Taisuke Inoue

Infection with hepatitis GB virus C in patients on maintenance hemodialysis.

BACKGROUND A recently discovered non-A-E hepatitis virus has been designated hepatitis GB virus C (HGBV-C), but little is known about its mode of transmission and its clinical manifestations. We studied 519 patients on maintenance hemodialysis to determine whether they were infected with HGBV-C. METHODS HGBV-C RNA was identified in serum by a reverse-transcription-polymerase-chain-reaction assay with nested primers deduced from a non-structural region. A nucleotide sequence of 100 bp in the nonstructural region was determined on HGBV-C clones. RESULTS HGBV-C RNA was detected on 3.1 percent of the patients on hemodialysis (16 of 519), as compared with 0.9 percent of healthy blood donors (4 of 448, P<0.03). None of the 16 patients had evidence of active liver disease, although 7 were also infected with hepatitis C virus. Eight patients with HGBV-C infection were followed for 7 to 16 years. In two patients the virus was present at the start of hemodialysis. One had a history of transfusion, and HGBV-C persisted over a period of 16 years; the other became free of HGBV-C after 10 years. In five patients, HGBV-C RNA was first detected 3 to 20 weeks after blood transfusion and persisted for up to 13 years. One patient with no history of transfusion was infected with an HGBV-C variant with the same sequence as in two of the patients with post-transfusion HGBV-C infections. CONCLUSIONS Patients on maintenance hemodialysis are at increased risk for HGBV-C infection. This virus produces persistent infections, which may be transmitted by transfusions but may also be transmitted by other means.

A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene

A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmonds classification.

Mutations in the envelope gene of hepatitis B virus variants co-occurring with antibody to surface antigen in sera from patients with chronic hepatitis B.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124-147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130-132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of lle or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

Activation of JNK and high expression level of CD133 predict a poor response to sorafenib in hepatocellular carcinoma

Background:Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer deaths worldwide. While sorafenib, a multikinase inhibitor targeting the Raf/extracellular signal-regulated protein kinase (ERK) pathway, has been shown recently to provide a survival advantage to patients with advanced HCC, a predictive biomarker has not been developed. We studied whether c-Jun N-terminal kinase (JNK), which promotes liver carcinogenesis in mice, affects therapeutic response to sorafenib in HCC patients.Methods:We collected pathological specimens from 39 patients with advanced HCC before starting sorafenib treatment, and measured JNK activity in HCCs.Results:In patients treated with sorafenib, the expression of phospho-c-Jun in HCC, as a read out of JNK activity, was significantly higher (P<0.001) in the non-responder group than in the responder group. c-Jun N-terminal kinase activation in HCC was associated with a decreased time to progression and a poor overall survival (P=0.0028 and P=0.0008, respectively).Conclusion:In addition, JNK activity was significantly correlated with CD133 expression level. Correspondingly, high expression level of CD133 was linked to a poor response to sorafenib. Furthermore, D-JNKi, a specific JNK inhibitor, reduced the growth of xenografted CD133+ cells in athymic mice. In conclusion, JNK activation was positively correlated with CD133 expression level and inversely correlated with the therapeutic response to sorafenib, suggesting that JNK activity may be considered as a new predictive biomarker for response to sorafenib treatment.

Deep sequencing analysis of variants resistant to the non-structural 5A inhibitor daclatasvir in patients with genotype 1b hepatitis C virus infection

Daclatasvir, a non‐structural (NS)5A replication complex inhibitor, is a potent and promising direct antiviral agent (DAA) for hepatitis C virus (HCV), being most effective in genotype 1b infection. Although it is known that genotype 1b viruses with Y93H and/or L31M/V/F mutations have strong resistance to daclatasvir, it is not known whether there are some clinical background conditions that favor the occurrence of HCV carrying those NS5A mutations.

DICKKOPF-4 and -2 genes are upregulated in human colorectal cancer.

To comprehensively screen for genetic events underlying colorectal cancer, we performed suppression subtraction hybridization analysis on an advanced colon cancer. Because Dickkopf‐4, a member of the Dickkopf family acting as a Wnt‐signaling modulator, was identified as one of the upregulated genes in this specimen, we investigated expression profiles of all the Dickkopf family members in 55 colorectal tumors (21 cancers and 34 adenomas). We also investigated mechanisms regulating the expression of Dickkopf‐4 in these cancers in vitro and in vivo. Compared with normal adjacent mucosae, Dickkopf‐4 (median 27.4, P < 0.01) and ‐2 (median 51.4, P < 0.01) were strongly expressed in colorectal cancers. The level of Dickkopf‐4 was positively correlated with fibroblast growth factor‐20 (rs = 0.61, P = 0.00017), a representative β‐catenin transcriptional target gene, and with the degree of nuclear accumulation of β‐catenin in colorectal tumors. Dickkopf‐4 was induced by activated β‐catenin in vitro. Reciprocally, recombinant Dickkopf‐4 significantly inhibited T‐cell factor/lymphocyte enhancer factor reporter activity stimulated by recombinant Wnt3a in human embryonic kidney 293 cells. We conclude that Dickkopf‐4 and ‐2 are significantly upregulated in most colorectal tumors, and that Dickkopf‐4 upregulation reflects activation of the Wnt/canonical pathway. (Cancer Sci 2009; 100: 1923–1930)

Effect of interferon on a nonenveloped DNA virus (TT virus) associated with acute and chronic hepatitis of unknown etiology

An unenveloped DNA virus named TT virus (TTV) has been reported in association with acute and chronic hepatitis of unknown etiology. The effect of interferon on TTV was evaluated in the patients with chronic hepatitis C who were coinfected with TTV. TTV DNA was determined by a polymerase chain reaction with heminested primers in the 96 patients with chronic hepatitis C who received interferon‐α (516 million units in 26 weeks) and followed for 24 months thereafter. TTV DNA was detected in 31 (32%) patients before therapy. TTV DNA became undetectable during interferon therapy and remained absent in 14 (45% of the 31 patients) through 24 months thereafter. The four patients with pretreatment TTV DNA titer ≥103/ml did not respond. These results indicate that TTV is sensitive to interferon, and the response would be inversely correlated with pretreatment viral titers. J. Med. Virol. 58:196–200, 1999.

Full-length genomic sequence of a hepatitis C virus genotype 2c isolate (BEBE1) and the 2c-specific PCR primers

SummaryWe sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.

Infection with GB virus C in leprous patients in Japan.

The detection of hepatitis C virus (HCV) in blood donors and patients with acute and chronic hepatitis has brought to the fore another virus or viruses which can be transmitted parenterally and induce liver disease. The RNA of a candidate virus designated GB virus C (GBV‐C) was determined by the polymerase chain reaction with primers deduced from a helicase‐like region in 229 leprous patients in Japan. GBV‐C RNA was detected in 12 (5.2%) patients, and HCV RNA in 41 (18%). Three patients were coinfected with GBV‐C and HCV. The nine patients infected with GBV‐C alone had aminotransferase levels lower than the three patients with the mixed infection or the 38 patients infected with HCV only (P < 0.001). Sequence comparison within 100 base pairs in the helicase‐like region suggested that two, three and three patients, respectively, would have been infected with three distinct strains of GBV‐C. These results indicate that patients with leprosy are at increased risk for infection not only with HCV, but also with GBV‐C, and that the infection with GBV‐C alone would not induce hepatic injuries as severe as HCV infection.

Infection with hepatitis G virus and its strain variant, the GB agent (GBV-C), among blood donors in Japan

BACKGROUND: The purpose of the study was to survey the epidemiology of recently reported non‐A through ‐E hepatitis virus designated hepatitis G virus (HGV) and its strain variant, the GB agent (GBV‐C). STUDY DESIGN AND METHODS: Pilot samples from 2461 blood donors in Japan, randomly selected to form cohorts with different levels of alanine aminotransferase (ALT) and markers of hepatitis B virus or hepatitis C virus (HCV) infection, were tested for RNA of HGV/GBV‐C by reverse transcription‐polymerase chain reaction with nested primers deduced from the 5′‐noncoding region. RESULTS: HGV/GBV‐C RNA was detected in 23 (7.4%) of the 361 donors with anti‐HCV and HCV RNA. This detection is more frequent than that in donors without elevated ALT levels (< or = 45 U/L) or markers of HCV or hepatitis B virus infection (15/1303; 1.2%) (p < 0.001), donors with ALT values between 46 and 99 U per L (0/108) (p < 0.002), donors with ALT values > or = 100 U per L (5/361; 1.4%), and donors with anti‐HCV but without detectable HCV RNA (1/93; 1.1%) (p < 0.05). CONCLUSION: More than 1 percent of Japanese blood donors were infected with HGV/GBV‐C, and the prevalence was much higher in those with HCV RNA. Should persistent infection with HGV/GBV‐C induce any hepatotoxic sequelae, either alone or in concert with the other hepatitis viruses, screening of blood units for HGV/GBV‐C would deserve consideration.