Showing papers by "Masashi Mizokami published in 2005"

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Abstract: Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15–1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture–generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.

A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon.

Abstract: Blood samples (n=544) from two different populations (Pygmies and Bantus) in Cameroon, West Africa, were analysed. Serological tests indicated that the anti-hepatitis C virus (HCV) prevalence in Bantus (20?3%) was higher than that in Pygmies (2?3%,P<0?0001), whereas the distribution of hepatitis B virus (HBV) serological markers was equally high in both populations: in total, 9?4, 17?3 and 86?8% for HBsAg, anti-HBs and anti-HBc, respectively. HBV genotype A (HBV/A) and HBV/E were predominant (43?5% each) in both populations, and HBV/D was found in a minority (13%). The preS/S region was sequenced in nine cases (five HBV/A and four HBV/E) and the complete genome in six cases (four HBV/A and two HBV/E). Subsequent phylogenetic analysis revealed that the HBV/A strains were distinct from the subtypes (subgenotypes) described previously, Ae (A2) and Aa (A1), and in the preS/S region they clustered with previously reported sequences from Cameroon. Based on the nucleotide difference from Aa (A1) and Ae (A2), more than 4% in the complete genome, the Cameroonian strains were suggested to represent a new subtype (subgenotype), designated HBV/Ac (A3). A high (3?9%) nucleotide divergence in HBV/Ac (A3) strains suggested that the subtype (subgenotype) has a long natural history in the population of Cameroon. One of the HBV/Ac (A3) strains was found to be a recombinant with an HBV/E-specific sequence in the polymerase reverse transcriptase domain. Further cohort studies will be required to assess detailed epidemiological, virological and clinical characteristics of HBV/Ac (A3), as well as its recombinant form.

Nonhepatic Cell Lines HeLa and 293 Support Efficient Replication of the Hepatitis C Virus Genotype 2a Subgenomic Replicon

Abstract: The hepatitis C virus (HCV) genotype 2a subgenomic replicon can replicate in two human non-hepatocyte-derived cell lines, HeLa and 293, with in vitro-transcribed replicon RNA. Sequencing analysis revealed that mutations in HCV-derived regions were not essential for replication in these cells, as some clones displayed no mutations.

Detection of Anti-Hepatitis C Virus Effects of Interferon and Ribavirin by a Sensitive Replicon System

Abstract: Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 μg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 μg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.

Hepatitis B Virus Genotype C Prevails Among Chronic Carriers of the Virus in Korea

Abstract: Hepatitis B virus (HBV) is one of the major causative agents of chronic liver diseases in Korea. HBV has been classified into 8 genotypes by a divergence of >8% in the entire genomic sequence, and have distinct geographic distributions. There are limited data on the relevance between HBV genotypes and clinical outcomes in Korea. To investigate the clinical feature relating to HBV genotype in Korea, a total 120 serum samples with HBsAg (65 from Seoul and 55 from the other city in Korea) were obtained from each 30 chronic HBV carriers with asymptomatic carrier (ASC), chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). HBV genotype was determined by either enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against genotype-specific epitopes in the preS2-region or the direct sequencing of small S gene. HBV genotypes were determined in 105 (87.5%) of 120 samples. HBV genotype C was identified in all HBV carriers with ASC, CH, LC, and HCC. Genotypes A, B, D, E, F and G were not detected in any of them. Genotype C HBV prevails predominantly among chronic carriers of the virus in Korea, irrespective of their clinical stages of liver disease and geographic origin.

Impact of occult hepatitis B virus infection on efficacy and prognosis of interferon-alpha therapy for patients with chronic hepatitis C.

Abstract: Background/Aims: It is reported that some patients with undetectable hepatitis B surface antigen (HBsAg) have serum hepatitis B virus (HBV) DNA in patients with chronic hepatitis C (HCV). The aim of this study was to elucidate the impact of occult HBV infection on the efficacy and prognosis of interferon-α (IFN) therapy in HCV patients. Methods: One hundred and forty HCV patients without HBsAg who received IFN therapy were studied. Serum HBV DNA was quantified by real-time detection polymerase chain reaction. Results: Of 140 patients, 11 (7.9%) were HBV DNA-positive before IFN therapy. The serum HBV DNA levels ranged from 106 to 884 copies/ml. Four of these 11 patients showed a sustained virologic response by IFN, compared with 39 of 129 without HBV DNA (P=NS). Interestingly, two of the 11 patients developed hepatocellular carcinoma (HCC) after therapy, compared with 16 of 129 without HBV DNA (P=NS). In the serial study, serum HBV DNA was transiently undetectable during and after IFN; however, most became positive during follow-up. Conclusions: Occult HBV infection may not have a significant impact on response to IFN therapy for chronic HCV and development of HCC after therapy. Occult HBV may be sensitive to IFN although HBV is not completely eradicated.

Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan

Abstract: Hepatitis B virus (HBV) genotypes B (HBV/B) and C (HBV/C) are prevalent in Asia. Recently HBV/B has been classified into two subtypes, HBV/Ba which is ubiquitously found in Asia, and HBV/Bj which is specific in Japan. In addition, the frequency of positive HBeAg has been reported to be higher in patients with HBV/Ba than those with HBV/Bj. However, little is known about the differences between patients with various genotypes who developed hepatocellular carcinoma (HCC). In 296 serum samples of HCC patients collected from all over Japan, HBV genotypes were determined with the restriction fragment length polymorphism. HBV/A was detected in 1.0%, HBV/Ba in 4.4%, HBV/Bj in 7.4%, and HBV/C in 86.5%. In the Tohoku district and Okinawa, HBV/Ba, HBV/Bj and HBV/C were found in 6.7, 40.0 and 48.9%, compared to 4.0, 1.6 and 93.2% in the other districts in Japan. HBV/Bj patients were more frequently found in the group older than 65 years while HBV/Ba patients were found in all age groups. The frequency of positive HBeAg in HBV/Bj patients was significantly low compared to that in the other patients. More than 60% of the patients with HCC had cirrhosis as the underlying liver diseases. However, in HBV/Ba patients aged 50 years or younger, 80% of them had chronic hepatitis, while 87.5% of those aged older than 50 years had cirrhosis. These data suggest that great differences exist among patients with HCC infected with different genotypes.

Distribution of HBV genotypes among HBV carriers in Benin:phylogenetic analysis and virological characteristics of HBV genotype E.

Abstract: AIM: To determine the distribution of Hepatitis B virus (HBV) genotypes in Benin, and to clarify the virological characteristics of the dominant genotype. METHODS: Among 500 blood donors in Benin, 21 HBsAg-positive donors were enrolled in the study. HBV genotypes were determined by enzyme immunoassay and restriction fragment length polymorphism. Complete genome sequences were determined by PCR and direct sequencing. RESULTS: HBV genotype E (HBV/E) was detected in 20/21 (95.2%), and HBV/A in 1/21 (4.8%). From the age-specific prevalence of HBeAg to anti-HBe seroconversion (SC) in 19 HBV/E subjects, SC was estimated to occur frequently in late teens in HBV/E. The comparison of four complete HBV/E genomes from HBeAg-positive subjects in this study and five HBV/E sequences recruited from the database revealed that HBV/E was distributed throughout West Africa with very low genetic diversity (nucleotide homology 96.7-99.2%). Based on the sequences in the basic core promoter (BCP) to precore region of the nine HBV/E isolates compared to those of the other genotypes, a nucleotide substitution in the BCP, G1757A, was observed in HBV/E. CONCLUSION: HBV/E is predominant in the Republic of Benin, and SC is estimated to occur in late teens in HBV/E. The specific nucleotide substitution G1757A in BCP, which might influence the virological characteristics, is observed in HBV/E.

Corrigendum: Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Abstract: Nat. Med. 11, 791–796 (2005) The chimpanzee used in this study is referred to as X0215 in several parts of the text and supplementary information. Its correct number is X0205.

Quantification of red blood cell fragmentation by the automated hematology analyzer XE-2100 in patients with living donor liver transplantation.

Abstract: The fragmented red cell (FRC) is a useful index for diagnosing and determining the severity of thrombotic thrombocytopenic purpura (TTP), thrombotic microangiopathy (TMA) and other similar conditions, as it is found in peripheral blood in patients with these diseases. The FRC expression rate has conventionally been determined by manual methods using smear samples. However, it is difficult to attain accurate quantification by such methods as they are time consuming and prone to a great margin of error. With cases of living donor liver transplantation, the current study examined the possibility of using a multi-parameter automated hematology analyzer, the XE-2100 (Sysmex Corporation) for FRC quantification. While there was a notable correlation between the manual and automated measurements, the manual measurement resulted in higher values. This suggested remarkable variations in judgment by individuals. The FRC values had a significant correlation with the reticulocyte count, red blood cell distribution width (RDW), fibrin/fibrinogen degradation products (P-FDP) and lactate dehydrogenase (LDH) among the test parameters, and this finding was consistent with the clinical progression in patients. The automated method can offer precise measurements in a short time without inter-observer differences, meeting the requirement for standardization. The determination of FRC count (%) by the XE-2100 that enables early diagnoses and monitoring of TTP or TMA will be useful in the clinical field.

Novel type of hepatitis B virus mutation: replacement mutation involving a hepatocyte nuclear factor 1 binding site tandem repeat in chronic hepatitis B virus genotype E.

Abstract: The genetic diversity of hepatitis B virus (HBV) strains has evolved through mutations such as point mutations, deletions or insertions, and recombination. We identified and characterized a novel type of mutation which is a complex of external insertion, deletion, and internal duplication in sequences from one of six patients with chronic hepatitis B virus genotype E (HBV/E). We provisionally named this mutation a “replacement mutation”; the core promoter upstream regulatory sequence/basic core promoter was replaced with a part of the S1 promoter covering the hepatocyte nuclear factor 1 (HNF1) binding site, followed by a tandem repeat of the HNF1 site. A longitudinal analysis of the HBV population over 6 years showed the clonal change from wild-type HBV/E to replacement-mutant type, resulting in a lower hepatitis B (HB) e antigen titer, a high HBV DNA level in serum, and progression of liver fibrosis. In an in vitro study using a replication model, the replacement-mutant HBV showed higher replication levels than the wild-type HBV/E replicon, probably mediated by altered transcription factor binding. Additionally, this HNF1 site replacement mutation was associated with excessive HB nucleocapsid protein expression in hepatocytes, in both in vivo and in vitro studies. This novel mutation may be specific to HBV genotype E, and its prevalence requires further investigation.

Classifying genotype F of hepatitis B virus into F1 and F2 subtypes.

Abstract: AIM: To explore the propriety of providing hepatitis B virus (HBV) genotypes F and H with two distinct genotypes. METHODS: Eleven HBV isolates of genotype F (HBV/F) were recovered from patients living in San Francisco, Japan, Panama, and Venezuela, and their full-length sequences were determined. Phylogenetic analysis was carried out among them along with HBV isolates previously reported. RESULTS: Seven of them clustered with reported HBV/F isolates in the phylogenetic tree constructed on the entire genomic sequence. The remaining four flocked on another branch along with three HBV isolates formerly reported as genotype H. These seven HBV isolates, including the four in this study and the three reported, had a sequence divergence of 7.3-9.5% from the other HBV/F isolates, and differed by >13.7% from HBV isolates of the other six genotypes (A-E and G). Based on a marked genomic divergence, falling just short of >8% separating the seven genotypes, these seven HBV/F isolates were classified into F2 subtype and the former seven into F1 subtype provisionally. In a pairwise comparison of the S-gene sequences among the 7 HBV/F2 isolates and against 47 HBV/F1 isolates as well as 136 representing the other six genotypes (A-E and G), two clusters separated by distinct genetic distances emerged. CONCLUSION: Based on these analyses, classifying HBV/F isolates into two subtypes (F1 and F2) would be more appropriate than providing them with two distinct genotypes (F and H).

Pyogenic granuloma of the stomach successfully treated by endoscopic resection after transarterial embolization of the feeding artery

Abstract: Pyogenic granulomas represent the aquisition of vasodilative granulation tissue in the skin or mucosa. They are extremely rare in the alimentary tract, other than in the oral cavity. Here, we report a case of pyogenic granuloma arising from the gastric mucosa. An 82-year-old man was admitted to our hospital because of melena of more than 3 months, duration. Esophagogastroduodenoscopy (EGD) revealed a 30-mm-diameter semipedunculated lesion with an irregular surface in the fundus of the stomach. During hospitalization, the patient’s anemia worsened due to loss of blood from the lesion, with the level of hemoglobin declining to 6 g/dl, and a blood transfusion was required. Because radiological and endoscopic findings indicated the lesion was hypervascular, transarterial embolization of the nutritional artery of the lesion was performed before endoscopic resection of the lesion. One week after the embolotherapy, endoscopic mucosal resection was performed, without any complications, such as massive bleeding. Histological studies of the resected specimen revealed many capillaries of various sizes, lined with plump endothelial cells, and accompanied by acute and chronic inflammatory infiltrates. On the basis of these observations, the lesion was diagnosed as a pyogenic granuloma. One year later, the patient was asymptomatic and there was no evidence of tumor recurrence on follow-up EGD.

Hepatic necroinflammation and fibrosis in patients with genotypes Ba and C, core-promoter and precore mutations.

Abstract: Summary. The role of infection with hepatitis B virus (HBV) genotypes on liver histology is largely unknown. The aim of study was to investigate the relationships between HBV genotypes (B, C), core-promoter (CP) and precore mutants and liver histology in 66 patients. Liver biopsies were scored by histologic activity index (HAI). HBV genotypes were determined by enzyme-linked immunosorbent assay (ELISA). Eighteen (27.3%) and 48 patients (72.7%) had genotype B (all were subtype Ba) and C, respectively. Forty-seven (71.2%) and 27 (40.9%) had CP and precore mutations, respectively. Patients with genotype C compared with subtype Ba had higher median scores of HAI-necroinflammation (HAI-NI) (7 vs 3), HAI-fibrosis (HAI-F) (1 vs 0) and total HAI (8.5 vs 3) (all P < 0.03). Patients with CP mutations compared with wild-type had higher median scores of HAI-NI (7 vs 3), HAI-F (3 vs 0) and total HAI (9 vs 3) (all P < 0.03). Forty patients (83.5%) with genotype C had CP mutations. Age and alanine aminotransferase levels were positively correlated with HAI scores while albumin levels were negatively correlated (P < 0.01 for all, except albumin levels and HAI-F, P = 0.08). There was no association between precore mutations and HAI scores. Multivariate analysis indicated that higher alanine aminotransferase (ALT) levels were associated with higher HAI scores (P < 0.04) and CP mutations were associated with higher HAI-NI (P = 0.034), but not with HAI-F score (P = 0.3). CP mutations were associated with more severe necroinflammation. The association between genotype C and poor histology was probably because of the association between genotype C and CP mutations.

New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0.

Abstract: AIM: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HCV MONITOR test v2.0 (microwell version).

Prevention of vertical transmission of Group B Streptococcus

Abstract: Group B Streptococcus (GBS) are pathogens that involve a risk of vertical transmission. They are the infecting organism in approximately one quarter of all cases of neonatal sepsis and meningitis, making prevention of GBS infection an important goal. The United States Centers for Disease Control and Prevention (CDC) recommends administration of antibiotic prophylaxis to GBS-colonized pregnant women at least 4 hours before delivery, but the time of antibiotic prophylaxis administration is not generally reported in Japan. The purpose of the present study was to identify the care provided to GBS-colonized pregnant and intrapartum women in order to prevent of vertical transmission of GBS. The subjects were women (n=150) judged during pregnancy to have been colonized by GBS, who delivered vaginally at one of two hospitals between January 2000 and December 2004, and their neonates (n=151). The relation between the care provided and GBS transmission was analyzed. GBS was transmitted to the neonates of 18 of the 150 women (transmission rate 12.0%). The relation between transmission to the neonate and time between administration of antibiotic prophylaxis and delivery was investigated, and transmission to the neonate was found to be significantly greater when it was less than 3.5 hours (transmission to 9 neonates of 53 women) than more than 3.5 hours (transmission 4 neonates of 83 women) (p < 0.05). The time between admission and delivery was significantly shorter in the cases of transmission (p < 0.05). This indicates the need for thorough health guidance for expectant mothers, especially multipara, during pregnancy regarding the timing of admission for delivery, in order to ensure sufficient time between administration of antibiotic prophylaxis and delivery.

Pulsed field gel electrophoresis analysis conditions for molecular epidemiological identification of vertical Group B Streptococcus infection.

Abstract: Group B Streptococcus(GBS)is a pathogen with a risk of vertical infection through the birth canal, but reports have also been made of horizontal infection in the nursery. Pulsed field gel electrophoresis (PFGE)enables molecular epidemiologic investigation of the infection route, but has the drawback of taking too much time. Conditions for PFGE analysis for GBS vary greatly. We optimized variables for identifying vertical GBS infection by molecular epidemiology.

A case of autoimmune hepatitis superimposed by Ukon-induced liver injury

Abstract: 症例は50歳の女性. 黄疸を伴う急性肝炎にて入院し, 臨床経過, 肝組織検査, 診断基準より慢性肝障害に合併したウコンによる薬物性肝障害と診断した. ウコンの服用中止後も肝障害が遷延したためプレドニゾロンを使用. プレドニゾロンは奏効し肝障害改善後退院となった. 外来にてプレドニゾロンを中止したが, 中止後50日目に再び肝障害を認めたため2回目の入院となった. 肝組織検査, 診断基準に基づき検討したところ, 2回目の肝障害は自己免疫性肝炎であった. 1回目の肝障害と2回目の肝障害を再検討したが, ウコンによる薬物性肝障害がtriggerとなって自己免疫性肝炎が誘導された可能性と, もともと自己免疫性肝炎が存在しウコン内服によりなんらかの影響を受けて急性増悪した可能性が考えられた.

A Case of Gastrointestinal Stromal Tumor with Internal Tandem Duplication in the 3′-Terminal of the KIT Juxtamembrane Domain

Abstract: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors originating from the gastrointestinal tract and are responsible for approximately 80% of all the gastrointestinal mesenchymal neoplasms. Most GISTs show immunologically positive for both CD34 and KIT (CD117), which is a characteristic also of the interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells. Therefore, GISTs are now considered to originate from the ICC or be progenitors that differentiate toward a pacemaker cells phenotype (1‐4). KIT is a type III receptor tyrosine kinase encoded by the protooncogene c-kit, and its ligand is stem-cell factor. Activating mutations of the KIT gene—which lead to ligand-independent activation of the tyrosine kinase of KIT—play a fundamental role in the development of these tumors (5, 6). It has been reported that most mutations consist of in-frame deletions or a single amino acid substitution within exon 11 that codes for the juxtamembrane domain (6), and rarely in exon 9 (extracellular region) as well as exon 13 (first part of the split kinase domain) (5). Diagnosis of malignant GISTs has been problematic among clinicians and pathologists. Traditionally, mitotic index, tumor size, and tumor location are considered to be the key predictive factors. KIT-activating mutations are now also proposed as a predictive factors of prognosis (5, 7‐9). Recently, internal tandem duplication (ITD) in