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Biomedical subjects

Tsuyoshi Yamashiro

Publications and source records attributed to Tsuyoshi Yamashiro.

14 recordsLinked to original sources
PubMed2006

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3.

BACKGROUND: Interferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system. METHODS: An HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE). RESULTS: The baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% +/- 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels. CONCLUSIONS: IRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.

Blotting, Western↗
PubMed2005

Clinical usefulness of biochemical markers of liver fibrosis in patients with nonalcoholic fatty liver disease.

AIM: Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD), and progresses to the end stage of liver disease. Biochemical markers of liver fibrosis are strongly associated with the degree of histological liver fibrosis in patients with chronic liver disease. However, data are few on the usefulness of markers in NAFLD patients. The aim of this study was to identify better noninvasive predictors of hepatic fibrosis, with special focus on markers of liver fibrosis, type VI collagen 7S domain and hyaluronic acid. METHODS: One hundred and twelve patients with histologically proven NAFLD were studied. RESULTS: The histological stage of NAFLD correlated with several clinical and biochemical variables, the extent of hepatic fibrosis and the markers of liver fibrosis were relatively strong associated. The best cutoff values to detect NASH were assessed by using receiver operating characteristic analysis: type VI collagen 7S domain > or =5.0 ng/mL, hyaluronic acid > or =43 ng/mL. Both markers had a high positive predictive value: type VI collagen 7S domain, 86% and hyaluronic acid, 92%. Diagnostic accuracies of these markers were evaluated to detect severe fibrosis. Both markers showed high negative predictive values: type VI collagen 7S domain (> or =5.0 ng/mL), 84% and hyaluronic acid (> or =50 ng/mL), 78%, and were significantly and independently associated with the presence of NASH or severe fibrosis by logistic regression analysis. CONCLUSION: Both markers of liver fibrosis are useful in discriminating NASH from fatty liver alone or patients with severe fibrosis from patients with non-severe fibrosis.

Adult↗
PubMed2004

Metabolic syndrome is directly associated with gamma glutamyl transpeptidase elevation in Japanese women.

AIM: This study aimed to determine whether metabolic syndrome is directly or indirectly, through fatty liver, associated with elevated gamma-glutamyl transpeptidase (GGT) levels in Japanese women. METHODS: From 4 366 women who received their annual health check-up, 4 211 women were selected for analysis. All 4 211 women were negative for both hepatitis B surface antigen and antibody to hepatitis C virus. Clinical and biochemical variables were examined by using univariate and multivariate analysis. RESULTS: A raised GGT level (>68 IU/L) was seen in 258 (6.1%) of the 4 211 women. In univariate analysis, all variables examined (age, body mass index, blood pressure, hemoglobin concentration, fasting blood glucose, glycosylated hemoglobin A1c, cholesterol, triglyceride, and uric acid) were associated with the elevated GGT level, whereas in multivariate analysis, four variables (age > or =50 yr, hemoglobin > or =14 g/dL, triglyceride > or =150 mg/dL, and presence of diabetes) were significantly and independently associated with raised GGT level. Clinical variables predicting the presence of ultrasonographic evidence of fatty liver were also examined by multivariate analysis; four variables were associated with the presence of fatty liver: BMI > or =25 kg/m(2), hemoglobin > or =14 g/dL, triglyceride > or =150 mg/dL, and uric acid > or =7 mg/dL. There was no significant association between the raised GGT level and the presence of fatty liver. Hypertriglyceridemia was significantly and independently associated with both the raised GGT level and the presence of fatty liver. CONCLUSION: Metabolic syndrome seemed to be directly, not indirectly through fatty liver, associated with the raised GGT level in Japanese women.

Adult↗
PubMed2004

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha.

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Amino Acid Sequence↗
PubMed2004

Quantitation of the level of hepatitis delta virus RNA in serum, by real-time polymerase chain reaction--and its possible correlation with the clinical stage of liver disease.

Some hepatitis B virus (HBV) carriers with chronic hepatitis delta virus (HDV) superinfection show progressive chronic hepatitis, whereas others show no apparent signs of liver disease. In the present study, we established a sensitive method for the quantitation of the level of HDV RNA in serum on the basis of real-time reverse-transcription polymerase chain reaction (RT-PCR), to clarify the role that the level of HDV RNA in serum plays in the diverse natural course of clinical manifestation. In 48 subjects who were positive for hepatitis B surface antigen and for anti-hepatitis delta antibody, the levels of HDV RNA in serum were quantitated by RT-PCR. The levels of HBV DNA in serum were determined by a transcription-mediated amplification assay. The levels of HDV RNA in serum of subjects with chronic hepatitis and of subjects with liver cirrhosis were significantly higher than those in asymptomatic carrier subjects. The levels of HBV DNA in serum did not differ significantly among these 3 groups. In conclusion, HDV RNA quantification by real-time RT-PCR is possibly a useful tool for understanding the pathophysiology of HDV infection.

Adult↗
PubMed2004

Specific inhibition of hepatitis C virus replication by cyclosporin A.

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.

Antiviral Agents↗
PubMed2004

Regulation of hepatitis C virus replication by interferon regulatory factor 1.

Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Base Sequence↗
PubMed2003

Monitoring low level hepatitis B virus by a newly developed sensitive test.

We have recently developed a sensitive quantitative test for hepatitis B virus (HBV) DNA using a real-time polymerase chain reaction (HBV RTD DIRECT), which can detect HBV DNA levels as low as 10(0.7) copies/ml. The aim of this study was to explore the significance of viremia changes below the detection limit of the other currently developed sensitive assays, transcription-mediated amplification and hybridization protection assay (TMA-HPA) and Amplicor HBV Monitor. The subjects consisted of 11 patients with chronic liver disease type B who showed undetectable test results of HBV DNA by TMA-HPA or Amplicor HBV Monitor during the observation period. A total of 150 serial serum samples were examined for viremia level by HBV RTD DIRECT: 139 were positive and 11 were negative. HBV RTD DIRECT could detect viremia in 72 of 78 serum samples negative for HBV DNA by TMA-HPA, or in 38 of 43 serum samples negative for HBV DNA by Amplicor HBV Monitor. The HBV DNA level was gradually increased from its lowest level before the spontaneous reactivation of hepatitis or the emergence of YMDD mutant during lamivudine treatment. However, such a phenomenon was not revealed by either TMA-HPA or the Amplicor HBV Monitor test.

Journal Article↗
PubMed2003

Epidemiology of primary biliary cirrhosis among women with elevated gamma-glutamyl transpeptidase levels in Okinawa, Japan.

We investigated the conditions among women who had an asymptomatic increase in serum gamma-glutamyl transpeptidase (gamma-GTP) levels, and the prevalence of primary biliary cirrhosis (PBC), in the general population. Among 4048 women who received their annual health check-up, 241 showed an elevated gamma-GTP level and were invited to participate in this study. Of the 241 women, 122 participated and were examined thoroughly, including for antimitochondrial antibody (AMA) and by using liver biopsy to make a clinical diagnosis. Six (4.9%) of the 122 women were AMA positive: five were diagnosed and one was suspected of having PBC. Another two women had the criteria of PBC despite being AMA negative. PBC was detected in 5.7% (95% confidence interval (CI), 1.6-9.9%) of asymptomatic women with raised gamma-GTP levels who were 6.0% of all 4048 women examined. The estimated prevalence of PBC in our area was 3400 per million women mainly over 40 years and 840 per million in the whole population. In 44% of women, the cause of chronic gamma-GTP elevation was unknown; they usually showed mild and non-specific histological change differing from their liver biochemical test results.

Journal Article↗
PubMed2003

Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease.

We determined the sequence of the hepatitis delta virus (HDV) genome in 40 Japanese patients, most of whom were from the Miyako Islands, Okinawa, Japan. Consensus sequences from 33 HDV full genomes out of a total of 40 patients were determined by directly sequencing four partially overlapping PCR products. Phylogenetic tree analysis classified these 33 complete HDV genomes as HDV genotype I (two patients), genotype IIa (one patient) and genotype IIb (30 patients). Among the 30 genotype IIb patients, there were two clusters of genetic variants. One group consisted of six isolates showing significant homology with genotype IIb, previously reported from Taiwan. The other group consisted of 24 isolates, whose sequences formed a new genetic subgroup (genotype IIb-Miyako; IIb-M). When the genetic structures were compared in detail between IIb and IIb-M, characteristic variations were found in the C-terminal sequence of the large delta antigen-conferring packaging signal as well as the RNA editing site. Determination of subclasses of genotype IIb in a total of 37 patients, including seven HDV patients whose partial HDV sequence was determined, revealed eight patients with IIb and 29 patients with IIb-M. Although there was no significant difference in the clinical background or virological state of hepatitis B virus between these two groups, patients with genotype IIb-M showed greater progression of chronic hepatitis and cirrhosis than those with genotype IIb (P=0.0009). These data indicate the existence of a genetic subgroup of HDV genotype IIb, which is associated with different clinical characteristics and which could be related to genetic variations in functionally important parts of the HDV genome.

Adult↗
PubMed2002

Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa, Japan.

The present study was designed to examine the distribution of hepatitis B virus (HBV) genotypes among patients at various stages of chronic liver disease type B in Okinawa Prefecture, Japan, where the prevalence of hepatitis B surface antigen is the highest in Japan despite the lowest mortality rate from primary liver cancer. Serum samples from 227 HBV carriers were determined for HBV genotype by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Five of 227 sera were negative for HBV DNA by nested PCR and were excluded from the genotype analysis. Genotype B was predominant in asymptomatic carriers (45/67, 67%), whereas genotype C was predominant in chronic liver disease: 49% (50/103) in patients with chronic hepatitis, 63% (20/32) in patients with cirrhosis, and 60% (12/20) in patients with hepatocellular carcinoma. The distribution of genotype B decreased with increasing liver disease severity. However, this tendency was seen among patients aged less than 50 years old, whereas the prevalence of genotype B was similar among carriers with various liver diseases who were older than age 50. In conclusion, HBV genotype B was prevalent and less frequent among patients with advanced liver disease, particularly in patients aged less than 50 years. These findings suggest that the preponderance of genotype B is responsible for the low mortality rate of primary liver cancer associated with HBV seen in Okinawa Prefecture, despite having the highest HBV carrier rate in Japanese.

Adult↗
PubMedDate not supplied

Alanine aminotransferase (ALT) levels in a normal population and interferon therapy in chronic hepatitis C patients with normal ALT.

BACKGROUND/AIMS: The aims of this study were to determine the distribution of serum alanine aminotransferase levels in a normal population and to clarify whether interferon treatment is justified in HCV-infected patients with persistently normal alanine aminotransferase levels. METHODOLOGY: The distribution of alanine aminotransferase levels was examined among 949 normal subjects who were negative for hepatitis viruses, denied regular alcohol use. Nineteen patients with chronic hepatitis C and persistently normal alanine aminotransferase levels were treated with alpha interferon (six or ten million units thrice weekly for six months). RESULTS: Peaks of alanine aminotransferase distribution among the normal subjects were seen at 16-20 IU/L and 11-15 IU/L in males and females, respectively. Fourteen of the 19 patients who received interferon treatment had favorable factors of response to interferon (eight with low pretreatment virus load, four with HCV genotype 2 and two with both). A sustained virological response was achieved in eight (57%) of 14, and alanine aminotransferase levels decreased significantly to within the normal range after interferon treatment in six of eight. CONCLUSIONS: Patients with chronic hepatitis C and persistently normal alanine aminotransferase levels should be treated with high doses of interferon if they have favorable factors of response to interferon treatment.

Adult↗
PubMedDate not supplied

Clinical characteristics of patients with cryptogenic liver cirrhosis in Okinawa, Japan.

BACKGROUND/AIMS: The aims of the present study were to determine the prevalence of patients with cryptogenic liver cirrhosis, and to clarify the clinical characteristics of these patients, with special focus on obesity and type 2 diabetes mellitus. METHODOLOGY: For the case-control study, we selected age- and sex-matched control patients with cirrhosis of known etiology. RESULTS: Of 404 patients with liver cirrhosis, 40 (9.9%) were diagnosed as having cryptogenic cirrhosis. Of these 40 patients, 29 (73%) were female. The mean age was 66.1 +/- 8.9 (range 40-87 years old), and was significantly higher than that of either virus, or alcohol-related liver cirrhosis patients. Both obesity and type 2 diabetes mellitus were more common in patients with cryptogenic cirrhosis (53% and 40%, respectively) compared with the case-controls (20%, 18%, respectively). CONCLUSIONS: Cryptogenic liver cirrhosis accounted for approximately 10% of liver cirrhosis, and was associated with both obesity and type 2 diabetes mellitus. Nonalcoholic steatohepatitis sequelae may be the most important etiological factor in patients with cryptogenic liver cirrhosis in Okinawa, Japan, where virus-etiology is more predominant in liver cirrhosis compared with Western countries.

Adult↗