled DNA probe and digoxigenin detection system and by immunohis-tochemistry with an enhanced biotin-streptavidin technique. DNAs extracted from liver and serum specimens were analyzed by Southern- and slot-blot hybridization, respectively. Using the in situ techniques, we detected hepatitis B virus–specific DNA and antigens in 11 of 12 HBsAg-positive patients and in none of the 11 HBsAg-negative individuals. Replicative intermediates of hepatitis B virus DNA were detected by Southern-blot analysis in the same 11 HBsAg-positive patients, 6 of whom had no serological markers of hepatitis B virus replication. Therefore a good correlation was found between the results obtained by the in situ and Southern-blot hybridization analyses of tissue specimens. However, a lack of correlation was found between serum- and tissue-associated markers of viral replication. In addition, the simultaneous in situ detection analyses revealed that some hepatocytes containing high levels of viral DNA were devoid of detectable HBcAg, suggesting a mechanism by which the virus may escape immunological surveillance. These data provide evidence that liver-associated HBV replication may persist in the absence of serological markers of active hepatitis B virus replication in cirrhotic patients with advanced liver disease and demonstrate that the evaluation of liver- rather than serum-associated markers of viral replication provides a more accurate assessment of the virological events occurring in HBsAg-positive individuals. (HEPATOLOGY 1993;18:1032-1038).

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