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 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.

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.

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.

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.

[Biliary excretion and clinical efficacy of T-3262 (tosufloxacin tosilate) administered in the treatment of cholecystitis].

T-3262 (tosufloxacin tosilate), a new oral pyridone carboxylic acid agent, was investigated for its biliary excretion and clinical efficacy and safety to evaluate its usefulness in the treatment of cholecystitis. T-3262 was administered to a total of 4 healthy volunteers for 2 days at a dose of 150 mg every 8 hours, and A-, B- or C-bile were collected using the MELTZER-LYON method at 10-11 hours after the final administration. Bile concentrations of T-3262 in 3 cases were 0.33-2.05 micrograms/ml (A-bile), 6.13-9.50 micrograms/ml (B-bile) and 1.11-2.70 micrograms/ml (C-bile). Thus, T-3262 levels in B-bile were 15-34 times higher than serum levels (0.28-0.41 micrograms/ml). Only a trace of serum concentration of T-3262 was detected in another case with the concentration in B-bile was 0.132 micrograms/ml. A total of 10 patients with cholecystitis were treated with T-3262 at a dose level of 150 mg per dose 3 times daily for 1 to 20 days. The clinical efficacy was excellent in 1 case, good in 5 cases and fair in 2 cases and unevaluable in 2 cases, thus the clinical efficacy rate was 75%. Bacteriologically, Klebsiella pneumoniae, Enterococcus faecalis and Haemophilus parahaemolyticus were isolated from biles of 3 patients before treatment. Upon the treatment, E. faecalis was eradicated and K. pneumoniae was unchanged. The fate of H. parahaemolyticus was not known because of examination was not done after treatment. Side effects were observed in 2 cases with diarrhea in 1 case and epigastric pain in another case. But those symptoms disappeared after cessation of administration of T-3262. Abnormal laboratory test values were not observed.

[Clinical evaluation of endoscopic therapy for early gastric cancer by diathermic polypectomy, Nd; YAG laser irradiation and heater probe].

Forty early gastric cancers (37 cases) were treated endoscopically by diathermic polypectomy and Nd; YAG laser irradiation. In 23 cases of protruded early gastric cancer, 16 cases were treated by diathermic polypectomy only, 2 cases by Nd; YAG laser only and 5 cases underwent combination therapy with both procedures. Endoscopic treatment was successful in 20 cases, but surgery was performed in two cases with cancerous invasion or lymph vessel infiltration at their cut ends after polypectomy. Local recurrence occurred in a case of submucosal involvement 6 months after combination therapy. In excavated early gastric cancers, 17 lesions of 14 cases were treated by Nd; YAG laser irradiation. Although 13 lesions disappeared successfully after laser treatment, cancers remained in 3 lesions with submucosal involvement after several irradiations. Local recurrence was experienced in a lesion of poorly differentiated adenocarcinoma three months after treatment. In addition, a heat probe unit for hemostasis was applied for endoscopic treatment of early gastric cancer and found to be effective for cancer of the mucosal layer. Among other early gastric cancers, endoscopic treatment is indicated for differentiated adenocarcinomas which are less than 2 cm in size and confined within the mucosal layer.

[Effects of intraarterial infusion chemotherapy on liver function in patients with hepatocellular carcinoma].

Periodic infusion chemotherapy from the reservoir implanted by direct arterial puncture (Seldinger method) was performed in 21 cases of hepatocellular carcinoma. The effectiveness of treatment in terms of efficacy and liver function in about 12 patients who survived more than 6 months was investigated. Owing to the progressive liver atrophy and liver dysfunction with ascites, some cases had to delay infusion treatment. Thus, it is necessary to check liver function regularly.

[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.

[Treatment of fulminant hepatitis with highly reliable artificial liver support and administration of interferon].

We treated 10 patients with viral fulminant hepatitis (FH) and subacute hepatitis (SH) by highly reliable artificial liver support (ALS), the combination of plasma exchange (PE) and hemodiafiltration (HDF) using polymethyl methacrylate (PMMA) membrane. All patients regained clear consciousness by the ALS. Even the patients with long term hepatic failure up to for 108 days were sustained in a favorable clinical condition. Five patients finally survived. Interferon was administered to one case with type B FH with positive HBeAg, four cases with NANB FH and SH who were assumed to have persistent viral replication. Two of them showed favorable clinical responses and definite liver regeneration was confirmed. The intensive liver support which can sustain patient with severe fulminant hepatic failure accompanied by the administration interferon is believed to be the most effective treatment for FH and SH especially caused by NANB virus in our country.

[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.

[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.