bromochloroacetic-acid and Hepatitis-B

Pooled tongue coating samples from 64 hepatitis B patients and 24 healthy adults were studied and a major band of differential proteins was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The differential proteins in this band were identified and proved to be keratins by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and Western blot analysis. Furthermore, relative quantification of the identified keratins was performed via using stable isotopic labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), showing the higher expression level of these keratins in tongue coating samples of hepatitis B patients than healthy adults. These results provided additional information to understand the medical diagnosis depending on the tongue coating.

Topics: Adult; Female; Hepatitis B; Humans; Keratins; Male; Molecular Weight; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tongue

Liver fibrosis (LF) is the accumulation of extracellular matrix (ECM) proteins due to chronic liver injury. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to perform a comparative analysis of cytosolic and nuclear protein patterns of nontransgenic (NTg) and HBV transgenic (Tg) mice livers at early stages of fibrosis. We identified several candidate proteins, involved in a variety of pathways, which could be used as putative biomarkers for LF early detection.

Topics: Animals; Biomarkers; Cell Nucleus; Cytosol; Electrophoresis, Gel, Two-Dimensional; Hepatitis B; Keratins; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nuclear Proteins; Phosphorylation; Proteome; Superoxide Dismutase

It is still not clear whether oval cells demonstrate diverse morphology, immunophenotype or quantity in different human liver diseases. The aim of this study was to investigate these differences in hepatitis B virus (HBV)-positive and hepatitis C virus (HCV)-positive human liver cirrhosis (HLC).. Thirty-eight cases of HBV+ HLC and 32 cases of HCV+ HLC were investigated by light microscopy and immunohistochemistry for Hepatocyte, CK19, stem cell factor (SCF) and CD34. Five cases were also examined by transmission electron microscopy. Oval cells of similar morphology could be found in proliferating bile ductules in both groups. These cells coexpressed CK19 and Hepatocyte, but did not express SCF or CD34. Some of these cells exhibited a trend towards differentiation. There was no difference in the amount of oval cells between the two groups. The oval cell number was found to increase significantly with the progression of inflammation. A similar stem-like cell was not seen in the normal liver.. There are bipotential oval cells in both HBV+ and HCV+ HLC. The lack of difference in oval cells between the two groups suggests that they might play a similar biological role in the histogenesis of different liver diseases.

Topics: Adult; Aged; Antigens, CD34; DNA, Viral; Female; Hepacivirus; Hepatitis B; Hepatitis B virus; Hepatitis C; Hepatocytes; Humans; Immunohistochemistry; Keratins; Liver; Liver Cirrhosis; Male; Microscopy, Electron, Transmission; Middle Aged; RNA, Viral; Stem Cell Factor

To investigate whether cells with features similar to those of the oval cells of rodents and the small epithelial cells (SEC) recently described in certain human liver diseases, i.e. hepatic progenitor cells, also occur in human liver cirrhosis.. Surgical specimens from 35 cases of hepatitis B virus-positive cirrhosis (30 cases containing hepatocellular carcinoma) were investigated by immunohistochemical staining for cytokeratin 7 and albumin. Electron microscopic investigations, and immunoelectron microscopic investigations using the same antibodies and a double-labelling technique were performed in 15 and seven cases, respectively. SEC were observed in proliferated bile ductules, at the margins of regenerating nodules and in the fibrous septa in all cases of cirrhosis. The SEC were morphologically similar to the SEC described previously, and to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, scanty electron-dense or electron-lucent cytoplasm, a high nucleo-cytoplasmic ratio, tonofilaments and intercellular junctions. Immunoelectron microscopy revealed that the SEC co-expressed cytokeratin 7 and albumin. Both relatively undifferentiated SEC and SEC with morphological and immunophenotypical signs of differentiation towards biliary epithelial cells and hepatocytes were found.. SEC that exhibit morphological and immunophenotypical features of the SEC seen in certain other liver diseases are found in cirrhosis. These findings further support the hypothesis that a bipotent hepatic stem cell that may give rise to biliary epithelial cells and hepatocytes exists in the human liver.

Topics: Albumins; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Fluorescent Antibody Technique, Indirect; Hepatitis B; Hepatocytes; Humans; Keratin-7; Keratins; Liver Cirrhosis; Liver Neoplasms; Microscopy, Immunoelectron; Stem Cells

The clinicopathologic findings in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) positive for biliary markers, those related to the hepatic progenitor cells, were investigated. Cytokeratin (CK) 19 was reactive for HCCs only in patients with prior hepatitis B virus (HBV) infection. The proportions of patients with prior HBV infection and poorly differentiated HCC were significantly higher among those with CK 19-positive HCC than among those with CK 19-negative HCC. Some HCCs develop from the hepatic progenitor cells in patients with HCV infection and prior HBV infection, which may affect the clinicopathologic findings of HCV-related HCCs.

Topics: Aged; Bile Ducts; Carcinoma, Hepatocellular; Epithelial Cells; Female; Hepatitis B; Hepatitis B Antigens; Hepatitis B virus; Hepatitis C; Humans; Immunoenzyme Techniques; Keratins; Liver Neoplasms; Male; Middle Aged; RNA, Viral; Survival Rate

To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms.. Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied.. Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10).. Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Topics: Albumins; alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Division; Chromosomes; Clone Cells; Flow Cytometry; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Keratins; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Tumor Cells, Cultured; Virus Integration

Transactivation of cellular genes and functional inactivation of p53 by the hepatitis B virus (HBV) X gene-encoded protein (HBx) are proposed as alternative mechanisms for induction of hepatocellular carcinomas (HCCs) in chronic HBV infection. Using an immunohistochemical approach, we studied the expression of HBx in 39 explanted livers with HBV-associated disease. Because the data reported previously have been inconsistent, possibly due to the application of different antibodies, we compared results with 5 polyclonal and 6 monoclonal anti-HBx antibodies from five laboratories. Ten of the 11 antibodies reacted with recombinant HBx by Western blotting, but only 1 polyclonal and 2 monoclonal antibodies reacted specifically with HBx in tissue, and were thus suitable for immunohistochemistry. Three other polyclonal antibodies reacted with tissue components in addition to HBx. One polyclonal and 4 monoclonal antibodies did not recognize the HBx in the tissue. HBx was demonstrated in 16 of 30 (53.3%) cirrhotic livers and 10 of 18 (58.8%) HCCs by all specific antibodies. The expression of HBx, among three HBV antigens examined, was found to be preferentially maintained in HCC and the surrounding liver parenchyma, including focal or nodular preneoplastic lesions. However, the immunoreactivity was always limited to the cytoplasm of a small number of parenchymal and neoplastic cells. The role of X gene expression in HBV-associated human hepatocarcinogenesis remains to be established.

Topics: Animals; Carcinoma, Hepatocellular; Hepatitis B; Humans; Immunohistochemistry; Keratins; Liver; Liver Neoplasms; Rabbits; Trans-Activators; Viral Regulatory and Accessory Proteins

Hepatocytes of normal adult liver express cytokeratins (CKs) 8/18, but bile duct cells additionally contain CK7/19. We have previously demonstrated the frequent occurrence of foci of altered hepatocytes in association with hepatic tumors in humans and provided evidence for a preneoplastic nature of the focal lesions. In this study, we investigated the CK expression in both the preneoplastic lesions and extrafocal parenchyma. Sixty-seven explanted livers with cirrhosis or advanced fibrosis harboring preneoplastic focal lesions, with or without hepatitis B virus (HBV) infection, as well as 9 livers with HBV-associated fulminant hepatitis, were studied for the expression of CK7/8/14/18/19. Five livers from woodchucks infected with the woodchuck hepatitis virus (WHV) were also investigated. Glycogenotic clear hepatocytes were negative or weakly positive for CK8/18, while amphophilic hepatocytes were strongly positive for these CKs, the changes being associated with marked reduction and increase, respectively, of highly organized membranous components in their cytoplasm. This allows the distinct recognition of the clear-cell and clear-cell-dominant preneoplastic lesions in the human and woodchuck livers. In ground-glass hepatocytes expressing viral antigens, an unusual accumulation of CK8/18 was observed, but there was no evidence of preferential necrosis of ground-glass hepatocytes. Many CK7- and CK19-positive ductular (oval) cells were found in extrafocal liver tissue, but only rarely were they present within focal lesions.

Topics: Adult; Animals; Chronic Disease; Glycogen; Hepadnaviridae Infections; Hepatitis B; Humans; Immunohistochemistry; Keratins; Liver; Marmota; Microscopy, Electron; Precancerous Conditions

Chronic infection of woodchucks with woodchuck hepatitis virus (WHV) invariably leads, within 2-4 years, to the appearance of hepatocellular carcinoma (HCC). HCC is preceded by an extended period of chronic liver damage, probably resulting from the immune response to viral antigens. It may be that infection itself also induces changes in the hepatocyte population. To begin to identify some of the changes in the liver prior to the appearance of HCC, monoclonal antibodies (MAbs) were generated from mice immunized with hepatocytes from a woodchuck chronically infected with WHV or with a tumor lysate. Immunofluorescence microscopy was used to select MAbs that reacted with host markers whose patterns of expression would distinguish chronically infected from uninfected liver or from liver tumors. One of these MAbs (2F2) reacted strongly with a subset of hepatocytes in chronically infected liver; a similar staining pattern was not detected in uninfected or transiently infected liver. Evidence is presented that this strong staining reaction reflects the overexpression or accumulation of the hepatocyte-specific intermediate filament protein, cytokeratin K18, a protein previously implicated in cryptogenic cirrhosis of the liver in humans (Ku, N. O. , Wright, T. L., Terrault, N. A., Gish, R., and Omary, M. B. J. Clin. Invest. 99: 19-23, 1997). Double immunofluorescent staining with antibodies to K18 and M-envelope protein of WHV suggested that strong reactivity to K18 was limited to cells expressing high levels of one or both of the large viral-envelope proteins, M and L; however, high expression of these viral proteins was not always associated with a strong K18 staining reaction.

Topics: Animals; Antibodies, Monoclonal; Chronic Disease; Female; Hepatitis B; Hepatitis B Virus, Woodchuck; Immunohistochemistry; Keratins; Liver; Mice; Mice, Inbred BALB C

All interferons display antiviral properties, but gamma-interferon especially has an immunomodulatory effect and may induce autoimmune phenomena. Therefore the formation of autoantibodies was investigated in patients with chronic hepatitis B treated with gamma-interferon. Eleven patients (all HBs-Ag and HBe-Ag positive) were treated for 6 months with recombinant gamma-interferon. The following antibodies were tested: anti-nuclear antibodies, smooth muscle antibodies, anti-actin, anti-mitochondrial antibodies of subgroup anti-M2 and anti-M9 as well as naturally occurring antibodies, antibodies to liver-kidney microsomes, vascular endothelial cell antibodies, sarcolemmal antibodies, parietal cell antibodies, thyroglobulin antibodies and antibodies to laminin and keratin. All patients produced autoantibodies during therapy. The maximum antibody formation and the highest titres were observed in the period between the 3rd and 6th month after therapy began. The cumulative frequencies of the different antibody specificities were as follows: n = 6 anti-nuclear antibodies, n = 7 smooth muscle antibodies, n = 1 anti-actin, n = 12 antibodies to laminin or keratin, n = 6 endothelial cell antibodies/sarcolemmal antibodies, n = 6 anti-mitochondrial antibodies, n = 1 antibodies to liver-kidney microsomes, n = 2 thyroglobulin antibodies, n = 4 parietal cell antibodies. Antibodies persisted in six patients over a period of 3 months (two cases of parietal cell antibodies and one case of antibodies to liver-kidney microsomes) and were still detectable in three patients 6 months after therapy. In three patients new antibody formation occurred 1 month after therapy. So far, clinical signs of an autoimmune disorder have not appeared in any of the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Adolescent; Adult; Antibodies; Antibodies, Antinuclear; Antibody Formation; Antibody Specificity; Autoimmunity; Chronic Disease; Endothelium, Vascular; Female; Hepatitis B; Humans; Interferon-gamma; Keratins; Laminin; Male; Microsomes, Liver; Middle Aged; Mitochondria; Muscle, Smooth

Proliferation of a new population of epithelial cells with distinct structure, as well as cytokeratin and alpha-fetoprotein expression, was observed in nonneoplastic liver tissues from 14 cases (13 hepatitis B virus-positive) of human hepatocellular carcinoma. These cells were characterized by oval nuclei; scant, pale cytoplasm; small cell size; and cross-reaction with a monoclonal antibody against rat oval cells. These putative human oval cells were strongly positive for cytokeratin 19 and displayed considerable heterogeneity in alpha-fetoprotein and albumin expression. The oval cells were most prominent in actively regenerating nodules and in liver tissue surrounding the cancer. Oval cells and transitional types of cells appear to be the principal producers of alpha-fetoprotein in the regenerating liver. Cancer cells positive for cytokeratins 8, 18 and 19 were observed in half the hepatocellular carcinomas studied. The data suggest that a new cell population structurally similar to oval cells seen in early stages of chemical hepatocarcinogenesis in rats is consistently present in regenerating liver lesions associated with human hepatocellular carcinoma. Furthermore, it is possible that the proliferation of these oval-type cells may partly account for the elevation of serum alpha-fetoprotein frequently seen in precancerous stages of hepatitis B virus-associated human hepatocellular carcinoma.

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Division; Female; Hepatitis B; Humans; In Situ Hybridization; Keratins; Liver; Liver Neoplasms; Male; Middle Aged; Serum Albumin

An ABC immunohistochemical study of the expression of cytokeratin (CK19 and CK18) was carried out in 315 cases of HBV-caused hepatitis, early-cirrhotic and cirrhotic livers. It was shown that hepatocytes in 73% of chronic active hepatitis (CAH) (80/110) and 81% of early-cirrhotic and cirrhotic livers (117/144) expressed CK19 (the abnormal CK expression), which could be of help in differentiating CAH from chronic persistent hepatitis, subtype CAH (mild, moderate to severe type) and in determining the activity of early-cirrhotic and cirrhotic livers. The abnormal CK expression was shown to be closely related to the activity of liver disorders. The CK19 expression in hepatocytes had the closest relations with the piece-meal necrosis of hepatocytes, isolation of hepatocytes into groups by connective tissue, and fibrosis. It is suggested that CK19 expression may be one of the local reactions to the piece-meal necrosis of hepatocytes.

Topics: Chronic Disease; Hepatitis B; Hepatitis, Chronic; Humans; Immunohistochemistry; Keratins; Liver; Liver Cirrhosis

64 cases of chronic active hepatitis (CAH) and 84 cases of non-CAH liver diseases were studied with keratin stain. The capillary-like bile ductules (CLBD) were proliferating and their morphology was identical to that with Type V collagen stain reported before. CLBD proliferation were more marked in CAH than in other liver diseases, and it was considered to be one of the characteristics of CAH and could be used for differential diagnosis. The ultrastructure of CLBD was specific in morphology. The HBV-DNA in CLBD shown by the technique of in situ hybridization suggested that HBV might infect the cells of CLBD.

Topics: Bile Canaliculi; Hepatitis B; Hepatitis B Core Antigens; Hepatitis B Surface Antigens; Hepatitis, Chronic; Humans; Keratins; Liver