Comparative Immunohistochemical Detection of Caspase-3 Activation in Liver Biopsy Specimens of Patients with Mono and Mixed Infection
Keywords:
Apoptosis, Biopsy, Caspase-3, Immunohistochemistry, Mixed infection, Tuberculosis, Viral hepatitisAbstract
Background: Apoptosis is a genetically programmed form of a cell death that plays a major role in pathogenesis of chronic liver diseases. As Caspase-3 activation is required to produce apoptotic chromatin condensation and DNA fragmentation it is used in the study of apoptosis.
Subjects and methods: Liver biopsies of patients with heroin abuse and coinfection of tuberculosis, chronic hepatitis C, human immunodeficiency virus (TB, HCV, HIV) were investigated. For comparison liver biopsies of patients with chronic hepatitis B (HBV) were used. All biopsies were performed according to routine medical program, using standard Mengini procedure. Material was fixed in formalin, embedded in paraffin and cut 7 micrometers in thickness. For immunohistochemical detection endogenous peroxidase activity was blocked with 3% hydrogen peroxide and the sections were stained using Immunohistochemistry kit according to the manufacture’s guidelines. Cleaved Caspase-3 (Asp175) served as primary antibody. Biotinylated secondary antibody and streptavidin-conjugated peroxidase were used for detection using DAB as chromogen. Nuclei were counter stained with Harris Hematoxylin. Negative controls did not contain primary antibodies. In addition to the immunohistochemical study of Caspase-3 numerical score was established for each biopsy specimen both for grading of necroinflammatory activity (Knodell et al.) and stage of fibrosis (French METAVIR).
Results: Our investigation showed that positive reaction of Caspase-3 strongly varies in different liver biopsies; activation is revealed in nuclei and cytoplasm of hepatocytes and some Kupffer cells as well as in endotheliocytes of sinusoids. In portal tracts the activation occurs in some endotheliocytes of interlobular portal veins, in cells of lymphohistiocyte infiltrates. In liver biopsies of patients with coinfection the Caspase-3 expression was noticed in 30-50% of nuclei, whereas in liver biopsies of patients with HBV mono infection the index did not exceed 10%. Index of histological activity according to Knodell of patients with coinfection reached 12-15 balls, fibrosis stage according to METAVIR – F2; in patients with HBV these indexes were respectively 4 balls and F-1.
Conclusions: Our study demonstrated the expression of Caspase-3 was significantly higher in cases of chronic hepatitis with more severe histological necroinflammatory activity. Especially high activation of Caspase-3 was in liver biopsies of patients with coinfection and heroin abuse. Our results suggest high level of apoptosis in liver biopsies of such types of patients.
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