[A discussion on diagnosis and typing of viral hepatitis].

OBJECTIVE: To bring forward a suggestion about clinical diagnostic standard and clinical typing for chronic and severe hepatitis. METHODS: To make a comprehensive study on clinical features and pathology of 895 cases of severe and chronic hepatitis, liver cirrhosis after hepatitis based on the viral prevention and control plan laid down in 1995. RESULTS: The chronic hepatitis can still be divided into mild, moderate and severe types clinically, but the PTA should be changed for normol-71, 70-61, 60-51, the A/G value for normal, 1.5-1.3, 1.2-1.0 respectively. ALT, BIL, alpha-globulin are kept unchanged. The albumin value can be cut out from the reference indexes of clinical typing for chronic hepatitis. Acute severe hepatitis can be divided into early stage (taking edema as the main type) and late stage (taking necrosis as the main type); subacute severe hepatitis can be divided into ascite type, coma type and mixed type; if those lacking of coma and ascite with PTA about 60%. 50% can be treated as earlier stage. Subacute and chronic severe hepatitis still can be divided into early, middle and late stages. The disease course of subacute severe hepatitis may prolong to six months. Chronic severe hepatitis can be divided into type B (typical chronic hepatitis type) type C (liver cirrhosis type) and type c (acute liver failure type developed from chronic hepatitis and viral carriers). CONCLUSIONS: The original procedure of 1995 are feasible on the whole.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins.

AIM: To prepare hybridoma cell lines that secrete monoclonal antibodies against hepatitis C virus (HCV) recombinant proteins NS3 and NS5 and to evaluate their use in the study of HCV NS3 and NS5 antigen distribution in human liver tissue. METHODS: Hybridoma cell lines were generated using spleen cells from BALB/C mice immunized with recombinant NS3 and NS5 proteins, following conventional protocols. Antibody-secreting cells were screened by solid phase ELISA and cloned by limited dilution. The specificity of the monoclonal antibodies was determined by testing hybridoma culture supernatants by Western blots of E. coli expressing the recombinant HCV proteins and ELISA with HCV core and hepatitis B virus (HBV) antigens. The monoclonal antibodies were employed in immunohistochemistry studies to determine the distribution of HCV NS5 and NS3 antigens in 51 paraffin embedded human liver tissue samples. RESULTS: Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were generated and named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Only one of them, 2B6 (secreting antibodies against NS3 protein), cross-reacted with the C7 polypeptide, a different recombinant NS3 polypeptide. The rest of the cell lines showed no cross-reactivity with HCV core or HBV antigens. In addition, monoclonal antibodies against NS3 antigens did not cross-react with NS5 antigens, and vice versa. In immunohistochemistry studies, these monoclonal antibodies did not detect HCV antigens in specimens from patients infected only with HBV (n = 20). In HCV-infected specimens (n = 31), the rates of positive detection of NS3 and NS5 antigens were 51.6% (16/31) and 54.9% (17/31), respectively. Six of these 31 specimens were from patients infected only with HCV and half of them were positive for HCV NS3 and NS5 antigens. In specimens from patients co-infected with HBV and HCV (n = 25), the rates of NS3 and NS5 antigen positive detection were 52% (13/25) and 56% (14/25), respectively, which are similar to those obtained in samples from patients infected only with HCV. In specimens from chronic active cirrhosis patients, the rates of HCV NS3 and NS5 antigen detection were 70.6% (12/17) and 76.5% (13/17), respectively. CONCLUSION: We successfully prepared monoclonal antibodies that are specific against recombinant HCV NS3 and NS5 proteins and could be useful for clinical immunohistochemistry diagnosis.

[Detection of HBV DNA in extrahepatic tissue with PCR comparison with immunohistochemistry and in situ hybridization].

Formalin-fixed paraffin-embedded tissue, including gallbladder, kidney, spleen, adrenal gland, heart, testicle, pancreas, and liver from eighteen autopsied cases with HBV infection were studied with nested polymerase chain reaction (PCR) for detection of HBV DNA. The DNA sequence representing HBV infection was detected in the tissue of liver (100%), gallbladder (6 from 7, 85.7%), spleen (6 from 8, 75.0%), kidney (8 from 11, 72.7%) adrenal gland (4 from 6, 66.7%), heart (10 from 18, 55.6%), testicle (10 from 18, 55.6%), pancreas (6 from 11, 54.5%) respectively. The DNA sequence representing HBV replication was detected in 5 cases of liver tissue only. The findings of PCR was correlated with the result of immunohistochemistry and in situ hybridization. It is shown that HBV can infect extrahepatic tissue but do not replicate in it. We think these findings may explain that the harboring of hepatitis virus in extrahepatic tissue could serve as one of extrahepatic infective sources, but have little pathological consequence on the infected extrahepatic organs.

[Preparation of digoxigenin labelled probe and detection of HBV DNA in liver and extrahepatic tissue with in situ hybridization].

A new rapid technique for intrahepatic and extrahepatic HBV DNA detection by using digoxigenin (dig) labelled probe with in situ hybridization was developed. This technique has the advantage of being non-radioactive and a quick procedure yielding stable results and showing a clear background. 45 liver specimens were tested with this technique. Among the patients with positive intrahepatic HBsAg and HBcAg, positive detection of HBV DNA was highest (77.27%, 17/22). Some results were confirmed by PCR test. 19 extrahepatic specimens were detected with in situ hybridization. HBV DNA was seen clearly in the nuclei of myocardial cells, pancreatic islet cell, renal tubule epithelial cells and testicular spermatogenic cells. The results of this study might contribute to the study of molecular mechanism of HBV-induced injury in liver cells and extrahepatic tissue.

[Detection of HBV DNA in kidney by PCR].

Nested polymerase chain reaction (PCR) in two groups of primer was performed in formalin-fixed, paraffin-embedded kidney and liver tissues from 11 autopsies of HBV infected patients for HBV DNA detection. DNA sequence representing HBV infection was detected in all the 11 liver tissue specimens (100%) and in 8 kidney tissue specimens (72.7%). DNA sequence representing HBV replication was detected in only 5 liver tissue specimens. The PCR findings correspond with those obtained in immunohistochemistry studies and in situ hybridization, suggesting that HBV can infect the kidney but does not replicate in this organ and that the kidney pathology in HBV infected patients may be the result of immuno-intermediate injury from immunocomplex deposited in glomeruli.

[Distribution of hepatitis B virus in testicle tissue in patients with hepatitis B infection].

To study distribution of HBV in the testicle tissue, we used polyclonal antibody and ABC method in detecting autopsy testicle in 34 cases. The positive rate of HBsAg and HBcAg was 52.9% and 11.8% respectively. The positive cells of spermatogonium, primary spermatocytes, spermatid and sertoli cells were single or grouped in clusters form in the seminiferous tubule. Of the 6 cases studied with in situ hybridization, 2 had HBV DNA in spermatogenic cells in individual nucleus. The findings support morphologically the transmission of HBV via sexual contact.