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Laboratory diagnosis of hepatitis B virus infection by nucleic acid hybridization analyses and immunohistologic detection of gene products.

The laboratory diagnosis of hepatitis B virus (HBV) infection is based on two types of analyses: (i) Immunochemical detection in serum or liver of viral antigens or their respective antibodies; (ii) Demonstration of HBV DNA in serum or liver by nucleic acid hybridization techniques (dot and transfer blots, in situ hybridization). These hybridization analyses have proven to be more sensitive than immunochemical assays for the detection of viral markers and have revealed HBV DNA in serum and liver of patients without serologic evidence of HBV infection. With appropriate controls, probes and hybridization conditions the hybridization assays are exquisitely specific. They are particularly useful in studying the molecular biology of the virus, its extrachromosomal replication or integration into the cellular genome and transformation of the infected hepatocytes. Further, recent studies have identified new target cells for HBV in liver (bile duct epithelial cells, endothelial cells, smooth muscle cells in blood vessel walls), spleen, bone marrow and white blood cells. While serological and immunohistochemical assays are still the basis of routine laboratory diagnosis of HBV infection, nucleic acid hybridization provides novel information useful in establishing the etiology of certain cases of viral hepatitis without serologic evidence for known viral agents (so-called non-A, non-B hepatitis), for demonstrating active viral replication in infected individuals, and for selecting and monitoring patients undergoing antiviral therapy.

DNA, Viral↗

Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.

Animals↗

Hepatitis B viral nucleotide sequences in non-A, non-B or hepatitis B virus-related chronic liver disease.

The presence of serological markers of hepatitis B virus (HBV) infection and of hepatocellular HBV DNA were investigated in 19 HBsAg-negative patients with clinically and histologically significant chronic liver disease. Four cases negative for antibodies to HBsAg (anti-HBs), to the core antigen (anti-HBc), and to the e antigen (anti-HBe) were classified as non-A, non-B hepatitis. The remainder, positive for one or more of the three antibodies, were classified as hepatitis B. Histologic diagnosis was chronic active hepatitis in five, chronic persistent hepatitis in 11, micronodular cirrhosis in two, and fatty liver in one patient. The DNA extracted from limited amounts of liver biopsies, without cleavage by restriction endonucleases, was analyzed by the Southern blot technique for the presence of episomal HBV DNA. Autoradiographs showed a single band of less than 4.0 kilobase (kb) corresponding to the monomeric form of HBV DNA in five patients, several bands of larger forms (4.0 to 18.0 kb) in three patients, both the monomeric and the larger forms in eight patients, and no HBV DNA in three patients. While HBV DNA was detected in the hepatocellular DNA of six patients who underwent splenectomy, hybridization was negative with the DNA extracted from their spleens. The episomal viral DNA larger than 4.0 kb may represent concatemeric forms or free oligomers which could not be distinguished from rearranged and/or integrated viral DNA in the limited analyses of the hepatocellular DNA hydrolyzed with HindIII or EcoRI. Our observations suggest the presence of HBV-like agents in the liver of serologically HBsAg-negative patients with chronic liver disease.

Adolescent↗