Preclinical Evaluation of An Anti-HCV miRNA Cluster for Treatment of HCV Infection

We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17–92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection.     

Impact of HIV on HCV, GBV-C/HGV, and HBV infection

To investigate influence of HIV co-infection on HCV, we examined 58 Japanese hemophiliacs with chronic hepatitis C(CHC) including 25 patients co-infected with HIV. HCV RNA levels and liver function were not affected statistically by the presence of HIV. Further studies are necessary to evaluate the impact of HIV on the prognosis of CHC in hemophiliacs. We were the first to report a beneficial effect of GBV-C/HGV infection on the course of HIV disease and many studies were confirmed our results. Discussions of these important issues such as impact of HIV on GBV-C/HGV and HBV have been performed in this paper.     

Prospects of RNAi and microRNA-based therapies for hepatitis C

Background: RNA interference (RNAi) represents a promising new approach to combat viral infections, and recent developments in the field of gene therapy have increased the feasibility of clinical applications. Objective: to explore the utility of RNAi for the treatment of the ultimately life-threatening liver disease caused by hepatitis C virus (HCV), which affects approximately 170 million people worldwide. Methods: A review of current developments in liver-directed gene delivery and the potential application of RNAi for the treatment of HCV. In addition, the involvement of microRNAs (miRNA) in HCV infection and the potential therapeutic implications are emphasized. Conclusions: RNAi technologies have fuelled rapid progress in the basic understanding of HCV biology and revealed numerous new viral and host-cell factors as potential targets for therapy. Together with the improvement of gene delivery technology and the discovery of the critical role of miRNA in HCV infection, RNAi and miRNA-based antiviral strategies hold great promise for the future.     

Evaluating the consistency of differential expression of microRNA detected in human cancers

Differential expression of microRNA (miRNA) is involved in many human diseases and could potentially be used as a biomarker for disease diagnosis, prognosis, and therapy. However, inconsistency has often been found among differentially expressed miRNAs identified in various studies when using miRNA arrays for a particular disease such as a cancer. Before broadly applying miRNA arrays in a clinical setting, it is critical to evaluate inconsistent discoveries in a rational way. Thus, using data sets from 2 types of cancers, our study shows that the differentially expressed miRNAs detected from multiple experiments for each cancer exhibit stable regulation direction. This result also indicates that miRNA arrays could be used to reliably capture the signals of the regulation direction of differentially expressed miRNAs in cancer. We then assumed that 2 differentially expressed miRNAs with the same regulation direction in a particular cancer play similar functional roles if they regulate the same set of cancer-associated genes. On the basis of this hypothesis, we proposed a score to assess the functional consistency between differentially expressed miRNAs separately extracted from multiple studies for a particular cancer. We showed although lists of differentially expressed miRNAs identified from different studies for each cancer were highly variable, they were rather consistent at the level of function. Thus, the detection of differentially expressed miRNAs in various experiments for a certain disease tends to be functionally reproducible and capture functionally related differential expression of miRNAs in the disease.     

CD8 epitope escape and reversion in acute HCV infection

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.     

Expression of apoptotic markers BCL-2 and bax in chronic hepatitis C virus patients

ObjectivesHepatitis C viral infection(HCV) influence the susceptibility to apoptosis. This could lead to insufficient antiviral immune response and persistent viral infection.Design and methodsGroup 1: chronic HCV patients with liver cirrhosis and ascites. Group 2: chronic HCV patients without liver cirrhosis and group 3: healthy subjects as control group. Bcl-2 and Bax expression were evaluated by flowcytometry.ResultsHCV patients (with cirrhosis and ascites) had a statistically significantly low Bcl-2 expression, a significantly high Bax expression and a significantly decreased Bcl-2/Bax ratio compared with controls. While, the results are inverted in the other HCV group. Both groups of HCV, Bcl-2/Bax ratio showed a significant positive correlation with Bcl-2 and a significantly negative correlation with Bax.ConclusionsChronic HCV exhibit a deregulation of apoptosis with the disease progression. This provides an insight into the pathogenesis of chronic HCV infection, and may contribute to the therapy.     

Genomics of hepatitis B and C infections: diagnostic and therapeutic applications of microarray profiling

Microarray profiling offers many potential advances in diagnostic and therapeutic intervention in human disease because of its unparalleled ability to conduct high-throughput analysis of gene expression. However, limitations of this technique relate in part to issues regarding the various methodologies and experimental designs as well as difficulties in the interpretation of results. Despite this, microarray profiling has led to a better understanding of the molecular pathogenesis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Key events in clearance and the development of chronicity of HCV have been identified that may prove to have a role in the development of future treatments. In addition, pharmacogenomic studies of interferon-based treatment for chronic HCV and HBV have provided mechanistic insights into the therapeutic action of interferons. These advances have implications with respect to the development of improved therapeutic agents. New biomarkers for cancer screening and gene profiles with prognostic value for survival have also been developed for hepatocellular carcinoma, which frequently complicates chronic viral hepatitis. Thus, microarray profiling offers enormous potential for improvements in antiviral therapy and our understanding of blood-borne viral hepatitis.     

丙型肝炎病毒感染者胆囊疾病发生情况及临床分析

目的 研究丙型肝炎病毒（Hepatitis C virus, HCV）感染者胆囊疾病的发生情况及相关危险因素。方法 依据彩色超声报告结果将290例HCV感染者分为有胆囊疾病组169例,无胆囊疾病组121例,分析病程、体重指数（BMI）、负性情绪、酗酒史、血清总胆红素(TBIL) 、丙氨酸氨基转移酶(ALT) 、门冬氨酸氨基转移酶(AST)、γ 谷氨酞转肽酶(GGT)、血清白蛋白(ALB)、三酰甘油(TG)、胆固醇(TC)、HCV病毒载量（HCV RNA）、门静脉内径宽度(PVW)、脾脏厚度、腹水等临床资料,探讨HCV感染者发生胆囊疾病的危险因素。结果 290例HCV感染者胆囊疾病的总发生率为58.28%（169/290）,有胆囊疾病组病程、酗酒史、TG、GGT、PVW、脾脏厚度均高于无胆囊疾病组（P＜0.05）,酗酒,GGT,脾脏厚度是HCV感染者发生胆囊疾病的独立危险因素。结论 HCV感染者胆囊疾病的发生率为58.28%,酗酒、脾脏厚度,GGT是HCV感染者发生胆囊疾病的独立危险因素。     

MicroRNAs在恶性肿瘤分子诊断和预后预测中的应用

微小RNA（microRNA,miRNA）是近年来发现的一类长度为18～26个核苷酸的非编码小分子RNA,它在转录后水平调控基因的表达,其表达情况与机体众多生理病理状态密切相关。目前发现,恶性肿瘤组织和血液循环中存在不同于正常机体的特征性miRNA表达谱,通过测定这些miRNA的表达变化可能可以成为恶性肿瘤早期诊断和预后预测的重要手段。本文总结了miRNA在恶性肿瘤性疾病分子诊断领域的研究进展,为疾病分子诊断学研究及临床实践提供参考。     

Hepatitis C infection and type 2 diabetes in American-Indian women

OBJECTIVE: The aim of this study was to describe the association between hepatitis C virus (HCV) infection and type 2 diabetes among a group of American-Indian women who were screened for both conditions. RESEARCH DESIGN AND METHODS: The study population was a convenience sample of women who were receiving prenatal care. All women were systematically screened for both HCV and diabetes. RESULTS: A total of 426 women were included in the sample. HCV infection was detected in 13 (3.1% [95% CI 1.7-5.0]) and type 2 diabetes in 22 (5.2%, [3.3-7.6]) women. Women diagnosed with type 2 diabetes were more obese and had higher serum alanine aminotransferase activity compared with women without diabetes. Four of 13 (30.8% [10.6-58.7]) HCV-infected women and 18 of 413 (4.4% [2.7-6.7]) women without evidence of HCV infection had type 2 diabetes. (odds ratio 9.8 [95% CI 2.4-34.0], Fisher's exact test P = 0.003). In a logistic regression model, increasing age (10-year increments), obesity (by standard deviations from the mean BMI), and positive HCV status were each independently related to the diagnosis of diabetes. CONCLUSIONS: Among American-Indian women, type 2 diabetes is more common in those with than in those without HCV infection. This association and its potential mechanisms may have clinical implications. Investigation into the mechanisms linking HCV infection to the expression of type 2 diabetes may also help to define processes that promote the development of type 2 diabetes in susceptible individuals.     

慢性丙型肝炎合并肾脏疾病的研究进展

丙型肝炎病毒（HCV）感染导致的肝外损害以肾损伤多见,同时慢性肾脏病发展到终末期肾病行血液透析时可增加HCV感染风险。慢性丙型肝炎合并肾脏疾病可加速疾病的进程,造成患者的不良预后。该文对HCV致肾损伤的机制和慢性丙型肝炎合并肾脏疾病的直接抗病毒药物治疗等研究在近年所取得的进展进行综述,旨在加深临床医师对慢性丙型肝炎合并肾脏疾病患者行抗病毒治疗的认识,改善患者预后。     

Chemical modification and design of anti-miRNA oligonucleotides

Antisense techniques have been employed for over 30 years to suppress expression of target RNAs. Recently, microRNAs (miRNAs) have emerged as a new class of small, non-coding, regulatory RNA molecules that widely impact gene regulation, differentiation and disease states in both plants and animals. Antisense techniques that employ synthetic oligonucleotides have been used to study miRNA function and some of these compounds may have potential as novel drug candidates to intervene in diseases where miRNAs contribute to the underlying pathophysiology. Anti-miRNA oligonucleotides (AMOs) appear to work primarily through a steric blocking mechanism of action; these compounds are synthetic reverse complements that tightly bind and inactivate the miRNA. A variety of chemical modifications can be used to improve the performance and potency of AMOs. In general, modifications that confer nuclease stability and increase binding affinity improve AMO performance. Chemical modifications and/or certain structural features of the AMO may also facilitate invasion into the miRNA-induced silencing complex. In particular, it is essential that the AMO binds with high affinity to the miRNA 'seed region', which spans bases 2-8 from the 5'-end of the miRNA.     

Impact of specimen handling and storage on detection of hepatitis C virus RNA

Direct detection of hepatitis C virus (HCV) RNA in serum or plasma is useful for validating the performance of anti-HCV assays and for the discrimination of persons with persistent HCV infections from those with resolved infections. Quantitation of HCV RNA may also be useful for disease prognosis and therapeutic monitoring. Previous studies have reported detection of HCV RNA in 50 to 70 percent of blood donors who were positive on anti-HCV supplemental tests. There is concern that specimen processing and storage conditions might influence the stability, and hence the detectability, of HCV RNA. To address this concern, the rate of detection of HCV RNA by the polymerase chain reaction (PCR) using donor pilot tube sera (PTS) previously subjected to routine donor screening and supplemental testing was compared with HCV PCR results obtained with fresh-frozen plasma (FFP) derived from the same donations. All 16 anti-HCV supplemental test-positive donations evaluated were HCV RNA positive with FFP, whereas only 10 (62.5%) were positive with PTS (p = 0.024). None of 11 FFP or PTS samples from HCV enzyme immunoassay-reactive donations not confirmed by supplemental anti-HCV assays tested positive for HCV RNA. Direct comparison of sample type (serum vs. plasma) and various storage conditions using specimens from two seropositive donors showed that room-temperature storage results in marked reduction in HCV RNA signal, while replicate freezing and thawing caused a moderate reduction. These data indicate that well-controlled sample processing and storage conditions are critical to the sensitive and potentially quantitative analysis of HCV RNA.     

miRNA-29b suppresses prostate cancer metastasis by regulating epithelial-mesenchymal transition signaling

Prostate cancer remains the second leading cause of cancer deaths among American men. Early diagnosis increases survival rate in patients; however, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment are necessary for inhibiting prostate cancer disease progression. Here, we have shown that miRNA-29b (miR-29b) expression was lower in prostate cancer cells (PC3 and LNCaP) as compared with immortalized prostate epithelial cells. Between these two prostate cancer cell lines, metastatic prostate cancer PC3 cells displayed lower expression of miR-29b. We also observed a significant downregulation of miR-29b expression in human prostate cancer tissues as compared with patient-matched nontumor tissues. PC3 cells ectopically expressing miR-29b inhibited wound healing, invasiveness, and failed to colonize in the lungs and liver of severe combined immunodeficient mice after intravenous injection, while PC3 cells expressing a control miRNA displayed metastasis. Epithelial cell marker E-cadherin expression was enhanced miR-29b transfected in prostate cancer cells as compared with cells expressing control miRNA. On the other hand, N-cadherin, Twist, and Snail expression was downregulated in PC3 cells expressing miR-29b. Together these results suggested that miR-29b acts as an antimetastatic miRNA for prostate cancer cells at multiple steps in a metastatic cascade. Therefore, miR-29b could be a potentially new attractive target for therapeutic intervention in prostate cancer.     

Case report: Identification of recombinant HCV genotype 1b–2b by viral sequencing in two patients with treatment failure,who responded to re-treatment with sofosbuvir and daclatasvir

Hepatitis C virus (HCV) infection is a global health problem. HCV has been classified into seven genotypes and >67 subtypes. Genotyping is necessary to enable selection of appropriate treatments. The commercial molecular techniques currently used do not identify some HCV subtypes, mixed infections and recombinant forms. In this study, the core–E1 and NS5B regions were sequenced and phylogenetically analysed to identify infections by HCV recombinant genotype 1b–2b in two patients who had initially been diagnosed with HCV genotype 2 infection by reverse hybridization with a Versant HCV Genotype 2.0 Assay. Response to treatment was monitored by viral kinetics. Therapeutic failure occurred with initial treatment with PEGylated interferon-α2b and ribavirin, but the use of sofosbuvir and daclatasvir on a re-treatment regimen after reclassification of the infecting virus resulted in a sustained virologic response. The use of a sequencing approach in treatment-naïve infected patients could enable physicians to select the optimal therapy and avoid possible relapses and adverse reactions associated with antiviral therapy.     

Chronic hepatitis C and genotyping: the clinical significance of determining HCV genotypes

Chronic hepatitis C, attributed to infection with hepatitis C virus (HCV), is a global health problem. The overall prevalence of viral hepatitis worldwide is estimated to be 3-5% with over 175 million people infected with HCV. Clinically, HCV can establish a persistent, chronic infection contributing to progressive liver disease, including cirrhosis and hepatocellular carcinoma (HCC), requiring intensive treatment regimens, possible liver transplantation and long-term care. Due to the chronic nature of HCV infection and the tremendous burden on healthcare resources, clinicians and laboratorians have looked for key epidemiological, pathological and viral characteristics that may provide insight into disease progression, severity and response to therapy to permit the administration of effective therapeutic regimens as well as long-term management of infected individuals. Determination of viral genotype has been identified as one parameter that could provide direction in the clinical management of patients with chronic HCV infections. The following review provides background on determination of HCV genotypes and the relevance of viral genome characterization in the current clinical setting.