Pathological changes and clinical manifestations of 1020 children with liver diseases confirmed by biopsy.

BACKGROUND: Liver biopsy plays an important role in accurate diagnosis of various liver diseases in children and liver damages caused by systemic illnesses. This study was designed to evaluate the value of liver biopsy in diagnosis of liver diseases in children and explore the relationship between their pathological changes and clinical manifestations. METHODS: One-second liver biopsy was performed in 1023 pediatric patients with liver diseases at our department from 1983 to 2000. Diagnosis of viral hepatitis was based on the diagnostic criteria formulated by the Chinese Society of Infectious and Parasitic Diseases in 1995. Inflammatory changes of the liver were graded from 0 to 4 (G0-4). RESULTS: Liver biopsy was performed successfully in 1020 patients including 135 infants and young children, of whom 90% were hospitalized patients with chronic liver diseases. Hepatitis virus was the leading cause for chronic liver diseases, among which hepatitis B was detected in 75.4% of the patients. Sixty-nine patients showed liver impairment induced by disorders relevant to that metabolism, Wilson's disease, and glycogen storage disease. Liver inflammatory injury (

Screening of augmenter of liver regeneration-binding proteins by yeast-two hybrid technique.

OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we used yeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR. METHODS: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformed into yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in a 2XYPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of the plasmid from blue colonies. Analysis was performed by bioinformatics. RESULTS: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. One colony is a new gene with unknown function. CONCLUSION: The successful cloning of gene of ALR interacting protein has paved the way for studying the physiological function of ALR and associated proteins.

Interaction between hepatitis C virus core protein and translin protein--a possible molecular mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus.

AIM: To investigate the interaction between hepatitis C virus core protein and translin protein and its role in the pathogenensis of hepatocellular carcinoma and lymphoma. METHODS: With the components of the yeast two hybrid system 3, "bait" plasmids of HCV core the gene was constructed. After proving that hepatitis C virus core protein could be firmly expressed in AH109 yeast strains, yeast two- hybrid screening was performed by mating AH109 with Y187 that transformed with liver cDNA library plasmids-pACT2 and then plated on quadruple dropout (QDO) medium and then assayed for alpha-gal activity. Sequencing analysis of the genes of library plasmids in yeast colonies that could grow on QDO with alpha-gal activity was performed. The interaction between HCV core protein and the protein we obtained from positive colony was further confirmed by repeating yeast two - hybrid analysis and coimmunoprecipitation in vitro. RESULTS: A gene from a positive colony was the gene of translin, a recombination hotspot binding protein. The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro. CONCLUSION: The core protein of HCV can interact with translin protein. This can partly explain the molecular mechanism for hepatocellular carcinoma and lymphoma caused by HCV.

[Cloning of genes transactivated by hepatitis B virus X protein].

OBJECTIVE: To construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function. METHODS: The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast. RESULTS: The subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function. CONCLUSION: A subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

[Cut-off period of subclassification and pathological features of severe hepatitis based on clinical and pathological analyses].

BACKGROUND: To explore the cut-off period of subclassification and pathological features of severe hepatitis (SH). METHODS: Based on combined clinical and pathological analyses, the complete clinical and biopsy or autopsy liver tissues data from 196 cases of patients with severe hepatitis were investigated. Meanwhile, proliferative hepatocytes, cholangioepithelia and collagens were identified by a panel of monoclonal antibodies such as those against albumin, cytokeratin 18,19 and collagen I, III with immunohistochemical method. RESULTS: The clinical and pathological analyses indicated the cut-off periods of acute, subacute and chronic SH (ASH,SSH and CSH) were (13.4+/-7.2) d, (77.4+/-69.3) d and (80.5+/-63.2) d, respectively. Among all SH cases, one case of ASH patient presented clinical manifestation and pathological changes of ASH for 21 days, however, one patient with SSH was demonstrated 12 day course by histological examination. The time of cut-off period between ASH and SSH in child cases was shorter than that in adult cases. Histologically, ASH liver tissues showed massive and/or submassive necrosis caused by one attack, with congestive sinusoid frameworks and proliferative cholangioepithelium-like hepatocytes, while SSH liver tissues presented combined fresh and old submassive or massive necrosis caused by multiple attacks, accompanied by obviously proliferative bile ducts and sinusoid framework collapse.However, the pathological changes of CSH showed ASH- or SSH-like lesions on the background of chronic liver injury. CONCLUSION: Our data indicated that the cut-off period between ASH and SSH is in accordance with the Scheme of Viral Hepatitis Prevention and Therapy, China, published in 2000, but excluded a part of child SH cases. In our study, the authors found a few pathological features in ASH and SSH.

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

AIM: To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry. METHODS: The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody. RESULTS: The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B. CONCLUSION: We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.

Antitumor activities of human autologous cytokine-induced killer (CIK) cells against hepatocellular carcinoma cells in vitro and in vivo.

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P<0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P<0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.