Southern-blot analysis and simultaneous in situ detection of hepatitis B virus-associated DNA and antigens in patients with end-stage liver disease.
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 immunohistochemistry 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Han
K H
KH
Veterans Affairs Medical Center, Houston, Texas 77030.
Hollinger
F B
FB
Noonan
C A
CA
Solomon
H
H
Klintmalm
G B
GB
Genta
R M
RM
Yoffe
B
B
D015139:Blotting, Southern / Q000379:methods; D002908:Chronic Disease; D004279:DNA, Viral / Q000032:analysis*; D006509:Hepatitis B / Q000150:complications / Q000276:immunology / Q000382:microbiology*; D006511:Hepatitis B Antigens / Q000032:analysis*; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods; D008099:Liver / Q000737:chemistry / Q000276:immunology / Q000382:microbiology; D008107:Liver Diseases / Q000209:etiology / Q000382:microbiology*; D014779:Virus Replication
D004279:DNA, Viral; D006511:Hepatitis B Antigens