bromochloroacetic-acid and Hepatitis-C--Chronic

Keratin proteins form intermediate filaments, which provide structural support for many tissues. Multiple keratin family members are reported to be associated with the progression of liver disease of multiple etiologies. For example, keratin 23 (KRT23) was reported as a stress-inducible protein, whose expression levels correlate with the severity of liver disease. Hepatitis C virus (HCV) is a human pathogen that causes chronic liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. However, a link between KRT23 and hepatitis C virus (HCV) infection has not been reported previously. In this study, we investigated KRT23 mRNA levels in datasets from liver biopsies of chronic hepatitis C (CHC) patients and in primary human hepatocytes experimentally infected with HCV, in addition to hepatoma cells. Interestingly, in each of these specimens, we observed an HCV-dependent increase of mRNA levels. Importantly, the KRT23 protein levels in patient plasma decreased upon viral clearance. Ectopic expression of KRT23 enhanced HCV infection; however, CRIPSPR/Cas9-mediated knockout did not show altered replication efficiency. Taken together, our study identifies KRT23 as a novel, virus-induced host-factor for hepatitis C virus.

Topics: Carcinoma, Hepatocellular; Cell Line; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Hepatocytes; Host-Derived Cellular Factors; Host-Pathogen Interactions; Humans; Keratins; Keratins, Type I; Liver; Liver Cirrhosis; Liver Neoplasms; RNA, Messenger; Transcriptome; Virus Replication

We describe the histopathologic features of 2 cases of biliary neoplasia with extensive intraductal spread arising in liver cirrhosis. The prevalence of this type of biliary neoplasia may be 0.4% from the review of 468 cases of cirrhotic liver. Histologic analysis revealed that the micropapillary proliferation of the atypical biliary epithelium composed of columnar cells with enlarged nuclei diffusely extended superficially from the septal intrahepatic bile duct to the reactive ductules associated with liver cirrhosis. Both cases exhibited prominent fibrous or sclerotic stroma near the biliary lesion. Immunohistochemical analysis revealed a characteristic cytokeratin and mucin expression pattern (CK7++, CK19++, CK20+, MUC1+/-, MUC2-, MUC5AC+, MUC6-). The tumor cytoplasm was focally positive for laminin gamma2 together with linear staining of the basement membrane. Proliferative activity confirmed by Ki67 staining was relatively high. Both patients were disease-free for 3 years after the operation. We believe that the possibility of biliary neoplasia with extensive intraductal spread should be considered to be a variant of biliary intraepithelial neoplasia.

Topics: Bile Ducts, Intrahepatic; Biliary Tract Neoplasms; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Count; Cell Proliferation; Cholangiocarcinoma; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Keratins; Liver Cirrhosis, Alcoholic; Liver Transplantation; Male; Middle Aged; Mucins; Neoplasms, Multiple Primary; Treatment Outcome

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other.

Topics: Adult; Age Factors; Analysis of Variance; Cohort Studies; Disease Progression; Female; Gene Expression Regulation; Genetic Markers; Genetic Variation; Hepatitis C, Chronic; Humans; Keratins; Liver Cirrhosis; Liver Function Tests; Logistic Models; Male; Middle Aged; Probability; Prognosis; Retrospective Studies; Risk Assessment; Severity of Illness Index; Sex Factors

Apoptosis is implicated as a major pathogenic mechanism in chronic hepatitis B and C. Previous studies of the relationship between apoptotic rates and histological necroinflammatory activity have produced conflicting results. Hepatocyte apoptosis was assessed in liver tissue from 32 cases of chronic viral hepatitis, seven cases of hepatocellular carcinoma (HCC) and six cases of steatohepatitis as non-viral disease controls and eight cases of control liver. Apoptotic rates were measured using H&E morphological assessment and immunohistochemical staining with antibodies to activated caspase-3 and M30. Histological necroinflammatory activity of viral hepatitis cases was scored using the Knodell scoring system, and the cases were divided according to their score into group 1 (mean 2.43 +/- 0.48) and group 2 (mean 7.80 +/- 0.49). Apoptotic indices were significantly higher in group 2 than group 1 using H&E (11.53 +/- 2.70 vs. 0 +/- 0, P=0.015) and activated caspase-3 (22.01 +/- 5.27 vs. 1.79 +/- 1.79, P=0.03) methods but were not significantly higher with M30 (3.80 +/- 1.74 vs. 0 +/- 0, P=0.207). Apoptotic scores using an antibody to activated caspase-3 are significantly higher in cases of chronic viral hepatitis with greater histological necroinflammatory scores, supporting a central role for apoptosis in disease pathogenesis. This method offers an alternative to routine histological assessment for measuring disease activity.

Topics: Antibodies, Monoclonal; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspases; Fatty Liver; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Keratins; Liver Neoplasms; Retrospective Studies

In patients with chronic hepatitis C, alanine aminotransferase (ALT) levels do not accurately reflect the extent of liver inflammation. The discrepancy between ALT level and liver damage could be related to the mode of cell death. In the present study, we quantified serum levels of apoptotic cytokeratin 18 (CK-18) neoepitopes that are generated by activated caspases during apoptosis. Apoptotic CK-18 neoepitopes were quantified by enzyme linked immunosorbent assay in sera from patients with chronic hepatitis C and elevated ALT levels (n = 72), patients with chronic hepatitis C and persistently normal ALT levels (n = 27) and healthy controls (n = 19). Serum CK-18 neoepitope levels were strongly correlated with ALT (r = 0.659, P < 0.0001) and the histology activity index (r = 0.374, P < 0.001). Patients with chronic hepatitis C and persistently normal ALT levels had higher apoptotic CK-18 neoepitope levels than healthy controls (P = 0.03) but lower levels than patients with chronic hepatitis C and elevated ALT levels (P < 0.001). Highest serum CK-18 neoepitope levels were observed in patients with cirrhosis (P = 0.002). Hence apoptotic CK-18 neoepitopes in serum of patients with chronic hepatitis C are associated with ALT level and histological liver damage. Serum apoptotic CK-18 neoepitope levels are elevated both in patients with chronic hepatitis C and elevated ALT levels as well as in patients with normal ALT levels indicating that also patients with chronic hepatitis C and normal ALT have an increased hepatocyte loss by apoptosis.

Topics: Adult; Aged; Alanine Transaminase; Apoptosis; Biomarkers; Biopsy, Needle; Case-Control Studies; Chi-Square Distribution; Disease Progression; Female; Hepatitis C, Chronic; Humans; Immunohistochemistry; Keratins; Liver Function Tests; Male; Middle Aged; Probability; Prognosis; Risk Assessment; Sensitivity and Specificity; Severity of Illness Index; Statistics, Nonparametric

Oval cell proliferation is known to occur in experimental models of hepatic regeneration and carcinogenesis. Recent studies have suggested that activation of progenitor cells, representing the human counterpart of oval cells, may play a role in hepatic diseases. Therefore, we evaluated putative progenitor cells in chronic viral hepatitis.. Forty-one needle liver biopsy specimens from patients with chronic hepatitis B and 43 specimens from patients with chronic hepatitis C were examined histologically. The grade (histological activity index (HAI)) and stage (degree of fibrosis) were determined on routinely stained sections. The number of progenitor cells was assessed semiquantitatively on cytokeratin 7- (CK 7-) stained sections.. In both aetiological categories of chronic viral hepatitis, progenitor cell numbers were found to increase in parallel to the HAI, as well as to the stage of disease. Features suggestive of hepatocytic differentiation of progenitor cells were also noted on immunohistochemical stains for CK 7 and 'hepatocyte-specific' antigen.. In chronic hepatitis B and chronic hepatitis C, progenitor cell activation is correlated with the grade and stage of disease. Proliferating progenitor cells may play a role in hepatic regeneration occurring in this setting.

Topics: Cell Differentiation; Fibrosis; Hepatitis B, Chronic; Hepatitis C, Chronic; Hepatocytes; Humans; Immunohistochemistry; In Vitro Techniques; Keratin-7; Keratins; Liver; Stem Cells

Keratin 8 and 18 (K8/18) phosphorylation plays a significant and site-specific role in regulating keratin filament organization, association with binding proteins, and modulation of cell cycle progression. Keratin hyperphosphorylation correlates with exposure to a variety of stresses in cultured cells and in mouse models of liver, pancreatic, and gallbladder injury, and it is found in association with mouse and human Mallory bodies. We asked whether K8/18 phosphorylation correlates with human liver disease progression by analyzing liver explants and biopsies of patients with chronic noncirrhotic hepatitis C virus (HCV) or cirrhosis. We also examined the effect of HCV therapy with interleukin-10 on keratin phosphorylation. Using site-specific antiphosphokeratin antibodies we found keratin hyperphosphorylation on most K8/18 sites in all cirrhotic liver explants tested and in most liver biopsies from patients with chronic HCV infection. Immunofluorescence staining of precirrhotic HCV livers showed focal keratin hyperphosphorylation and limited reorganization of keratin filament networks. In cirrhotic livers, keratin hyperphosphorylation occurred preferentially in hepatic nodule cells adjacent to bridging fibrosis and associated with increased stress kinase activation and apoptosis. Histological and serological improvement after interleukin-10 therapy was accompanied by normalization of keratin hyperphosphorylation on some sites in 7 of 10 patients. In conclusion, site-specific keratin phosphorylation in liver disease is a progression marker when increased and a likely regression marker when decreased.

Topics: Biomarkers; Case-Control Studies; Disease Progression; Hepatitis C, Chronic; Hepatocytes; Humans; Interleukin-10; Keratin-18; Keratin-8; Keratins; Liver Cirrhosis; Mutation; Phosphorylation

Chronic hepatitis C virus (HCV) infection is characterized by inflammatory liver damage and is associated with a high risk of development of cirrhosis and hepatocellular carcinoma. Although histological examination of liver biopsies is currently the gold standard for the detection of early liver damage, there is a strong need for better noninvasive methods. We recently demonstrated that the proapoptotic activation of caspases is considerably enhanced in histological sections from HCV-infected liver tissue, suggesting an important role of apoptosis in liver damage. Here, we investigated whether caspase activation is detectable also in sera from patients with chronic HCV infection. Using a novel enzyme-linked immunosorbent assay that selectively recognizes a proteolytic neoepitope of the caspase substrate cytokeratin-18, we demonstrate that caspase activity is markedly increased in the sera of HCV patients. Interestingly, while 27% of patients with chronic HCV infection showed normal aminotransferase levels despite inflammatory and fibrotic liver damage, more than 50% of those patients exhibited already elevated serum caspase activity. Moreover, 30% of patients with normal aminotransferase but elevated caspase activity revealed higher stages of fibrosis. In conclusion, compared with conventional surrogate markers such as aminotransferases, detection of caspase activity in serum might be a more sensitive method of detecting early liver injury. Thus, measurement of caspase activity might provide a novel diagnostic tool, especially for patients with normal aminotransferases but otherwise undiagnosed histologically active hepatitis and progressive fibrosis.

Topics: Adult; Aged; Apoptosis; Biopsy; Case-Control Studies; Caspases; Cohort Studies; Enzyme Activation; Female; Hepatitis C, Chronic; Humans; Keratins; Liver; Liver Cirrhosis; Male; Middle Aged; Transaminases

Progenitor cell activation with subsequent maturation to hepatocytes and cells of the biliary lineage has been demonstrated in a variety of chronic liver diseases but the kinetics and magnitude of the progenitor cell response has not been adequately studied in detail in chronic hepatitis. We undertook this study to evaluate factors responsible for the progenitor cell/ductular response and further dissect the role of disease grade and stage as determinants of hepatocellular differentiation of bipotential progenitor cells in chronic hepatitis.. Cytokeratin 7 (and 19) stained biopsies from patients with chronic hepatitis C (n = 47), hepatitis B (n = 20), and autoimmune hepatitis (n = 20) were studied. Ploidy analysis and proliferation indices were evaluated in a subset of cases.. Ductular reactions were present in the majority of cases (97%), appeared early in disease, and correlated with disease activity, while progenitor cell derived hepatocyes appeared later in disease and their extent correlated with disease stage. Proliferation indices of all cell types correlated with disease activity.. Progenitor cell derived hepatocytes accrue in chronic hepatitis, possibly related to native hepatocellular dysfunction. However, the fate of these hepatocytes is unclear.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bile Ducts; Cell Division; Child; Child, Preschool; DNA; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; Hepatitis, Autoimmune; Hepatocytes; Humans; Immunohistochemistry; Infant; Keratin-7; Keratins; Liver; Liver Regeneration; Male; Middle Aged; S Phase; Stem Cells

Chronic hepatitis C virus (HCV) has been linked to extrahepatic autoimmune phenomena. In addition a variety of autoantibodies were found in patients with HCV. This study was performed to assesss the clinical relevance of antikeratin antibodies in rheumatoid arthritis (RA) and in patients with symmetric polyarthritis associated with hepatitis C infection. Serum antikeratin antibodies were evaluated in 3 different groups of patients; all were rheumatoid factor (RF) seropositive: Group 1: 31 patients with HCV associated symmetric polyarthralgia or arthritis. Group 2: 28 patients with RA (modified ACR criteria for probable RA). Group 3: 16 patients with autoimmune disorders other than RA. Seventeen healthy individuals matched for age and sex served as controls. In our study, 75 patients who were rheumatoid factor positive (measured by ELISA, the cutoff was established to 20 U/mL) were tested for antikeratin antibodies using an indirect immunofluorescence technique with 1:10 serum dilution. Antikeratin antibodies were detected in 18/28 (64%) patients with true RA and only 3/31 (9%) patients with HCV-related arthritis (p < 0.0001). Antikeratin antibodies were observed in 3/16 (18%) patients of group 3 (p < 0.05). Antikeratin antibodies were not found in the sera of the healthy controls.

Topics: Arthritis; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Case-Control Studies; Female; Fluorescent Antibody Technique, Indirect; Hepatitis C, Chronic; Humans; Immunoglobulin G; Keratins; Male; Rheumatoid Factor

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F(1)-ATP synthase beta-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.

Topics: Adult; Aged; Antibody Specificity; Autoantibodies; Calcium-Binding Proteins; Calreticulin; Carcinoma, Hepatocellular; Case-Control Studies; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Keratins; Liver Neoplasms; Male; Middle Aged; Nucleoside-Diphosphate Kinase; Proteome; Proton-Translocating ATPases; Ribonucleoproteins

Cytokeratin 19 fragment (CK19) levels in serum have already been documented as a useful tumour marker for lung cancer. In the present study, we hypothesize that CK19 may be increased in serum from patients with hepatoma.. We measured the CK19 levels in serum from patients with hepatoma and evaluated the correlation between CK19 level and each clinical parameter. We studied 70 patients diagnosed with hepatoma, and used 14 patients with chronic hepatitis C and 45 patients with liver cirrhosis as controls.. In 33 of 70 patients (47.1%) with hepatoma, the serum CK19 level was elevated to above the normal range. CK19 levels in serum from patients with hepatoma were significantly correlated with levels of alpha-fetoprotein and prothrombin induced by vitamin K absence for factor II (PIVKA-II). In 57 patients with hepatoma in whom both CK19 and alpha-fetoprotein were measured, only CK19 was elevated in seven patients (12.3%). Immunohistochemical studies using hepatoma tissues demonstrated that hepatoma cells were stained by anti-human CK19 antibody. We also demonstrated that the HepG2 cell line expressed CK1 9.. Our data demonstrate that hepatomas aberrantly express CK19, and that measurement of CK19 might be a useful tumour marker in diagnosing hepatoma.

Topics: Adult; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Biopsy; Carcinoma, Hepatocellular; Female; Hepatitis C, Chronic; Humans; Immunohistochemistry; Keratins; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Tumor Cells, Cultured

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease.

Topics: Cell Count; Disease Progression; Glutathione Transferase; Hemochromatosis; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Keratins; Leukocyte Common Antigens; Liver; Liver Diseases, Alcoholic; Pyruvate Kinase; Stem Cells