Isolation and characterization of a fibrogenic factor from CCl(4)-damaged rat liver.

A fibrogenic factor which stimulates collagen production without cell proliferation of rat skin fibroblast cultures was isolated from CCl4-damaged rat liver. (1) The factor was isolated from saline extracts of CCl4-induced fibrotic rat liver and fractionated by Sephadex G-50S gel filtration and DEAE-cellulose chromatography. The original extract produced a 6-fold increase in collagen synthesis and the active factor eluted from gel filtration columns in a region corresponding to 5000 daltons. (2) The active factor was destroyed by heat (57 degrees C, 30 min), phospholipase C digestion, but was insensitive to proteolytic enzymes or phospholipase A. Chemical analysis of the partially purified factor revealed relatively high quantities of phosphorus (3%) and low quantities of protein (13.3%), neutral sugar (1.9%) and uronic acid (4.9%). The possibility of this component being a complex phospholipid containing polypeptide is suggested. (3) Fibrogenic properties of the isolated factor was enhanced by apparent oxidation in air, to a more active, yet insoluble complex. Attempts to solubilize the oxidized product completely destroyed its biological activity.

Procollagen production by rat hepatocytes in primary culture.

Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3-5 days and declined thereafter. The rate of collagen production was approx. one-tenth that observed by rat skin fibroblasts of the same animals after 3-5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as alpha 1(I) and alpha2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are not major collagenous products of these cells.

HCV RNA monitoring for tailored regimens of interferon therapy in hepatitis C.

Sustained non-viraemia is prerequisite for a virological cure of hepatitis C virus (HCV) disease. We monitored serum HCV RNA in our patients during interferon therapy and for 6 months of follow-up. The rate of sustained responders (RNA-negative at the end of follow-up) differed significantly by whether or not RNA became sero-negative within 24 weeks of the initiation of therapy (71% vs 7%); and also by whether or not the RNA-seronegativity lasted for 12 weeks during therapy (88% vs 11%). Thus, by monitoring viraemia in individual patients, we can tailor the duration of therapy and minimize waste of interferon.

Hepatitis C virus (HCV) genotype 1b sequences from fifteen patients with hepatocellular carcinoma: the 'progression score' revisited.

The genome of hepatitis C virus (HCV) associated with hepatocellular carcinoma (HCC) may have some characteristics which would barely be found in those of HCV from asymptomatic carriers (ASC). We analyzed 15 HCC patients who were infected with HCV genotype 1b (HCV-1b) for complete nucleotide sequences of the viral genomes. Of the 15 isolates, three were sequenced up to the first nucleotide of the 5'UTR, and six were sequenced to encompass the X-tail at the 3' end: sequencing of at least three-quarters of the 5'UTR and entire polyprotein-ORF was accomplished in all 15 isolates. Analyses of these sequences together with those reported previously by Nagayama et al. [Hepatology; 31 (2000) 745] suggested that nine residues (nt 119 of 5'UTR and aa 90, 434, 938, 962, 1176, 1412, 2143, and 2774 of polyprotein) might be useful to discriminate HCC-type sequences from ASC-type ones. The 'progression score' was 1.4+/-0.9 in ASC versus 3.7+/-1.5 in HCC (P=3.87E-07) when calculated with the Nagayama et al.'s seven residues, but was 1.4+/-0.6 versus 4.6+/-1.9 (P=1.33E-09) with our nine residues: a greater difference between HCC and ASC was achieved in the latter system. Further analyses, by increasing the sample size and/or by extending the comparison to include entire 5'UTR and 3'UTR/X-tail, may thus contribute to define the 'progression score' more appropriately.

[Assay of serum collagen markers in chronic liver diseases and liver cancer].

In order to identify the most useful marker to diagnose hepatic fibrosis, type III procollagen peptide (P-III-P), laminin P1, and prolyl-hydroxylase (PH) in sera obtained from patients with liver diseases were simultaneously measured and compared with histological features of chronic hepatitis and with tumor size estimated on abdominal CT scan. Further more, the diagnostic accuracy of these markers was evaluated by using discriminant analysis. P-III-P and laminin P1 were closely correlated with the activity of chronic hepatitis. These two markers most accurately discriminated between the compensated stage of liver cirrhosis and the decompensated stage, and between liver cirrhosis and hepatocellular carcinoma. Laminin P1 was found to most clearly distinguish chronic hepatitis from liver cirrhosis. P-III-P was significantly correlated with the size of hepatocellular carcinoma. However, PH failed to discriminate among liver diseases, and showed no significant correlation with a liver tumor size. And none of the markers examined were correlated with a degree of hepatic fibrosis. From these results, the analysis of both serum P-III-P and laminin P1 is a useful approach to evaluate hepatic collagen metabolism.

Localization of type III procollagen aminopeptide antigenicity in hepatocytes from cirrhotic human liver.

Immunolocalization of type III procollagen (pro III) in normal and cirrhotic human liver was studied using rabbit antiserum specific for bovine type III procollagen aminopeptide. The material examined was deparaffinized, trypsin-treated hepatic tissue sections from 28 autopsy cases, including 19 cirrhotic and 9 normal liver donors. Immunostaining, performed by the unlabeled peroxidase-antiperoxidase antibody technique demonstrated that extracellular matrices corresponding to perisinusoidal reticulin, collagen in periportal areas, and blood vessel walls were the common sites of pro III antigenicity in both normal and cirrhotic liver. Moreover, in the cirrhotic liver, the fibrous septa of pseudolobules, and cytoplasm of hepatocytes and sinusoidal cells were positive when stained for pro III peptide. The differential counts of pro III positive cells in cirrhotic liver, however, revealed that the average ratio of these hepatocytes to sinusoidal cells was 25 to 1, indicating complete dominance of hepatocytes with respect to stainability for pro III peptide compared to sinusoidal cells. In hepatocellular carcinomas coexisting with cirrhosis, neoplastic cells also displayed pro III antigenicity. These data suggest that hepatocytes of cirrhotic liver and hepatocellular carcinoma cells play a significant role in type III collagen synthesis in vivo.

High concentrations of N-terminal peptide of type III procollagen in the sera of patients with various cancers, with special reference to liver cancer.

The concentrations of N-terminal peptide of type III procollagen in the sera of patients with various cancers were measured by radioimmunoassay. The mean value (with standard deviation) in the control group was 9.9 +/- 2.6 ng/ml. Serum levels exceeding 15 ng/ml were defined as positive, and it was found that 94% of 18 patients with primary liver cancer with cirrhosis, 88% of 8 patients with primary liver cancer without cirrhosis, 77% of 13 patients with metastatic liver cancer, 86% of 7 patients with recurrent breast cancer, 86% of 8 patients with colonic cancer, 75% of 8 patients with pancreatic cancer, 70% of 23 patients with stomach cancer, 51% of 35 patients with lung cancer, and 54% of 28 patients with uterine cancer showed positive levels. The concentrations showed great intersubject variations, probably reflecting the activity of tumor growth and/or invasion. The concentrations in the sera of patients with primary liver cancer with cirrhosis were generally higher than those in patients with liver cirrhosis alone or primary liver cancer without cirrhosis. This result suggested that the growth of primary liver cancer complicated by cirrhosis might be detected by serial measurements of this peptide in the serum of patients with liver cirrhosis. Present data suggested that this peptide is not cancer-specific, but assay of the peptide might be of value as an auxiliary means of detecting and monitoring various cancers, especially liver cancer.

Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan.

We identified hepatitis E virus (HEV) RNA in serum from a Japanese patient with acute hepatitis, who had never been abroad. The full-genome nucleotide sequence of the HEV isolate (JRA1) from this patient was composed of 7227 nucleotides excepting the poly(A) tail and had ORF1 coding for 1703 amino acids (aa), ORF2 coding for 660 aa, and ORF3 coding for 122 aa. This Japanese strain showed approximately 87% nucleotide similarity to human and swine strains reported from the United States, while it had only 73-76% similarity to Asian and Mexican strains. Here we report the characteristics of the HEV-JRA1 isolate, which might be the first example of an indigenous strain(s) of HEV in Japan.

Anti-fibrotic effect of malotilate on liver fibrosis induced by carbon tetrachloride in rats.

The effect of malotilate on liver fibrosis of rat induced by carbon tetrachloride (CCl4) was studied. CCl4 was given subcutaneously (1.5 ml/kg) to male Wistar rats twice a week for 11 weeks. Administration of malotilate (100 mg/kg) 5 times a week was started in the 5th experimental week and continued thereafter. The biochemical parameters in serum such as concentration of total protein and transaminase activities were improved by malotilate administration. The livers treated with CCl4 only were atrophic and markedly nodular, and histologically, severe collagen deposition and pseudolobular formation were observed. However, the livers treated with CCl4 and malotilate showed only slight accumulation of collagen fibers although hypertrophic and fatty metamorphosis was observed. Immunocytochemically, type I and type III collagens were stained in livers from rats treated with CCl4 only and rats treated with CCl4 and malotilate, but stainability was rather weak in the latter. The increased amount of hydroxyproline in the liver and that excreted into urine were markedly suppressed by malotilate administration. The levels of protocollagen prolyl hydroxylase activity in the liver were almost the same in CCl4-treated and CCl4 and malotilate-treated rats. However, the levels of collagenolytic enzyme activity were slightly higher in the latter. From these results, anti-fibrotic action of malotilate was clear and involvement of enhanced collagenolytic enzyme activity was suggested.

Clearance of serum hepatitis C virus RNA after interferon therapy in relation to virus genotype.

The effect of recombinant interferon-alfa on serum HCV RNA levels in Japanese patients with chronic hepatitis C was investigated. At 24 weeks of treatment, 41 (32.5%) of 126 patients lost HCV RNA from serum, and aminotransferases were normalized in 31 (75.6%) of these 41 cases. HCV genotypes were categorized into four types (Type I, II, III, IV); the frequencies among the patients were: Type I: 0%, Type II: 70.6%, Type III: 20.6%, and Type IV: 6.3%. At the end of the 24-week treatment, HCV RNA levels were remarkably decreased in Type III patients and became undetectable in 18 (69.2%) of 26. In contrast, only 18 (20.2%) of 89 patients with Type II and two of eight with Type IV lost HCV RNA from sera. The relation between HCV genotype (Type III) and response to IFN therapy was also confirmed using a logistic regression model. HCV genotype seems to be an important factor in determining the response to IFN in patients with chronic hepatitis C.