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ABSTRACT: To study the early stages of cell death in various types of chronic liver injury, liver biopsies from a total of 26 patients, including 7 with chronic hepatitis C(CHC), 4 with chronic hepatitis B(CHB), 7 with alcoholic liver disease (ALD), 4 with autoimmune or drug hepatitis (AI/DH), and 4 with primary biliary cirrhosis(PBC), were examined by an in situ nucleotidyl transferase assay (ISNTA), which detects DNA fragmentation. Positive nuclei in hepatocytes and sinusoidal lining cells were counted in all parenchymal areas, excluding triads and areas of fibrosis, using a computer with Sigmascan software. The number of positive hepatocytes/mm2 was similar in the biopsies of patients with CHC, CHB, ALD and AI/DH, but significantly lower in PBC. The number of positive sinusoidal lining cells/mm2 was significantly greater in biopsies with CHC compared to CHB, ALD, AI/DH and PBC. Double staining revealed that the ISNTA-positive sinusoidal lining cells were also CD68 positive, indicating that they were Kupffer cells. The frequency of ISNTA positivity did not correlate with serum AST or ALT levels, steatosis, cell swelling or cirrhosis. ISNTA-positive hepatocytes were more frequent than acidophilic bodies in every disease category. We conclude that apoptosis may be a common pathway of cell death in different liver diseases, that the high frequency of DNA fragmentation in Kupffer cells in CHC suggests that during chronic hepatitis C infection activated Kupffer cells may be subject to regulatory control by apoptosis and that ISNTA is more sensitive than acidophilic bodies in assessing the degree of cell injury in the liver.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 10/1997; 431(3):189-94. · 2.49 Impact Factor
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ABSTRACT: Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease that includes the presence of lymphoid infiltrates in portal tracts, high titer autoantibodies against pyruvate dehydrogenase-E2 (PDH-E2) and branched chain ketoacid dehydrogenase-E2 (BCKD-E2), and biliary tract destruction. The mechanism by which the autoimmune response is induced, the specificity of damage to the biliary epithelium, and the role of T cells in PBC are still unknown. To address these issues, we have taken advantage of a mouse mAb, coined C355.1, and studied its reactivity against a panel of liver tissue from normal subjects as well as a panel of liver specimens from patients with PBC, progressive sclerosing cholangitis, and chronic active hepatitis (CAH). C355.1, much like human autoantibodies to PDH-E2, reacts exclusively by immunoblotting with PDH-E2, binds to the inner lipoyl domain of the protein, and inhibits PDH-E2 activity in vitro. In addition, we have also attempted to develop cloned T cell lines that react with PDH-E2 and/or BCKD-E2 using liver biopsies from patients with PBC, compared with CAH. Although monoclonal C355.1 produced typical mitochondrial fluorescence on sections of normal liver, pancreas, lung, heart, thyroid, and kidney, it produced a distinct and intense reactivity when used to stain the bile ducts of patients with PBC. Nine of 13 PBC liver biopsies studied herein contained bile ducts on light microscopy, all of which reacted intensely at a 1:100 culture supernatant dilution of monoclonal C355.1. In contrast, although bile ducts of liver specimens from normals, CAH, and progressive sclerosing cholangitis also reacted with C355.1, such reactivity was exclusively mitochondrial and readily detectable only at a dilution of 1:2. More importantly, we generated CD4+, CD8-, alpha beta TCR+ cloned T cell lines from patients with PBC, but not from CAH, that produced IL-2 specifically in response to PDH-E2 or BCKD-E2.
The Journal of Immunology 02/1991; 146(1):89-94. · 5.79 Impact Factor
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ABSTRACT: Antimitochondrial antibodies, serological hallmarks of primary biliary cirrhosis, recently were found to be directed against the E2 subunits of mitochondrial dehydrogenase complexes (pyruvate, branched-chain ketoacid, and alpha-ketoglutarate dehydrogenases). The objectives of this study were to extend these findings and to determine whether purified immunoglobulin from the sera of patients with primary biliary cirrhosis inhibit activity of these dehydrogenase complexes in vitro. Sera were examined from 14 patients with primary biliary cirrhosis (13 mitochondrial antibody positive), 23 with rheumatic diseases and 30 with chronic active hepatitis (all 53 positive for mitochondrial antibodies by indirect immunofluorescence), 10 with alcoholic liver disease, and 5 normal controls. Antibodies against pyruvate dehydrogenase, branched-chain alpha-ketoacid dehydrogenase and alpha-ketoglutarate dehydrogenase complexes were detected by immunoblot and quantified by enzyme-linked immunosorbent assay. Of the 14 serum samples obtained from patients with primary biliary cirrhosis, 13, 11, and 2 samples tested positive by immunoblot for the E2 subunits of pyruvate, branched-chain ketoacid, and alpha-ketoglutarate dehydrogenase, respectively. In contrast, samples from subjects with rheumatic diseases, chronic active hepatitis, and alcoholic liver disease and control subjects tested negative for these antibodies. Serum immunoglobulin G with high titers of mitochondrial antibodies showed concentration-dependent inhibition of activity of the dehydrogenase complexes, and close correlation (r = 0.917, n = 13) was observed between inhibitory activity against pyruvate dehydrogenase complex and the reciprocal titer of immunoglobulin against this complex. These data suggest that such autoantibodies, besides serving as diagnostic markers for primary biliary cirrhosis, may have a pathogenic role by their ability to inhibit important mitochondrial enzymes.
Gastroenterology 08/1990; 99(1):187-94. · 11.68 Impact Factor
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ABSTRACT: Heme oxygenase, the rate controlling enzyme for heme catabolism, is inducible by a variety of treatments, some of which induce by a heme-dependent mechanism and others by a heme-independent mechanism. This work shows that, in cultured chick embryo liver cells, synergistic induction of heme oxygenase by iron, added with the phenobarbital-like drug, glutethimide was heme-dependent. Addition of an inhibitor of heme biosynthesis abolished the synergistic induction of heme oxygenase providing evidence for the heme-dependent mechanism of induction. Glutethimide and iron appeared to induce at the transcriptional level since both heme oxygenase mRNA and protein levels correlate with changes in heme oxygenase activity.
Biochemical and Biophysical Research Communications 05/1990; 168(1):176-81. · 2.48 Impact Factor
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ABSTRACT: This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.
Biochemical Journal 03/1989; 258(1):237-45. · 4.90 Impact Factor
