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A growing list of microRNAs (miRNAs) show aberrant expression patterns in hepatocellular carcinoma (HCC), but the regulatory mechanisms largely remain unclear. RNA editing catalyzed by members of the adenosine deaminase acting on the RNA (ADAR) family could target the miRNA precursors and affect the biogenesis process. Therefore, we investigate whether RNA editing could be one mechanism contributing to the deregulation of specific miRNAs in HCC. By overexpression of individual ADARs in hepatoma cells, RNA editing on the precursors of 16 miRNAs frequently deregulated in HCC was screened by a sensitive high-resolution melting platform. The results identified RNA precursors of miR-214 and miR-122 as potential targets edited by ADAR2. A subset of HCC showing elevated ADAR2 verified the major editings identified in ARAR2 overexpressed hepatoma cells, either with A-to-I or U-to-C changes. The unusual U-to-C editing at specific residues was demonstrated as being attributed to the A-to-I editing on the RNA transcripts complementary to the pri-miRNAs. The editing event caused a decrease of the RNA transcript complementary to pri-miR-214, which led to the decrease of pri-miR-214 and miR-214 and resulted in the increased protein level of its novel target gene Rab15. In conclusion, the current study discovered ADAR2-mediated editing of the complementary antisense transcripts as a novel mechanism for regulating the biogenesis of specific miRNAs during hepatocarcinogenesis. [ABSTRACT FROM AUTHOR]

Background & Aims: The World Health Organisation (WHO) Prevention & Control of Viral Hepatitis Infection: Framework for Global Action offers a global vision for the prevention and control of viral hepatitis. In October 2012, the Coalition to Eradicate Viral Hepatitis in Asia Pacific (CEVHAP) organised the North Asia Workshop on Viral Hepatitis in Taipei to discuss how to implement the WHO Framework in the North Asia region. This paper presents outcomes from this workshop. Methods: Twenty-eight representatives from local liver associations, patient organisations, and centres of excellence in Hong Kong, Japan, Korea, and Taiwan participated in the workshop. Findings: Priority areas for action were described along the four axes of the WHO Framework: (1) awareness, advocacy and resources; (2) evidence and data; (3) prevention of transmission; and (4) screening and treatment. Priorities included: axis 1: greater public and professional awareness, particularly among primary care physicians and local advocacy networks. Axis 2: better economic data and identifying barriers to screening and treatment uptake. Axis 3: monitoring of vaccination outcomes and targeted harm reduction strategies. Axis 4: strengthening links between hospitals and primary care providers, and secure funding of screening and treatment, including for hepatocellular carcinoma. Conclusions: The WHO Framework provides an opportunity to develop comprehensive and cohesive policies in North Asia and the broader region. A partnership between clinical specialists, primary care physicians, policy makers, and people with or at risk of viral hepatitis is essential in shaping future policies. [Copyright &y& Elsevier]

Background. The serum hepatitis B virus (HBV) surface antigen (HBsAg) level can predict hepatocellular carcinoma (HCC) development in hepatitis B e antigen (HBeAg)-negative patients with an HBV DNA level of <2000 IU/mL. However, little is known regarding how well the combination of both viral biomarkers stratifies HCC risk. Methods. A total of 2165 Taiwanese HBeAg-negative noncirrhotic patients were followed for 14.9 years. The predictive power of the HBsAg level for HCC was analyzed for different viral load ranges. Results. In patients with HBV DNA levels of 2000-19 999 IU/mL (intermediate viral load), a positive correlation between HBsAg level and HCC development was identified after adjustment for other risk factors (P = .002). In contrast, no association was found between HBsAg level and HCC in patients with higher viral loads. HBsAg level was subsequently included to stratify HCC risk in patients with low and intermediate viral loads. Receiver operating characteristic curve analysis showed that combining HBV DNA and HBsAg level better predicts 10-year HCC development as compared to using HBV DNA level alone in the overall cohort (P = .028). Conclusions. Serum HBsAg level helps stratify HCC risk in patients with intermediate viral loads. Combining HBV DNA and HBsAg levels better predicts HCC risk. [ABSTRACT FROM AUTHOR]

Background. The serum hepatitis B virus (HBV) surface antigen (HBsAg) level can predict hepatocellular carcinoma (HCC) development in hepatitis B e antigen (HBeAg)–negative patients with an HBV DNA level of <2000 IU/mL. However, little is known regarding how well the combination of both viral biomarkers stratifies HCC risk.Methods. A total of 2165 Taiwanese HBeAg-negative noncirrhotic patients were followed for 14.9 years. The predictive power of the HBsAg level for HCC was analyzed for different viral load ranges.Results. In patients with HBV DNA levels of 2000–19 999 IU/mL (intermediate viral load), a positive correlation between HBsAg level and HCC development was identified after adjustment for other risk factors (P = .002). In contrast, no association was found between HBsAg level and HCC in patients with higher viral loads. HBsAg level was subsequently included to stratify HCC risk in patients with low and intermediate viral loads. Receiver operating characteristic curve analysis showed that combining HBV DNA and HBsAg level better predicts 10-year HCC development as compared to using HBV DNA level alone in the overall cohort (P = .028).Conclusions. Serum HBsAg level helps stratify HCC risk in patients with intermediate viral loads. Combining HBV DNA and HBsAg levels better predicts HCC risk. [ABSTRACT FROM AUTHOR]

Abstract: This research reports the design, analysis, integration, and test of a prototype of a real-time convective polymerase chain reaction (RT-cPCR) machine that uses a color charged coupled device (CCD) for detecting the emission of fluorescence intensity from an RT-cPCR mix in a microliter volume glass capillary. Because of its simple mechanism, DNA amplification involves employing the cPCR technique with no need for thermocycling control. The flow pattern and temperature distribution can greatly affect the cPCR process in the capillary tube, a computational fluid dynamics (CFD) simulation was conducted in this study for the first time to estimate the required period of an RT-cPCR cycle. This study also tested the PCR mix containing hepatitis B virus (HBV) plasmid samples by using SYBR Green I fluorescence labeling dye to assess the prototype performance. The measured results from the image-processing scheme indicate that the RT-cPCR prototype with a CCD-based fluorometer can achieve similar DNA quantification reproducibility compared to commercial machines, even when the initial DNA concentration in the test PCR mix is reduced to 10copies/μL [Copyright &y& Elsevier]

Background & Aims: Immunoprophylaxis reduces but does not completely eradicate hepatitis B virus (HBV) transmission. This prospective study aims at assessing the rate and risk factors of maternally transmitted HBV infection. Methods: We enrolled 303 mother-infant pairs with positive maternal hepatitis B surface antigen (HBsAg) under current immunization program. Maternal viral load was determined by a real-time PCR-based assay. The children were tested for HBsAg at 4–8months and/or 1–3years of age. Rates of HBV infection were estimated using a multivariate logistic regression model. Results: HBeAg-positive mothers (81/303, 26.7%) had higher viral loads than HBeAg-negative mothers (7.4±1.9 vs. 2.7±1.4 log10 copies/ml, p <0.0001). Ten children, born to HBeAg-positive mothers with high viral load (median, 8.4; range, 6.5–9.5log10 copies/ml), were chronically infected. After adjustment for maternal age, birth type, factors related to maternal-fetal hemorrhage, gestational age, infant gender, birth weight, timeliness of vaccination, and feeding practice, maternal viral load was significantly associated with risk of infection (adjusted odds ratio for each log10 copy/ml increase, 3.49; 95% confidence interval (CI), 1.63–7.48; p =0.001). The predictive rates of infection at maternal viral load levels of 7, 8, and 9log10 copies/ml were 6.6% (95% CI, 0.5–12.6%; p =0.033), 14.6% (95% CI, 5.6–23.6%; p =0.001), and 27.7% (95% CI, 13.1–42.4%; p <0.001), respectively. Conclusions: Additional strategies to further reduce transmission should be considered in mothers with a viral load above 7–8log10 copies/ml. [ABSTRACT FROM AUTHOR]

Objectives: Hepatitis E virus (HEV) is an emerging pathogen. We evaluated the seroprevalence of HEV infection among swine farmers and the general population in Taiwan, a nonendemic country. Methods: We conducted a cross-sectional seroepidemiologic study in rural Taiwan in 2012 and 2013. The study included swine farmers, health examination attendees, pregnant women, and students. A commercial enzyme-linked immunosorbent assay was used to detect immunoglobulin G (IgG) and IgM against HEV. Pertinent information was collected using a questionnaire. Results: In total, 660 participants were enrolled in the study, including 156 swine farmers, 314 health examination attendees, 100 pregnant women, and 90 students. IgG anti-HEV was detected in 29.5% of swine farmers, 11.5% of health examination attendees, 2% of pregnant women, and 1.1% of students. Two subjects were positive for IgM anti-HEV. Logistic regression analysis revealed that swine farmers had an approximately 3.5-fold increased risk (odds ratio [OR], 3.46; 95% confidence interval [CI], 1.91–6.27; p<0.0001) for being seropositive for IgG anti-HEV as compared to the general population. Age was positively associated with seropositive rate (OR, 1.07 per year; 95% CI, 1.05–1.09; p<0.0001). Conclusion: HEV infection is prevalent in Taiwan. The seroprevalence of HEV infection is high in swine farmers and in the elderly population. [ABSTRACT FROM AUTHOR]

Background. The long-term evolution and outcomes of infection with a hepatitis B virus (HBV) surface antigen (HBsAg) gene mutant (hereafter, “HBsAg-mutant HBV”) among immunized children remain unclear.Methods. Ninety-five HBsAg-positive children aged 0.5–18.4 years (mean age, 5.8 years) who did not respond to postnatal immunoprophylaxis were prospectively followed for 1–22.8 years (mean follow-up, 8.5 years). Clinical, serologic, and virologic features were compared between 38 untreated HBV e antigen (HBeAg)–positive children carrying HBsAg-mutant HBV (group 1) and 49 children carrying wild-type HBV (group 2). HBsAg-mutant HBV was examined in 20 immunized children presenting with HBV-related hepatocellular carcinoma (HCC).Results. The initial alanine aminotransferase (ALT) level, the maximal ALT level during the HBeAg-positive phase of HBV infection, the cumulative incidence of HBeAg seroconversion (P = .0018), and the rate of low serum HBV DNA load (defined as <104 copies/mL) at the last visit (P = .006) were higher in group 1 than in group 2. A higher frequency of HBV genotype C and a higher ALT level during surface mutant viremia were observed in codon 110–129 mutants than in codon 144–145 mutants. None of the 95 patients developed cirrhosis or HCC. HBsAg-mutant HBV was detected in 3 of 8 (38%) HBV DNA-positive children with HCC.Conclusions. HBeAg-positive immunized children carrying HBsAg-mutant HBV may develop hepatitis activity, HBeAg seroconversion, and a low viremic state earlier than those carrying wild-type HBV. Continuous monitoring of children with wild-type HBV infection and those with HBsAg-mutant HBV for possible development of HCC is needed. [ABSTRACT FROM PUBLISHER]

Hepatocellular carcinoma ( HCC) presents with a high burden of disease in East Asian countries. Intermediate-stage HCC as defined by the Barcelona Clinic Liver Cancer ( BCLC) staging system poses a clinical challenge as it includes a heterogeneous population of patients that can vary widely in terms of tumour burden, liver function and disease aetiology. Intermediate HCC patients often have unsatisfactory clinical outcomes with repeated transarterial chemoembolization ( TACE, due to non-response of the target tumour or the development of further metastasis indicating progressive disease. In September 2011, an Expert Panel Opinion on Interventions in Hepatocellular Carcinoma ( EPOIHCC) was convened in HK in an attempt to provide a consensus on the practice of TACE. To that end, current clinical practice throughout Asia was reviewed in detail including safety and efficacy data on TACE alone as well as in combination with targeted systemic therapies. This review summarises the evidence discussed at the meeting and provides expert recommendation regarding the available therapeutic options for unresectable intermediate stage HCC. A key consensus of the Expert Panel was that in order to improve patient outcomes and long-term survival, the possibility of using TACE in combination with targeted agents given systemically should be explored. While the currently available clinical data is promising, the expected completion of several pivotal phase II and III RCTs will provide further evidence in support of the rationale for combination therapy regimens. [ABSTRACT FROM AUTHOR]

Background and Purpose Previously, we have shown that sorafenib sensitizes hepatocellular carcinoma ( HCC) to apoptosis induced by TNF-related apoptosis-inducing ligand (TNFSF10; TRAIL). Here, we report that sorafenib and SC-49 sensitize HCC cells to CS-1008, a novel anti-human death receptor 5 (TNFRSF10B) antibody. Experimental Approach HCC cell lines ( PLC5, Huh-7, and Hep3B) were treated with CS-1008 and/or sorafenib and analysed in terms of apoptosis and signal transductions. Key Results SC-49 is a sorafenib derivative, which is devoid of kinase inhibitory activity. Both sorafenib and SC-49 down-regulated the phosphorylation of STAT3 at Tyr705 and subsequently reduced the levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in CS-1008-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to CS-1008 in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effects of sorafenib and SC-49 on CS-1008-induced apoptosis, indicating that inhibition of STAT3 mediates the enhancing effects of these compounds when combined with CS-1008. Importantly, inhibition of SHP-1 by adding a specific SHP-1 inhibitor reduced the effects of SC-49 and CS-1008 on p-STAT3 and apoptosis, whereas co-treatment of CS-1008 with SC-49 increased the activity of SHP-1. These data indicate that the combined effects of CS-1008 and SC-49 on HCC are mediated by SHP-1. Moreover, the combination of CS-1008 and SC-49 inhibited HCC xenograft tumour growth in vivo. Conclusions and Implications Sorafenib and its derivative SC-49 sensitize HCC cells to the antitumour effects of CS-1008 through SHP-1-dependent inactivation of STAT3. [ABSTRACT FROM AUTHOR]

Despite the clinical utility of genetic diagnosis to address idiopathic sensorineural hearing impairment (SNHI), the current strategy for screening mutations via Sanger sequencing suffers from the limitation that only a limited number of DNA fragments associated with common deafness mutations can be genotyped. Consequently, a definitive genetic diagnosis cannot be achieved in many families with discernible family history. To investigate the diagnostic utility of massively parallel sequencing (MPS), we applied the MPS technique to 12 multiplex families with idiopathic SNHI in which common deafness mutations had previously been ruled out. NimbleGen sequence capture array was designed to target all protein coding sequences (CDSs) and 100 bp of the flanking sequence of 80 common deafness genes. We performed MPS on the Illumina HiSeq2000, and applied BWA, SAMtools, Picard, GATK, Variant Tools, ANNOVAR, and IGV for bioinformatics analyses. Initial data filtering with allele frequencies (<5% in the 1000 Genomes Project and 5400 NHLBI exomes) and PolyPhen2/SIFT scores (>0.95) prioritized 5 indels (insertions/deletions) and 36 missense variants in the 12 multiplex families. After further validation by Sanger sequencing, segregation pattern, and evolutionary conservation of amino acid residues, we identified 4 variants in 4 different genes, which might lead to SNHI in 4 families compatible with autosomal dominant inheritance. These included GJB2 p.R75Q, MYO7A p.T381M, KCNQ4 p.S680F, and MYH9 p.E1256K. Among them, KCNQ4 p.S680F and MYH9 p.E1256K were novel. In conclusion, MPS allows genetic diagnosis in multiplex families with idiopathic SNHI by detecting mutations in relatively uncommon deafness genes. [ABSTRACT FROM AUTHOR]

Background & Aims: Quantitative HBsAg has been recognized to assist in the management of chronic hepatitis B virus (HBV) infection. However, its role in disease monitoring of HBeAg-negative patients remains unclear. We aimed to investigate the longitudinal HBsAg change in HBeAg-negative carriers with HBV genotype B or C infection. Methods: This is a retrospective cohort study conducted in a university hospital. Treatment-naïve HBeAg-negative carriers followed for more than 3 years were recruited. Their hepatitis activities were categorized by longitudinal HBV-DNA levels into high viral-load (HVL: HBV-DNA >/ = 2000 IU/mL persistently), low viral-load (LVL: HBV-DNA <2000 IU/mL persistently) and fluctuated viral-load (FVL: HBV-DNA between HVL and LVL). The baseline and end-of-follow-up (EOF) HBsAg levels were quantified for analyses. Results: We recruited 187 patients with a median follow-up of 8 years. LVL patients had a significantly lower HBsAg at baseline and EOF and a significantly greater annualized HBsAg decline compared with the FVL and HVL. The longitudinal HBsAg change was independent of genotype B or C. The lower baseline HBsAg level predicted the HBsAg decline and HBsAg loss, whereas the higher baseline HBV-DNA predicted the hepatitis flare. A baseline HBsAg <50 IU/mL predicted subsequent HBsAg loss with a sensitivity of 82% and specificity of 67%. The annualized HBsAg decline appeared non-linear, and accelerated as the HBsAg level lowered (0.054, 0.091, 0.126 log10 IU/mL in patients with baseline HBsAg >1000, 100–999, <100 IU/mL, respectively, P for trend = .014). Conclusions: In genotype B or C HBeAg-negative carriers, baseline HBsAg levels correlate with future disease activities and help to predict HBsAg decline or loss. Inactive carriers with lower baseline HBsAg levels have a greater and accelerating HBsAg decline over time, regardless of HBV genotypes. [ABSTRACT FROM AUTHOR]

Abstract: In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1–B5 and C1–C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa–IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1–2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development. [Copyright &y& Elsevier]

Background. It is unclear whether hepatitis B e antigen (HBeAg) seroconversion induced by nucleos(t)ide analogues (NUC) has a prognosis that is similar to that of spontaneous HBeAg seroconversion.Methods. A total of 148 noncirrhotic NUC-induced HBeAg seroconverters were consecutively enrolled. A historical control of 407 noncirrhotic spontaneous HBeAg seroconverters was also recruited. We compared the rates of HBeAg seroreversion and HBV reactivation between these 2 cohorts.Results. There were 1652.8 and 465.2 person-years of follow-up for spontaneous and NUC-induced HBeAg seroconverters, respectively. Compared with NUC-induced seroconverters, spontaneous seroconverters were younger when achieving HBeAg seroconversion. We thus compared these 2 cohorts according to their age at HBeAg seroconversion. In patients achieving HBeAg seroconversion before 30 years of age, NUC-induced seroconverters had a higher 2-year HBeAg seroreversion rate than spontaneous seroconverters (12.0% vs 2.9%; P = .004) and were at a higher risk of HBV reactivation (hazard ratio, 4.6; 95% confidence interval, 1.5–14.4). Using multivariate analysis, NUC-induced HBeAg seroconversion remained a risk factor of both endpoints in young HBeAg seroconverters.Conclusion. NUC-induced HBeAg seroconverters may not have durable response after stopping therapy. For patients achieving HBeAg seroconversion before 30 years of age, the risk of HBeAg seroreversion and HBV reactivation is higher in NUC-induced seroconverters than spontaneous HBeAg seroconverters. [ABSTRACT FROM AUTHOR]

Abstract: Obatoclax is a linear oligopyrrole compound which antagonizes the antiapoptotic effects of the Bcl-2 family. Herein we describe the synthesis of obatoclax derivatives by replacement of the pyrrole and indole ring of obatoclax with thiophene, furan and thiazolidinedione. The in vitro cytotoxicity of the newly synthesized compounds is evaluated against hepatocellular carcinoma cells. Pyrrole and indole substituents of obatoclax analogues exhibited potent inhibition of cell growth. Among the tested compounds, 5d and 5e were active at 6.3 and 13.2 μM against PLC5 cells. Further assays confirmed a correlation between cell death, and p-STAT3 inhibition and SHP-1 activation by these analogues. [Copyright &y& Elsevier]

Background Naturally occurring pre-S deletion mutants have been identified in hepatitis B virus ( HBV)-related hepatocellular carcinoma ( HCC). Aims This study investigated whether specific deletions within the pre- S region were associated with HCC development. Methods The virologic characteristics of 56 HBV chronic carriers and 112 age-matched patients with HBV-related HCC were examined. Results The HCC patients had a significantly higher frequency of high viral load, basal core promoter mutation and pre-S deletion than chronic carriers. Sequencing analysis showed that the deleted regions were clustered mainly in the C terminus of pre-S1 (70.5%) and the N terminus of pre-S2 (72.7%) in HCC patients. Immuno-epitope mapping of these pre-S deletion sequences showed that all the deletion regions encompassed T- and B- cell epitopes and the B-cell epitope at amino acid 1-6 of pre-S2 was significantly deleted in HCC patients (60.0% vs. 0.0%; P = 0.036). Functional mapping of these deletion mutants showed that most of HCC patients lost one or more functional sites and the deletion of site for viral secretion (aa 1-5 of pre-S2 domain) was significantly detected in HCC patients than chronic carriers (62.5% vs. 0.0%; P = 0.029). Computational protein function prediction indicated that these mutants may have different molecular functions and participate in other biological processes compared with wild-type pre-S. Conclusions Deletion of B-cell epitope at amino acid 1-6 of pre-S2 region and the site for virion secretion are significantly associated with the development of HCC in HBV carriers. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we demonstrated that the multiple kinase inhibitor sorafenib mediates the repression of phospho-STAT3 in hepatocellular carcinoma cells. In this study, we used this kinase-independent mechanism as a molecular basis to use sorafenib as scaffold to develop a novel class of SHP-1-activating agents. The proof of principle of this premise was provided by SC-1, which on replacement of N-methylpicolinamide by a phenylcyano group showed abolished kinase activity while retaining phospho-STAT3 repressive activity. Structural optimization of SC-1 led to compound 6, which repressed phospho-STAT3 through SHP-1 activation and inhibited PLC5 cell proliferation at sub-micromolar potency. In light of the pivotal role of phospho-STAT3 in promoting tumorigenesis and drug resistance, this novel SHP-1-activating agent may have therapeutic relevance in cancer therapy. [Copyright &y& Elsevier]

Background: Eltrombopag is an oral thrombopoietin-receptor agonist. This study evaluated the efficacy of eltrombopag for increasing platelet counts and reducing the need for platelet transfusions in patients with thrombocytopenia and chronic liver disease who are undergoing an elective invasive procedure. Methods: We randomly assigned 292 patients with chronic liver disease of diverse causes and platelet counts of less than 50,000 per cubic millimeter to receive eltrombopag, at a dose of 75 mg daily, or placebo for 14 days before a planned elective invasive procedure that was performed within 5 days after the last dose. The primary end point was the avoidance of a platelet transfusion before, during, and up to 7 days after the procedure. A key secondary end point was the occurrence of bleeding (World Health Organization [WHO] grade 2 or higher) during this period. Results: A platelet transfusion was avoided in 104 of 145 patients who received eltrombopag (72%) and in 28 of 147 who received placebo (19%) (P<0.001). No significant difference between the eltrombopag and placebo groups was observed in bleeding episodes of WHO grade 2 or higher, which were reported in 17% and 23% of patients, respectively. Thrombotic events of the portal venous system were observed in 6 patients who received eltrombopag, as compared with 1 who received placebo, resulting in the early termination of the study. The incidence and severity of other adverse events were similar in the eltrombopag and placebo groups. Conclusions: Eltrombopag reduced the need for platelet transfusions in patients with chronic liver disease who were undergoing elective invasive procedures, but it was associated with an increased incidence of portal-vein thrombosis, as compared with placebo. (Funded by GlaxoSmithKline; ELEVATE ClinicalTrials.gov number, NCT00678587.) [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma (HCC). LCL161 showed differential effects on apoptosis in four HCC cell lines, and the endogenous level of Bcl-2 determined the sensitivity of HCC cells to LCL161. Cytotoxicity and apoptosis were observed in sensitive PLC5 and Hep3B cells that express lower levels of Bcl-2, but not in resistant Huh-7 and SK-Hep1 cells with higher Bcl-2 expression. Down regulation of Bcl-2 by small interference RNA overcame the resistance to LCL161 in Huh-7, and the apoptotic effect was rescued in Bcl-2-expressing Hep3B. To test the hypothesis that Bcl-2 determines the sensitivity of HCC cells to LCL161, we assayed the biological effect of SC-2001, a novel Bcl-2 inhibitor derived from obatoclax, in LCL161-resistant cell lines. Huh-7 cells co-treated with LCL161 and SC-2001 showed a significant dose-dependent apoptotic effect demonstrated by sub-G1 assay and cleavage of PARP. Furthermore, the combination index (CI) of LCL161 and SC-2001 showed a convincing synergism in resistant Huh-7. In addition, the combinational therapy showed significant growth inhibition in Huh-7-bearing xenograft tumors. Notably, down regulation of Bcl-2 was observed in a tumor sample treated with LCL161 and SC-2001. In conclusion, targeting Bcl-2 with SC-2001 overcomes drug resistance to LCL161 in HCC cells thus suggesting a new anti-IAP combinational therapy for HCC. [Copyright &y& Elsevier]

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-c production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocellular carcinoma (HCC) often displays resistance to recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Dovitinib, a multiple tyrosine kinase inhibitor, and tigatuzumab, a novel humanized anti-human death receptor 5 (DR5) agonistic antibody, are both under clinical investigations in HCC. Here, we report that dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through inhibition of signal transducers and activators of transcription 3 (STAT3). Our data indicate that HCC cells showed significant resistance to TRAIL- and tigatuzumab-induced apoptosis. The combination of dovitinib and tigatuzumab restored the sensitivity of HCC cells to TRAIL- and tigatuzumab-induced apoptosis. Dovitinib down-regulated phospho-STAT3 (Tyr705) (p-STAT3) and subsequently reduced the protein levels of STAT3-regulated proteins, Mcl-1, survivin and cylcin D1, in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA-interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the sensitizing effect of dovitinib on TRAIL-induced apoptosis. Importantly, silencing SHP-1 by RNA-interference reduced the effects of dovitinib and TRAIL on p-STAT3 and apoptosis, whereas co-treatment of TRAIL and dovitinib increased the activity of SHP-1. Moreover, in vivo the combination of tigatuzumab and dovitinib inhibited Huh-7 xenograft tumor growth. In conclusion, dovitinib sensitizes resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through a novel machinery: SHP-1 dependent STAT3 inhibition. [Copyright &y& Elsevier]

Depletion of β-catenin impairs regeneration of the rapid turn-over gut epithelial cells, but appears dispensable for that of the slow turn-over mature hepatocytes in mice until 1 y of age. As the life span of mature murine hepatocytes is about 400 d, we studied conditional β-catenin knockout mice (Alb-Cre;Ctnnb1flx/flx) until 20 mo of age to determine the function of β-catenin in the postnatal liver. β-catenin was absent from the hepatocytes of β-catenin knockout mice 4 wk after delivery. From 9 mo of age, hepatocytes were gradually replaced by newly formed β-catenin-positive hepatocytes, which constituted about 90% of hepatocytes at 18-20 mo of age. This process was accompanied by active proliferation of bile duct/ductule cells. β-catenin-positive hepatocytes exhibited elevated proliferation activity and expression of progenitor cell markers, but lower albumin and Cre. This might explain their intact β-catenin protein, and suggest their origins from hepatic progenitor cells. Liver tumors arose spontaneously from β-catenin-positive cells, and tumorigenesis was accelerated by hepatitis B X protein. These results indicate β-catenin critical for the regeneration of mature hepatocytes. Failure to regenerate mature hepatocytes results in proliferation of hepatic progenitor cells that are able to maintain liver function but are predisposed to form liver tumors. [ABSTRACT FROM AUTHOR]

Background & Aims: Recently, we reported that sorafenib sensitizes hepatocellular carcinoma (HCC) cells to TRAIL through the inhibition of signal transducer and activator of transcription 3 (STAT3). Here, we report that sorafenib inhibits HCC via a kinase-independent mechanism: SHP-1 dependent STAT3 inactivation. Methods: SC-1 is a sorafenib derivative that closely resembles sorafenib structurally but with no kinase inhibition activity. HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib or SC-1 and apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with Huh-7 xenografts. Results: SC-1 showed similar effects to sorafenib on growth inhibition and apoptosis in all tested HCC cell lines. SC-1 down-regulated phosphorylation of phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested HCC cells. Expression of STAT3-driven genes, including Cyclin D1 and Survivin, was also repressed by SC-1. Luciferase reporter assay confirmed the inhibition of transcriptional activity of STAT3 in both sorafenib-treated and SC-1-treated cells. Ectopic expression of STAT3 in PLC5 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 up-regulated SHP-1 activity. Knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced apoptosis induced by SC-1. Finally, SC-1 reduced Huh-7 tumor growth significantly in vivo, which was associated with down-regulation of p-STAT3 and up-regulation of SHP-1 activity. Conclusions: STAT3 is a major kinase-independent target of sorafenib in HCC. [ABSTRACT FROM AUTHOR]

Abstract: This study investigates the feasibility of using mobile phones cameras for DNA detection. DNA amplification uses the convective polymerase chain reaction (cPCR) technique due to its simple mechanism, which requires no thermal cycling control. Fluorescence increment analysis and information entropy analysis are employed separately to determine whether the test samples contain target DNA (Positive) or not (Negative). The fluorescence increment method uses the brightness of the captured images before and after DNA amplification to calculate ΔF. ΔF values above a threshold level indicate that the test sample is positive. The information entropy method defines the probability, P(C/X), which indicates whether the fluorescence image tends towards a specific shape. If a DNA template is successfully amplified, the captured fluorescence image should be a perfect circle. P(C/X) provides a threshold of 0.5 to identify a circle and values above 0.5 indicate the test sample is positive. Experimental results show that P(C/X) is more effective than ΔF for determining DNA detection results. The information entropy analysis method is applied to ten mobile phones of three different brands equipped with camera sensors, which have pixel numbers ranging from 120M to 800M. The clinical evaluation study (n =60) for screening hepatitis B virus (HBV) plasmid samples shows that the accuracy rate of all models of mobile phones ranges from 85% to 100%. This illustrates that successful DNA detection can be achieved using the most widely deployed electronic device. [Copyright &y& Elsevier]

Abstract: STAT3 is a transcription factor that modulates survival-directed transcription. It is persistently activated in many human cancers. Literature has shown that sorafenib, Raf kinase inhibitor, reduces Phospho-STAT3 and induces cell death. A series of sorafenib derivatives were synthesized as new inhibitors for STAT3. Urea, sulfonamide, and carboxamide linkers brought out different SARs from the end of sorafenib. Urea and carboxamide linked derivatives showed greater inhibition against STAT3 activity than sulfonamide linked derivatives. In particular, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophenoxy)phenyl)urea (1), a urea linker, was as potent as sorafenib in reducing P-STAT3 level and cell death but no inhibition for Raf activity. Such result provides a new lead for the design of STAT3 inhibitors. [Copyright &y& Elsevier]

Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4tm1Dontuh/tm1Dontuh mice) homozygous for the c.919-2A.G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4tm1Dontuh/tm1Dontuh mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4tm1Dontuh/tm1Dontuh mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4tm1Dontuh/tm1Dontuh mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4tm1Dontuh/tm1Dontuh mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a4-/- mice and Slc26a4loop/loop mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains. [ABSTRACT FROM AUTHOR]

Summary: Background: Therapy for chronic hepatitis B reduces the risk of progressing to hepatocellular carcinoma (HCC); however, there is no suitable and accurate means to assess risk. This study aimed to develop and validate a simple scoring system to predict HCC risk in patients with chronic hepatitis B. Methods: The development cohort consisted of 3584 patients without cirrhosis from the community-based Taiwanese REVEAL-HBV study (of whom 131 developed HCC during follow-up), and a validation cohort of 1505 patients from three hospitals in Hong Kong and South Korea (of whom 111 developed HCC during follow-up). We used Cox multivariate proportional hazards model to predict risk of HCC at 3, 5, and 10 years. Variables included in the risk score were sex, age, serum alanine aminotransferase concentration, HBeAg status, and serum HBV DNA level. We calculated the area under receiver operating curve (AUROC) and calibration of predicted and observed HCC risk. Findings: A 17-point risk score was developed, with HCC risk ranging from 0·0% to 23·6% at 3 years, 0·0% to 47·4% at 5 years, and 0·0% to 81·6% at 10 years for patients with the lowest and highest HCC risk, respectively. AUROCs to predict risk were 0·811 (95% CI 0·790–0·831) at 3 years, 0·796 (0·775–0·816) at 5 years, and 0·769 (0·747–0·790) at 10 years in the validation cohort, and 0·902 (0·884–0·918), 0·783 (0·759–0·806), and 0·806 (0·783–0·828), respectively, after exclusion of 277 patients in the validation cohort with cirrhosis. Predicted risk was well calibrated with Kaplan-Meier observed HCC risk. Interpretation: A simple-to-use risk score that uses baseline clinical variables was developed and validated. The score accurately estimates the risk of developing HCC at 3, 5, and 10 years in patients with chronic hepatitis B. Clinicians can use this score to assess risk of HCC in patients with chronic hepatitis B and subsequently make evidence-based decisions about their clinical management. Funding: The Academia Sinica; the National Health Research Institute, Taiwan; and Bristol-Myers Squibb. [Copyright &y& Elsevier]

Abstract: Hepatitis B virus (HBV) infection is endemic in many Asian countries. Among many transmission routes, transfusion is the one that should be prevented. The first major success in enhancing transfusion safety came with the implementation of hepatitis B surface antigen (HBsAg) in the early 1970s. However, the studies quoted in this review demonstrate that transmission by blood components negative for HBsAg can still occur in the acute phase of infection during the seronegative window period, or during chronic stages of infeetion (i.c. “occult” HBV infection, OHB). OHB is defined as the presence of HBV DNA in blood or liver tissues in patients negative for HBsAg, with or without any HBV antibodies. Because of limitations in current blood screening practices, OHB is an overlooked source of HBV transmission. For policy development on screening for HBV infection in blood donors, it would be useful to assess the relative contribution of the above two sources of transfusion-transmitted HBV infection from HBsAg-negative donations. New screening policy should be evaluated on the basis of available data or newly designed studies. While anti-HBc screening can climinate residual risk of occult HBV transmission by transfusion in low-endemic areas, it would not be practical in most parts of the world where the prevalence of anti-HBc is >10% as too many otherwise healthy donors will be ineligible. On the contrary, studies mentioned in this paper indicate that nucleic acid amplification test (NAT) or new HBsAg tests of enhanced sensitivity would be effective in the screening of blood donors for OHB in highly endemic countries. However, the cost-effectiveness of blood screening tests is a major concern in Asia. We therefore have systemically reviewed the literature on prevalence and infectivity of OHB in Asian countries and the possible role of NAT for identifying blood donors in the pre-HBsAg window phase or in later stages of OHB. [Copyright &y& Elsevier]

Background & Aims: Sorafenib, a multi-kinase inhibitor with anti-angiogenic activity, was recently approved for the treatment of advanced hepatocellular carcinoma (HCC). Metronomic chemotherapy using tegafur/uracil (4:1molar ratio), an oral fluoropyrimidine, has been shown to enhance the anti-tumor effect of anti-angiogenic agents in preclinical models. This phase II study evaluated the efficacy and safety of combining metronomic tegafur/uracil with sorafenib in patients with advanced HCC. Methods: Patients with histologically- or cytologically-proven HCC and Child-Pugh class A liver function were treated with sorafenib (400mg twice daily) and tegafur/uracil (125mg/m2 based on tegafur twice daily) continuously as first-line therapy for metastatic or locally advanced disease that could not be treated by loco-regional therapies. The primary endpoint was progression-free survival (PFS). Results: The study enrolled 53 patients. Thirty-eight patients (72%) were hepatitis B surface antigen-positive. The median PFS was 3.7months (95% C.I., 1.9–5.5) and the median overall survival was 7.4months (95% C.I., 3.4–11.4). According to RECIST criteria, 4 patients (8%) had a partial response and 26 patients (49%) had a stable disease. Major grade 3/4 toxicities included fatigue (15%), abnormal liver function (13%), elevated serum lipase (10%) hand-foot skin reaction (HFSR) (9%), and bleeding (8%). HFSR was the major adverse event resulting in dose reduction (19%) or treatment delay (21%). Conclusions: Metronomic chemotherapy with tegafur/uracil can be safely combined with sorafenib and shows preliminary activity to improve the efficacy of sorafenib in advanced HCC patients. [Copyright &y& Elsevier]

Background & Aims: Previously we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. Here we report that combined treatment with sorafenib and bortezomib shows synergistic effects in HCC. Methods: HCC cell lines (PLC/PRF/5, Huh-7, and Hep3B) were treated with sorafenib and/or bortezomib and analyzed in terms of apoptosis signal transduction. In vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. Results: Pretreatment with sorafenib enhanced bortezomib-induced apoptotic cell death by restoring bortezomib’s ability to inactivate Akt in PLC/PRF/5 cells. Knocking down Akt1 by RNA-interference overcame apoptotic resistance to bortezomib in PLC/PRF/5 cells and ectopic expression of active Akt in HCC cells abolished the bortezomib sensitizing effect of sorafenib, indicating Akt inactivation plays a key role in mediating the combinational effects. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of phospho-Akt (P-Akt) expression induced by co-treatment with sorafenib and bortezomib, and 1, 9 di-deoxy-forskolin, a PP2A agonist, restored bortezomib’s effect on P-Akt and apoptosis. Importantly, silencing of PP2A by RNA-interference reduced the apoptotic effect induced by sorafenib–bortezomib co-treatment, indicating that PP2A is indispensable for mediating the effects of these drugs. Notably, sorafenib with bortezomib increased PP2A activity in PLC/PRF/5 cells without altering protein levels of PP2A complex or the interaction between PP2A and Akt proteins. Finally, sorafenib plus bortezomib significantly suppressed PLC/PRF/5 xenograft tumor growth, down-regulated P-Akt expression, and up-regulated PP2A activity. Conclusions: The combination of sorafenib and bortezomib shows synergy in HCC through PP2A-dependent Akt inactivation. [Copyright &y& Elsevier]

Abstract: In this study, a new polymerase chain reaction (PCR) system incorporated with a fluorescence detection module and a micro PCR chip has been demonstrated to allow for on-line detection of infectious diseases. A new flow-through PCR chip was developed to reduce cooling and heating times for a PCR process. It is comprised of two micro modules for thermal and microfluidic control. The microfluidic control module in this chip adopts three serpentine-shape micropumps to rapidly transport DNA samples and PCR reagents through three reaction chambers. Using this approach, one can randomly adjust cycle numbers and reaction times of the DNA samples for each reaction, thus optimizing a PCR process. The micro thermal control module consists of three individual array-type heaters and temperature sensors to modulate three specific temperatures for three thermal steps of a PCR process. The optical detection system is then used for detection and quantification of the amplified products. The amplification and detection of detection genes associated with two viruses, specifically hepatitis B virus (HBV) and hepatitis C virus (HCV), has been performed to demonstrate the capabilities of the developed system. The miniaturized PCR system has been successfully used for DNA amplification and quantification and is a promising new tool for molecular diagnosis. [Copyright &y& Elsevier]

Background/Aims: Hepatocellular carcinoma recurrence after curative treatment adversely influences clinical outcome. It is important to explore adjuvant therapies. This phase II/stage 1 multi-center, randomized trial investigated the safety, optimal dosage and preliminary efficacy of PI-88, a novel heparanase inhibitor, in the setting of post-operative recurrence of HCC according to a Simon’s 2-stage design. Methods: Three groups were included: one untreated arm (Group A) and two PI-88 arms (Group B: 160mg/day; Group C: 250mg/day). Treatment groups received PI-88 over nine 4-week treatment cycles, followed by a 12-week treatment-free period. Safety and optimal dosage were assessed. Results: Overall, 172 patients were randomized and 168 were included in the intention-to-treat (ITT) population. Treatment-related adverse effects included cytopenia, injection site hemorrhage, PT prolongation, etc. Four serious adverse events were possibly related to PI-88 treatment. One (1.8%) group B patients and six (10.5%) group C had hepatotoxicity-related withdrawals. Among the ITT population, 29 patients (50%) in Group A, 35 (63%) in Group B, and 22 (41%) in Group C remained recurrence-free at completion. Calculated T1 value suggested 160mg/day treatment satisfied the criteria for the next stage of the trial. Conclusions: PI-88 at 160mg/day is optimal and safe, and shows preliminary efficacy as an adjunct therapy for post-operative HCC. [Copyright &y& Elsevier]

Background/Aims: Patients infected with hepatitis C virus (HCV) genotype 2 or 3 usually respond better to interferon (IFN) treatment than those infected with genotype 1. In this study, we investigated whether the non-structural 5A protein (NS5A) of HCV genotypes 1 and 2 (1b-NS5A and 2a-NS5A, respectively) exerted differential counteractivities against IFN treatment. Methods: We compared the inhibitory effects of 1b-NS5As and 2a-NS5As on IFN activity. We also investigated the replication inhibition of HCV subgenomic replicons containing 1b-NS5A or 2a-NS5A in response to IFN treatment. Results: 1b-NS5As exerted more profound inhibitory effects on IFN activity than 2a-NS5As. The replication of the 2a-NS5A-containing replicons was more sensitive to IFN treatment than that of the 1b-NS5A-containing replicons. Deletion of the interferon sensitivity-determining region/protein kinase R-binding domain (PKR-BD), the V3 domain, or the C-terminus region of NS5A significantly abrogated its anti-IFN activity. Domain swapping between 1b-NS5A and 2a-NS5A in the V3 domain and/or the C-terminus region resulted in a transfer of their anti-IFN activity. Conclusions: 1b-NS5As exert higher magnitudes of IFN antagonism than do 2a-NS5As. The V3 and the C-terminus regions are responsible for the differential anti-IFN effects. This phenomenon may partly explain the genotype-linked differences in the response of HCV to IFN treatment. [Copyright &y& Elsevier]

Abstract: Enteroviruses (EVs) are among the most common pathogens in humans. EV71 infections have caused devastating enterovirus-associated outcomes in children globally. In this study, eleven EV71 isolates in Taiwan during 2006–2007 were selected for N-terminal VP1 gene analysis. A fragment of 403bp on VP1 gene was sequenced and a phylogenetic analysis was performed. In addition, the full-length genome sequencing was carried out on two selected isolates. The results showed that subgenogroups of B5 and C5 had circulated and become predominant in Taiwan over the specified 2 years. Moreover, glutamic acid and threonine are found conservative at positions 43 and 58 on VP1 for genogroup B; however they are replaced by lysine and alanine, respectively, for genogroup C. To our knowledge, this is the first report describing the circulation of these two EV71 subgenogroups in Taiwan. [Copyright &y& Elsevier]

Accurate epidemiological data on common deafness genes are essential to improve the efficiency and to reduce the cost of molecular diagnosis. They may depend on several factors, including a clear delineation of the source of patients being studied. In the present study, we hypothesize that patients with idiopathic sensorineural hearing loss recruited from different sources might reveal discrepancies in the epidemiological results of genetic screening, because patients from different sources might demonstrate distinct clinical or audiologic features and thus result in biased selection of subjects. To elucidate the relative importance of common deafness genes in Taiwanese and to verify our hypothesis, we conducted a prospective project screening mutations in GJB2, SLC26A4 and mitochondrial 12S rRNA gene in a total of 420 Taiwanese families with idiopathic bilateral sensorineural hearing loss, of which 325 families were recruited from hospitals and 95 from hearing rehabilitation facilities. Allele frequencies of common mutations in these three genes and distributions of the corresponding genotypes were then compared between the two groups. The allele frequencies of mutations in SLC26A4, GJB2 and mitochondrial 12S rRNA in the probands of the 420 families were 14.4, 21.7 and 3.8%, respectively. The allele frequency of SLC26A4 mutations in the hospital group was significantly higher than that in the rehabilitation facility group (16.2 vs. 8.4%, chi(2)-test, p < 0.05), whereas no difference in the frequencies of GJB2 mutations and mitochondrial 12S rRNA mutations was found between the two groups. Distributions of probands classified by SLC26A4 genotypes were also different between the two groups (chi(2)-test, p < 0.05). Accordingly, a discrepancy in the genetic screening results might exist between different sources of idiopathic hearing-impaired patients. Further analysis of audiological results and construction of a logistic regression model showed that different audiological features, namely hearing levels and hearing loss patterns, might be responsible for the unequal distributions of mutations and probands between the hospital and rehabilitation facility groups. [ABSTRACT FROM AUTHOR]

Background and Aims: Although insulin resistance affects liver fibrosis progression and treatment response in chronic hepatitis C (CHC), the relationship between chronic hepatitis C virus (HCV) infection and insulin resistance (IR) remains to be firmly established. We thus studied the impact of host, metabolic and viral factors on IR in CHC patients. Methods: A total of 162 CHC patients with complete clinical data were enrolled. Among them, 94 received histological examinations. Quantitative HCV RNA was assayed by a real-time polymerase chain reaction (PCR) assay. Genotyping was performed by reverse transcription PCR with type-specific primers. The pretreatment IR index was determined using homeostasis model assessment (HOMA), and an index value of more than 2.4 was designated IR. Unadjusted and adjusted association of the HCV RNA level and IR was further analysed. Results: In multivariate linear regression analysis, a dose–response relationship was observed between the log10 HCV RNA level and the presence of IR. IR was positively correlated with body mass index, triglyceride, HCV RNA and alanine aminotransferase levels, but negatively correlated with adiponectin level. Subgroup analysis stratified by HCV genotype showed that there was a trend towards a higher HOMR-IR index value and lower adiponectin levels in genotype 1 patients. Histological analysis showed that IR was positively associated with the severity of hepatic steatosis. Conclusions: Our data indicate that higher HCV RNA levels are associated with the presence of IR in CHC patients. Further studies are needed to clarify the interplays between HCV infection, IR and adiponectin in an attempt to develop new adjuvant therapy for CHC. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a worldwide health issue that has started receiving attention but is still poorly understood. However, the hepatitis B virus (HBV) and the hepatitis C virus (HCV) are known to be two major causative agents of HCC. They differ in their modes of infection, their treatment options, their genomes and their carcinogenic abilities. However, both share a link with HCC through alterations of the host genome. In order to continue in our search for the mechanisms behind viral hepatocarcinogenesis, the individual entities (HBV, HCV, HCC and host), their natural history, treatment options and genomic properties must be further understood. Additionally, an understanding of the genomics, the link between the entities, is crucial for the success of the ongoing search for therapeutic options for HCC. Similar to most types of cancer, hepatocarcinogenesis is a multistep process involving different genetic alterations that ultimately lead to malignant transformation of the hepatocyte. As technology advances and research continues, the genetic changes and influences among these entities will prove essential to improved diagnostic and therapeutic options. It remains a challenge to provide a clear picture of the connection between virus and cancer. We review (i) the epidemiological link between HBV/HCV infection to HCC; (ii) prevention and control of chronic hepatitis B or C in reducing HCC risk; and (iii) genetic characters of viruses and hosts and the mechanisms associated with HCC susceptibilities, with the intention of providing a direction for future research and treatment. [ABSTRACT FROM AUTHOR]

Abstract: Influenza B viruses were predominant in Taiwan during the 2004–2005 epidemic and both Victoria and Yamagata lineage viruses co-circulated. A reassortant influenza B virus that contained a Victoria lineage hemagglutinin (HA) gene and Yamagata lineage neuraminidase (NA) gene appeared first in 2002 and became predominant during the 2004–2005 epidemic. During the 2006–2007 epidemic, an influenza B outbreak occurred in Taiwan and only Victoria lineage viruses circulated. We characterized the viruses isolated in the 2006–2007 epidemic and found that the HA genes of influenza B viruses from that epidemic were highly similar to those from the 2004–2005 epidemic. We also analyzed the NA genes of isolates from the 2006–2007 epidemic and found that they all belonged to the Yamagata lineage and formed a new genetic subclade. Comparison of isolates from the 2004–2005 and 2006–2007 epidemics revealed four substitutions, N220K, E320D, K343R and E404K in NA genes. Although the HA sequences from the 2006–2007 epidemic were similar to those from the 2004–2005 epidemic, the NA sequences differed, suggesting distinct patterns of evolution of the HA and NA genes from 2004–2007 in Taiwan. This study emphasizes that the evolution of the NA genes may contribute to reemergence of influenza B viruses. [Copyright &y& Elsevier]

Risk factors for development of non-alcoholic steatohepatitis include obesity, especially central adiposity, glucose intolerance or type 2 diabetes mellitus (T2DM), and dyslipidemia. Non-alcoholic fatty liver disease (NAFLD) is now considered a manifestation of metabolic syndrome. During the last two decades, NAFLD has become the most common chronic liver disease in North America and Europe, but until recently was thought to be uncommon (perhaps due to the lack of study) in Asia. Fatty liver can be identified on imaging modalities (ultrasonography, computed tomography scans, and magnetic resonance imaging) with high sensitivity, but steatohepatitis and fibrosis cannot be distinguished. Thus, an inherent drawback in studying the epidemiology of NAFLD is the lack of definitive laboratory tests, no uniform definition—with different studies using cut-off values of alcohol consumption from <20 g/week to 210 g/week, and case selections where biopsy was used for definition. In studies outside the region, the prevalence of NAFLD varies from 16% to 42% by imaging, and 15–39% of liver biopsies. The major risk factors for NAFLD, central obesity, T2DM, dyslipidemia, and metabolic syndrome, are now widely prevalent and are increasing geometrically in the Asia–Pacific region. It is therefore not surprising that NAFLD is common in this region. Estimates of current prevalence range from 5% to 30%, depending on the population studied. Central obesity, diabetes, and metabolic syndrome are the major risk factors. To date, however, data on the natural history and impact of NAFLD causing serious significant chronic liver disease are lacking and there is a need for prospective, cooperative studies. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to compare the clinical features and survival in patients with hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) and hepatitis C virus-related hepatocellular carcinoma (HCV-HCC). A total of 2820 HCC patients were included. The mean age of HBV-HCC patients was 52.5±13.4 years, while it was 64.8±8.7 years in HCV-HCC patients. The male/female ratio was 7.8 in HBV-HCC, while it was 1.7 in HCV-HCC. The mean α-fetoprotein level in HBV-HCC was 11,661±22,805ng/mL, while it was 5079±15,005ng/mL in HCV-HCC. The mean tumour size was 6.4±4.1cm in HBV-HCC, while it was 4.6±3.1cm in HCV-HCC. The median survival was 11.1 months in HBV-HCC, while it was 23.9 months in HCV-HCC. Compared with HBV-HCC patients, HCV-HCC patients were older, had a lower male/female ratio, lower white blood cell count, lower serum albumin level, higher serum ALT level, lower serum α-fetoprotein level, smaller tumour size and survived longer. [Copyright &y& Elsevier]

We investigated whether infection with a novel human coronavirus (HCoV), called "New Haven coronavirus" (HCoV-NH)--which is similar to and likely represents the same species as another novel HCoV, HCoV-NL63--is associated with Kawasaki disease (KD) in Taiwan. Fifty-three patients with KD were enrolled in our study. Serum, peripheral-blood mononuclear cells, nasopharyngeal aspirates, throat swabs, and rectal swabs from these patients were assayed for HCoV-NL63 by real-time reverse-transcriptase (RT) polymerase chain reaction (PCR), and the throat swabs, nasopharyngeal aspirates, and rectal swabs were also assayed for HCoV-NH by RT-PCR. All PCR results were negative for both HCoV-NL63 and HCoV-NH; therefore, we did not find any association between HCoV-NH infection and KD in Taiwan. [ABSTRACT FROM AUTHOR]

Background/Aims: The infectivity of occult hepatitis B virus (HBV), defined as HBsAg-negative but HBV DNA-positive, after transfusion has been low but not negligible. To address this, we investigated the incidence of post-transfusion HBV infection after receiving screened blood units in Taiwan. Methods: Consecutive HBV-naïve (anti-HBc-negative) recipients with normal ALT were followed for HBV DNA and serologic markers before and after transfusion. Among 4448 blood recipients, 467 (10.5%) were anti-HBc-negative. Post-transfusion 6-month follow-up was completed for 327. We identified 5 (1.5%) who developed hepatitis B viremia 1 week after transfusion. Three were children who later seroconverted to anti-HBc but with normal ALT indicating subclinical acute infection, despite all had anti-HBs from previous vaccination. One had transient transfusion-transmitted HBV without seroconversion to anti-HBc and one possibly had occult HBV infection. Our findings suggested the possibility that occult HBV infection was transmissible by transfusion. The incidence of post-transfusion acute HBV infection was 0.9% (100 per million units) in naïve recipients in Taiwan, a figure 7∼40-fold higher than in developed countries. Moreover, some vaccinated children with anti-HBs were still susceptible. Conclusions: Therefore, despite active immunization, sensitive screening assays for occult HBV infection such as nucleic acid amplification test could be considered in endemic areas. [Copyright &y& Elsevier]

Lin C-L, Liao L-Y, Wang C-S, Chen P-J, Lai M-Y, Chen D-S, Kao J-H. Basal core-promoter mutant of hepatitis B virus and progression of liver disease in hepatitis B e antigen-negative chronic hepatitis B.Liver International: 2005: 25: 564–570.© Blackwell Munksgaard 2005The long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are distinct from those in HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in this special clinical setting remain largely unknown. We thus investigated the association of hepatitis B virus (HBV) genotypes as well as precore/basal core-promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative chronic hepatitis B in Taiwan.HBV genotypes and sequences of precore and basal core-promoter regions of the HBV genome were determined in 174 HBeAg-negative chronic HBV infection patients including 62 inactive carriers and 112 with different stages of liver disease.HBV carriers with older age (>50 years) (odds ratio, 9.09; 95% confidence interval (CI), 3.22–25,P<0.001) and basal core-promoter mutant of HBV (odds ratio, 4.12; 95% CI, 1.41–12.03,P=0.01) were associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). The gender-related risk factors associated with the development of liver cirrhosis and HCC were further analyzed, and basal core-promoter mutant was only associated with the development of liver cirrhosis and HCC in male carriers (odds ratio, 4.35; 95% CI, 1.30–14.52,P=0.02).The risk of development of liver cirrhosis and HCC is significantly increased in patients with advanced age as well as with basal core-promoter mutant of HBV. In addition, basal core-promoter mutant might contribute to the gender difference of the progression of liver disease in HBeAg-negative chronic hepatitis B in Taiwan. [ABSTRACT FROM AUTHOR]

Mah Y-H, Kao J-H, Liu C-J, Chen C-L, Chen P-J, Lai M-Y, Chen D-S. Prevalence and clinical implications of HFE gene mutations (C282Y and H63D) in patients with chronic hepatitis B and C in Taiwan.Liver International DOI: 10.1111/j.1478-3231.2005.01055.x.© Blackwell Munksgaard 2005The implication of hemochromatosis (HFE) gene mutations in chronic viral hepatitis remains controversial. We therefore studied the prevalence of HFE mutations and their impact on the progression of chronic viral hepatitis in Taiwan.H63D and C282Y mutations were screened by using polymerase chain reaction followed by restriction fragment length polymorphism in 152 chronic hepatitis B patients with various stages of liver disease, 87 chronic hepatitis C patients with various stages of liver disease, and 49 healthy controls. The distribution of each allele frequency was then compared among different groups of patients and in various stages of liver disease.All three groups of patients were C282Y wild type and the majority of H63D mutations were heterozygotes. Although statistically not significant, allele frequencies of H63D mutation in hepatitis B-related liver cirrhosis (6%) and hepatitis C-related liver cirrhosis (9.1%) were higher than those in healthy control (2%). After adjustment for age and sex, hepatitis B patients with H63D heterozygosity had a higher likelihood of cirrhosis than those with H63D wild type (odds ratios (OR): 3.2, confidence interval (CI): 0.49–20.5,P=0.22). Similarly, hepatitis C patients with H63D homozygosity had a higher likelihood of cirrhosis compared with those with H63D wild type (OR: 2.35, CI : 0.19–28.5,P=0.52).Almost all Taiwanese are C282Y wild type. H63D heterozygote and homozygote, occurring in less than 5% of the subjects, tended to be associated with the development of liver cirrhosis, irrespective of viral etiology. Screening for H63D mutation might be considered in patients with chronic viral hepatitis in Taiwan. [ABSTRACT FROM AUTHOR]

Both viral titer and genotype of hepatitis B virus (HBV) play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. In this study, a method was developed to determine both parameters in a single-tube reaction.The method contains two consecutive steps, the first step used real-time PCR for quantification and second step used melting curve analysis for genotyping. For accurate quantification, the PCR primers and hybridization probes were selected from highly conserved regions to ensure the equivalent amplification and hybridization of all genotypes of HBVs. Within the sensor probe there exists signature single nucleotide polymorphisms (SNPs), which could effectively differentiate different HBV genotypes by showing different melting temperatures.The quantification results showed great consistency with the commercial assays in linear range from 102 to 1011copies/ml. By comparison with the traditional restriction fragment length polymorphism (RFLP) methods, 99% of samples were accurately genotyped by current assay, and with a higher detection rate. In addition, this method can detect mixed HBV infections.Currently, this methodology can be applied to areas prevalent with HBV genotypes B and C, providing an efficient alternative for clinical diagnosis and large-scaled longitudinal studies. [Copyright &y& Elsevier]

Vaccinia vaccines have become important vectors for antigen-specific immunotherapy. Calreticulin has been shown to enhance MHC class I presentation of linked peptide/protein and may be useful for antigen-specific cancer treatment. An innovative vaccine administering antigen linked to calreticulin via a vaccinia vector may generate a potent antigen-specific antitumor response. We tested the efficacy of linking calreticulin (CRT) to model antigen human papilloma virus type 16 (HPV-16) E7 in the context of a vaccinia vaccine (Vac-CRT/E7). Intraperitoneal vaccination of C57BL/6 mice with Vac-CRT/E7 led to a dramatic increase in E7-specific IFN-γ-secreting CD8+ T cells and a potent antitumor effect against E7-expressing tumors compared to immunization with Vac-E7 or Vac-CRT. When compared to other chimeric vaccinia vaccines employing various intracellular targeting strategies previously developed in our lab, Vac-CRT/E7 elicited the highest number of E7-specific CD8+ T cells. Thus, vaccination with vaccinia expressing CRT linked to a tumor antigen may represent an advantageous strategy for cancer immunotherapy. [Copyright &y& Elsevier]

Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-P1 in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs. [Copyright &y& Elsevier]

To determine whether the two domains of hepatitis C virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins—the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3–NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3–NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein, R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3–NS4A. Together, these results suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities. [Copyright &y& Elsevier]

For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis, we conducted anchorage-independent growth (AIG) assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells. After cloning of genes by differential subtractive chain reactions (DSC) from strong AIG to AIG negative cells, we sequenced 2304 clones and identified 245 genes. After four stringent criteria for selection of transforming genes among DSC clones, our results of quantitative RT-PCR analysis indicated that six genes, DDX3, EIF3S2, CLIC1, HDGF, GPC3, and HSPCA were overexpressed in 64%, 62%, 60%, 58%, 49%, and 47%, respectively, of 45 hepatocellular carcinoma (HCC) tissues. The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (>100-fold), DDX3 and CLIC1 were moderate, GPC3 and HSPCA were weak, and HDGF was none in forming colonies in soft agar. Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes. In addition, our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HCC. [Copyright &y& Elsevier]

Since early March 2003, the severe acute respiratory syndrome (SARS) coronavirus (CoV) infection has claimed 346 cases and 37 deaths in Taiwan. The epidemic occurred in two stages. The first stage caused limited familial or hospital infections and lasted from early March to mid-April. All cases had clear contact histories, primarily from Guang-dong or Hong Kong. The second stage resulted in a large outbreak in a municipal hospital, and quickly spread to northern and southern Taiwan from late April to mid-June. During this stage, there were some sporadic cases with untraceable contact histories. To investigate the origin and transmission route of SARS-CoV in Taiwan's epidemic, we conducted a systematic viral lineage study by sequencing the entire viral genome from ten SARS patients. SARS-CoV viruses isolated from Taiwan were found closely related to those from Guang-dong and Hong Kong. In addition, all cases from the second stage belonged to the same lineage after the municipal hospital outbreak, including the patients without an apparent contact history. Analyses of these full-length sequences showed a positive selection occurring during SARS-CoV virus evolution. The mismatch distribution indicated that SARS viral genomes did not reach equilibrium and suggested a recent introduction of the viruses into human populations. The estimated genome mutation rate was &ap;0.1 per genome, demonstrating possibly one of the lowest rates among known RNA viruses. [ABSTRACT FROM AUTHOR]