Mutations in the non-structural protein 5A gene in patients with chronic hepatitis C virus 1b infection during repeated interferon treatment.

It has been previously reported that the non-structural region 5A (NS5A) of the hepatitis C virus (HCV) includes an interferon sensitivity determining region (ISDR) and that amino acid substitutions in this region are closely associated with the response to interferon (IFN) treatment. We assessed the clinical significance of serial changes of amino acid sequences in the ISDR during repeated IFN treatment in patients with chronic hepatitis C (genotype 1b), related to serum HCV RNA load. During treatment, additional amino acid substitutions in the ISDR were observed in four of eight patients (50% 2/5 of complete responders (CR); 2/3 of non-responders (NR). However, comparing these amino acid substitutions to wild-type ISDR, the number of amino acid mutations was limited to only one amino acid identified in two CRs. The virus load changed regardless of the amino acid substitutions in the ISDR during treatment, and the wild-type and intermediate type (with less than three amino acid substitutions) showed wide variations in virus load. These data indicate that amino acid mutations in the ISDR, which indicate the switch to mutant-type do not occur easily even during repeated IFN treatment, and the additional amino acid substitutions in the ISDR are not a sensitive marker during repeated IFN treatment. In cases where virus load is used as a marker of response to repeated UN treatment, serial examinations are necessary to determine the precise virus load levels.

Amino acid mutations in the interferon sensitivity determining region and serum virus load in hepatitis C virus carriers with long-term normal ALT levels.

The amino acid mutations in a part of the non-structural region 5A (NS5A) of the hepatitis C virus (HCV) genome, called the interferon sensitivity determining region (ISDR), can affect the response to interferon (IFN) treatment. We analyzed the serial changes of the amino acid substitutions in the ISDR during the natural course of patients with sustained long-term normal alanine aminotransferase (ALT) levels in relation to the changes in virus load, and assessed the clinical significance of ISDR in the natural course and IFN treatment. The subjects were nine patients infected with HCV (genotype 1b) who had been examined for serum ALT levels every month for more than 1 year and had well-sustained normal levels. The amino acid sequence of the ISDR was determined by the direct sequencing method, and the number of amino acid mutations was evaluated in comparison with the prototype (HCV-J). Quantitation of serum HCV RNA levels was conducted by the Amplicor-monitor method (Nihon Roche). On the initial analysis of the ISDR, six patients were determined to have no mutations, and three patients had one or two mutations. However, an increased number in amino acid mutations compared with the wild type during the follow-up period was confirmed in only one patient, and that increase was limited to within two amino acids. Virus load changed regardless of the changes in amino acid substitutions in the ISDR. The ISDR was therefore inferred to be a stable region unrelated to the virus load in patients with well-sustained normal ALT levels. Additional changes of amino acid sequence in this region were not a sensitive marker for determining whether IFN treatment is indicated.

Hepatitis viral infection in Behçet's disease.

To elucidate the role of hepatitis viruses in the pathogenesis of Behçet's disease (BD), we measured hepatitis viral markers (anti-hepatitis A (anti-HA), HBsAg, anti-HBs, anti-HBc) and viral nucleic acids (hepatitis B virus (HBV)-DNA, hepatitis C virus (HCV)-RNA, GB virus C (GBV-C)-RNA, TT virus (TTV)-DNA) in the sera of 68 BD patients along with 76 blood donors matched for age and sex. Positivity of anti-HA in patients with BD (36.8%) was lower than that in blood donors (68.0%). Both anti-HCV and HCV-RNA were detected in only one (1.5%) patient with BD and in none of the blood donors. The prevalence ratios of HBsAg, anti-HBs, anti-HBc in both groups were similar (2.9:0, 16.2:15.8 and 17.7:19.7%, respectively). However, serum HBV-DNA was detected more frequently in BD patients (8/68; 11.8%) than in blood donors (2/76; 2.6%) (P<0.05). The prevalence of GBV-C-RNA was also higher in patients with BD (4/68; 5.9%) compared with blood donors (0%). However, characteristics and clinical features are similar between GBV-C-RNA-positive and -negative groups. With respect to the prevalence of TTV-DNA, there was no significant difference between BD patients (23.5%) and blood donors (30.3%). Our study indicates that HBV and GBV-C infection might be related to BD, although the role of these viruses remains to be investigated.

Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus.

The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475-1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/deltaX transcript promoter, mapped to nucleotides 1525-1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567-1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743-1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625-1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696-1685, 1781-1766, and 1801-1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene.

A combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than is ursodeoxycholic acid monotherapy.

Inhibitors of 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase have been reported to decrease the cholesterol saturation index (CSI) in duodenal bile in humans and to prevent formation of cholesterol gallstones in animal studies. We performed a prospective study to evaluate the role of HMG-CoA reductase inhibitors as gallstone-dissolving agents. Fifty patients with radiolucent gallstones in a gallbladder opacifying at drip infusion cholecystography were treated with either 10 mg/day simvastatin plus 600 mg/day ursodeoxycholic acid (group 1, n=26) or 600 mg/day ursodeoxycholic acid alone (group 2, n=24) for 12 months. The ratio of solitary to multiple gallstone cases was 21:29. Plasma lipid levels were assessed and ultrasonographic examination of the gallbladder was performed at baseline and at 3-month intervals during treatment. Duodenal bile sampling was performed in five patients in each group at baseline and after 12 months of treatment. Plasma cholesterol decreased significantly in group 1 but not in group 2. In solitary gallstone cases, no significant difference in dissolution rates was observed between groups 1 (3 of 9, 33%) and 2 (4 of 12, 33%). In contrast, the dissolution rate in multiple gallstone cases was significantly higher in group 1 (12 of 17, 71%) than in group 2 (3 of 12, 25%) (p < 0.01). Bile cholesterol saturation index was significantly decreased (p < 0.01) but did not significantly differ between the two groups. These results suggest that combination therapy with simvastatin and ursodeoxycholic acid is more effective for cholesterol gallstone dissolution than ursodeoxycholic acid monotherapy in patients with multiple gallstones.

Analysis of the X gene promoter of woodchuck hepatitis virus.

During the course of woodchuck hepatitis virus (WHV) replication three virus-specific mRNA transcripts that encode four essential proteins are produced. The transcripts are 3.6, 2.3, and 0.7 kb in size. The 3.6-kb transcript serves as the replicative intermediate as well as the template for translation of the nucleocapsid and polymerase proteins. The 2.3-kb mRNA serves as the template for translation of the virus envelope proteins. Both the 3.6- and 2.3-kb transcripts are polyadenylated and are readily found in the cytoplasm of infected hepatocytes. However, the 0.7-kb transcript, specific for the X gene, accumulates in the nucleus of infected cells and is polyadenylated poorly in hepatocytes. Thus, while it is likely that the 0.7-kb transcript is the template for translation of the X protein, it is possible that it also has a function at the RNA level to regulate virus replication or gene expression. In order to characterize the WHV X promoter we cloned the region of the WHV8 genome encompassing the viral enhancer through the amino terminus of the X gene into the vector pSV0CAT. We transfected Huh-7 and WLC-3 cells with the WHV X promoter construct, along with a plasmid encoding human growth hormone to control for transfection efficiency, and assayed for the presence of chloramphenicol acetyl transferase activity. We found that the WHV X promoter was about one-half as active as the well-studied simian virus 40 early or Rous sarcoma virus promoters. Next, we made a series of 5' and 3' deletion mutants and mapped the WHV X promoter to a 21-nucleotide domain (1482-GGGGAAGCTGACGTCCTTTCC-1502) which is approximately 100 bp downstream of the corresponding promoter in hepatitis B virus. Further analysis, using oligonucleotide-directed mutagenesis, demonstrated that the essential nucleotides comprising the WHV X promoter are located in a 10-nucleotide domain near the initiation codon of the X gene. Mutation of either nucleotide T at position 1490 or G at position 1491 within this domain was sufficient to reduce the level of promoter activity by 100-fold. Thus, we have defined the important nucleotides within the promoter of the WHV X transcript which is a first step in understanding the role of this transcript in WHV replication and gene expression.

Humoral inhibitor of rat hepatocyte DNA synthesis from patients with fulminant liver failure.

Sera and ultrafiltrates (relative molecular mass < 10,000 Da) from patients with fulminant liver failure inhibit hepatocyte DNA synthesis in vivo. In this study the effects of ultrafiltrates from pooled sera from fulminant liver failure patients in the United Kingdom and plasma ultrafiltrates from fulminant liver failure patients in Japan have been investigated in primary cultured rat hepatocytes, with incubation for up to 72 hr. Both types of ultrafiltrate inhibited the incorporation of [3H]thymidine into acid-precipitable material and reduced the cell labeling index as determined on autoradiography in hepatocytes stimulated by epidermal growth factor and insulin compared with normal sera/plasma ultrafiltrates. The inhibitory effects observed were dose dependent, reversible when the fulminant liver failure ultrafiltrate was removed and were not associated with increased release of lactate dehydrogenase or suppression of protein synthesis as assessed on the basis of the incorporation of [3H]leucine. The effects appeared to be specific for hepatocytes; in preliminary experiments DNA synthesis was not inhibited in cultured fibroblasts (NIH 3T3 cells). These experiments are further evidence of the presence of an inhibitory factor of relative molecular mass less than 10,000 Da in the blood of patients with fulminant liver failure.

A simple method for evaluating prothrombin time in severe liver disease.

A measurement of prothrombin time by a new, whole blood capillary system was evaluated for use in severe liver disease, such as fulminant and chronic hepatic failure. The measurement required a single drop of fresh, whole blood and was easily performed at bedside. Results were available within 5 minutes after collection of the blood samples. Good correlation was observed between prothrombin time values determined by the rapid method and those determined with the laboratory method (r = .89). The laboratory method was used as a reference. The whole blood system may be especially helpful in emergency situations, when central laboratory services are not available.

Prognostic value of hepatic volumetry in fulminant hepatic failure.

Serial hepatic volumetry calculated from the liver area on abdominal computed tomography was performed in 19 patients with fulminant hepatic failure to determine a relationship between liver volume and prognosis. All patients received intensified artificial liver support comprised of plasma exchange and hemodiafiltration using high-performance membranes, and 10 patients survived. Liver volume was significantly larger in survivors than in nonsurvivors, both in an initial volumetry performed at the onset of coma and in subsequent volumetry performed 10-20 days after the onset of coma. The difference became more significant in the subsequent volumetry because of the recovery of liver size in some of the survivors and progressive liver shrinkage in all nonsurvivors. All patients with a liver volume greater than 656 ml at 10-20 days after the onset of coma survived, whereas all but one patient with a liver volume less than that died. Multivariate analysis revealed only liver volume in subsequent volumetry had discriminatory power upon prognosis among six prognostic factors. These observations imply that in order to obtain an accurate prediction of fulminant hepatic failure by hepatic volumetry, serial studies at least until 10-20 days after the onset of coma are necessary.

[Per oral gallstone dissolution therapy].

In this paper the author describe present status and problems of Per oral cholesterol gallstone dissolution therapy by bile acid. 1) Both ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) decrease the cholesterol output in bile, but their action mechanism somewhat differs. 2) Clinical results of UDCA treatment in our hospital during past 17 years are as follows. Complete dissolution rate Approx. 20% Partial dissolution rate Approx. 20% 3) In the attempt to increase the complete dissolution rate, bedtime administration method (one time administration instead of 3 times, every day), combination therapy of UDCA and CDCA and UDCA plus simvastatin (HMG-CoA reductase inhibitor) were tried. The results of these trial were not superior compared with ordinary UDCA therapy (3 times after each meal administration). 4) The author concluded from the results that small size (< 1 cm in diameter), Ia in ultrasonographic classification, floating by oral cholecystography, and radiolucent gallstones are the most suited for bile acid dissolution therapy.

Development of reliable artificial liver support (ALS)--plasma exchange in combination with hemodiafiltration using high-performance membranes.

A new artificial liver support system (ALSS) consisting of plasma exchange (PE) in combination with hemodiafiltration (HDF) using high-performance membranes of polymethyl methacrylate (PMMA) and cellulose triacetate (CTA) was developed to efficiently remove middle molecules from plasma and treat fulminant hepatic failure (FHF) complicated by the onset of hepatic coma. Twenty-seven patients with FHF due to viral hepatitis, two with type A (HA), nine with type B (HB), and 16 with type non-A, non-B (NANB) underwent therapy with this new ALSS over the last five years. Three patients with an exacerbation of chronic HB and 15/16 with type NANB hepatitis were treated with interferon (IFN) also. Of these, 25 patients (92.6%) regained consciousness and 15 (55.6%) [1/2 (50%) with type A, 6/9 (66.7%) with type B and 8/16 (50%) with type NANB hepatitis] survived. Including four patients who survived with intensive care and plasma exchange alone, 19/31 (61.3%) patients survived. Because of its biocompatibility, both survivors and nonsurvivors could be sustained with the ALSS without complications for long periods (19.3 days for the survivors and 32.4 days for nonsurvivors). With this ALSS the ability to sustain life for such prolonged periods allows hepatic regeneration to occur and result in patient survival. It is anticipated that this new ALSS will not only be of value in cases of fulminant hepatic failure but that it may also play a role in sustaining life for those awaiting liver transplantation.

[Effects of intra-arterial infusion chemotherapy with radiation therapy for advanced hepatocellular carcinoma].

Eight cases were treated with periodic arterial infusion therapy from reservoir and four cases with arterial infusion therapy plus 30 Gy's radiation therapy for hepatocellular carcinoma (HCC). The anatomical extent of the tumor was E3 and E4, respectively. We evaluated these forms of therapy from the viewpoint of tumor characteristics, survival time and therapeutic effects. There was no effective case of life prolongation and most of the reservoir only treated cases died a short time after therapy. But no severe complication was observed in reservoir plus radiation cases. One case showed a response, and another case tumor necrosis. The results indicate that this method is effective for advanced HCC.

Reactivation of precore mutant hepatitis B virus leading to fulminant hepatic failure following cytotoxic treatment.

Three hepatitis B virus carriers who were HB(e)Ag negative and having normal liver function developed fulminant hepatitis with evidence of HBV replication following intensive chemotherapy for non-Hodgkin's lymphoma. Each was continuously negative for HB(e)Ag. Analysis of the precore region of HBV isolated from each demonstrated that the HBV of each had a point mutation in the precore region that inhibited the synthesis and the release of hepatitis B(e) antigen. This observation suggests that all HB carriers receiving either immunosuppressive or cytotoxic therapy should be monitored closely even if standard assays suggest that viral replication is not present. Sudden enhanced replication of a HBV mutant as a result of such therapy can be a cause of either very severe hepatitis or occasionally fulminant hepatitis.

[Relation between Helicobacter pylori and intractable gastric ulcer--PAS positive intramucosal mucus as an index].

Helicobacter pylori (Hp) is considered to be one of the causes of gastric mucosal injury. Using biopsy specimens from the gastric mucosa of patients with gastritis or gastric ulcer, the intramucosal mucus was quantified by computer image analysis to evaluate its relationship with Hp. In gastric mucosa positive for Hp, the mucus content within the gastric mucosa was significantly decreased. Ammonia was administered based on its assumed role in decreasing the mucus content of the gastric mucosa, and resulted in a decrease in rats to whom it was administered. Based on these results, cases of intractable gastric ulcer were studied. In these intractable cases, Hp was present significantly more often than in other cases and the intramucosal mucus content was significantly lower. These findings suggest that Hp may be a factor in the resistance of gastric ulcer to treatment.