OBJECTIVES: DNA polymerase (pol) of Hepatitis B virus (HBV) includes 3 different domains such as terminal protein (TP), reverse transcriptase (RT) and RNase H. Humoral immune responses to each of these proteins have not been well documented previously, although antibody to pol was detected in serum of patients with chronic hepatitis B. We have constructed TP (amino acids 1-182), RT (amino acids 346-685) and RNase H (amino acids 690-832). METHODS: By ELISA using each protein expressed in E. coli as antigens, the corresponding antibodies were tested in serum from 40 patients with type B viral chronic liver diseases. (20 HBeAg-positive and 20 HBeAg-negative). As negative controls, sera from 3 healthy young men were used. With the mean values of the OD, which were tested 4 times per each test sample and 3 times per each control sample, we considered to be positive if the mean OD of each test sample is 2-fold or higher than that of controls. RESULTS: Five of 40 sera (12.5%) contained one or two different antibodies detectable by this method: 4 of 20 HbeAg-positive sera (20%) and 1 of 20 HbeAg-negative sera (5%). Anti-TP, anti-RT and anti-RNase H antibodies were detected in 2.5% (1/40), 10% (4/40) and 7.5% (3/40), respectively. Among 4/20 HbeAg-positive ELISA-positive sera, anti-TP, anti-RT and anti-RNase H were positive in 5% (1/20), 20% (4/20) and 10% (2/20), respectively, while 1 HBeAg-negative ELISA-positive sera were positive only for anti-RNase H. CONCLUSIONS: These results suggest that the corresponding antibody responses to individual recombinant peptides derived from 3 domains of DNA polymerase may tend to be detected more frequently in HBeAg-positive sera than in HBeAg-negative sera from various patients with type B viral chronic liver diseases.
Adult ; Antibodies, Antinuclear/analysis* ; Biological Markers/analysis ; DNA Polymerase II/immunology* ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Surface Antigens/analysis* ; Hepatitis B Virus/immunology* ; Hepatitis B, Chronic/immunology* ; Human ; Male ; Middle Age ; Odds Ratio ; Reference Values ; Substances: DNA Polymerase II ; Substances: Hepatitis B Surface Antigens ; Substances: Biological Markers ; Substances: Antibodies, Antinuclear

BACKGROUND/AIMS: Oxidative stress is known to play a role in the pathogenesis of a certain liver diseases such as alcoholic liver disease, metal storage disease, and ischemia/reperfusion injury. Recently oxidative stress(lipid peroxidation) has also been implicated in hepatic fibrosis, which is now regarded as a common response to chronic liver injury regardless of its nature. Development of fibrosis and cirrhosis are the major complications of chronic hepatitits B. So we aimed to detect lipid peroxidation in chronic hepatitis B and to investigate its potential role in the pathophysiology of the disease. METHODS: The subjects were histologically-proven 56 patients, including fatty liver(FL, n=8), healthy HBsAg carrier(n=6), chronic persistent hepatitis(CPH, n=8), mild chronic active hepatitis(CAH-m, n=10), severe CAH(CAH-s, n=16), and liver cirrhosis(LC, n=8). All patients were serologically HBsAg-positive except those with FL. Lipid peroxidation was detected in serum and liver specimen with TBARS(thiobarbituric acid-reacting substances) assay. Western blot and immunohistochemical stain of liver specimen were also performed, using polyclonal antibody against malondialdehyde (MDA). RESULTS: 1. There were no significant differences in serum TBARS levels among groups(p= 0.24). 2. The mean tissue TBARS level(nmol/g) was significantly higher in CAH-s group(175.4+ 41.5) than in other groups(FL 54.0+ 6.4, Carrier 51.1+ 15.9, CPH 63.9+ 2.9, CAH-m 68.9+ 7.9, LC 22.6+ 5.1) (p<0.05). 3. Tissue TBARS levels correlated with serum ALT levels(r=0.5934, p<0.05). 4. Western blot showed MDA bands only in CAH-s group. 5. Immunohistochemistry showed a strong MDA stain around portal and periportal area in CAH-s group, but weak or no stain in other groups. CONCLUSIONS: This study shows that lipid peroxidation can be detected in situ and commonly occurs in severe chronic hepatitis B. Oxidative stress may be related to active necroinflammatory change of the liver and contribute to the progression of the disease in chronic hepatitis B.
Blotting, Western ; Fibrosis ; Hepatitis B Surface Antigens ; Hepatitis B, Chronic ; Humans ; Immunohistochemistry ; Lipid Peroxidation* ; Liver Diseases* ; Liver Diseases, Alcoholic ; Liver* ; Malondialdehyde ; Oxidative Stress ; Thiobarbituric Acid Reactive Substances