Short peptide nucleic acids (PNA) inhibit hepatitis C virus internal ribosome entry site (IRES) dependent translation in vitro

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) which governs the initiation of protein synthesis from viral RNA represents an ideal target for antisense approaches. Using an original bicistronic plasmid, we first established that sequence and translational activity of HCV IRESs cloned from six patients, whether responders or not to combination therapy, were conserved. We then tested the hypothesis that antisense molecules, i.e. short peptide nucleic acids (PNA), could inhibit HCV translation by binding to the highly conserved IIId or IV loop regions of the IRES. Five 6–10 mer PNAs were designed. They strongly inhibit HCV IRES-driven translation in a rabbit reticulocyte lysate assay. This inhibition was highly specific since corresponding PNAs with only one mismatch were inactive. Short phosphorothioate oligonucleotides of same sequence were unable to inhibit HCV translation. PNA molecule was shown to have anti-HCV activity in Huh-7.5 cells when electroporated with a full-length HCV genome construct. Using oligonucleotide as carrier, PNA was also transfected in HCV replicon-harboring cells and in JFH1 infected Huh-7.5 cells.     

广东地区丙型肝炎患者HCV基因亚型分析

目的 了解广东地区丙型肝炎患者HCV的基因亚型分布特征,为丙型肝炎的治疗与预防提供依据.方法 采用巢式逆转录-聚合酶链反应扩增广东省194例丙型肝炎患者HCVNS5B基因区域,特异性PCR产物测序后通过美国洛斯拉莫斯国家实验室HCV数据库的Blast程序进行HCV基因亚型分析.结果 194例丙型肝炎患者中有99例为单纯HCV感染者、95例为HIV/HCV共感染者.丙型肝炎患者HCV共有8种基因亚型,其中1b亚型占42.3％(82/194)、6a亚型占35.0％(68/194)、3a亚型占11.9％(23/194)、3b亚型占6.7％(13/194)、2a亚型占2.6％(5/194)、1a亚型占0.5％(1/194)、4a亚型与0.5％(1/194)、5a亚型占0.5％(1/194).丙型肝炎患者中的单纯HCV感染者HCV有7种基因亚型,以1b亚型为主,占67.7％(67/99);丙型肝炎患者中的HIV/HCV共感染者HCV有5种基因亚型,以6a亚型为主,占53.7％(51/95).结论 广东地区丙型肝炎患者HCV基因亚型呈现多样性,主要基因亚型为1b亚型和6a亚型;丙型肝炎患者中的单纯HCV感染者与HIV/HCV共感染者HCV主要基因亚型不同.     

针对HBVX基因的反义核酸抑制HBV表达的体外研究

目的筛选反义脱氧寡核苷酸 (ASON)抗乙型肝炎病毒 (HBV)的有效靶向位点。方法设计合成针对HBVX翻译起始位点的ASON即序列Ⅰ ,并设非互补序列作对照即序列Ⅱ ,以人肝癌细胞系 (2 .2 .15 )为细胞模型 ,应用ELISA动态观察不同浓度 (1、5、10 μmol/L)ASON一次性用药对HBV基因表达的抑制作用 ,用MTT比色法观察ASON对细胞代谢的影响 ,以苔盼蓝染色计数观察ASON对细胞活性的影响。结果ASON可有效地抑制HBV基因的表达 ,在 1、5、10 μmol/L浓度时对HBsAg的抑制率分别为 6 3%、45 %和 5 7% ,对HBeAg的抑制率分别为 38%、41%和 5 6 % ;而对照序列Ⅱ对HBsAg和HBeAg的抑制率均低于 12 %。MTT法表明ASON对 2 .2 .15细胞代谢无影响(P >0 .0 5 ) ,苔盼蓝染色计数表明ASON对 2 .2 .15细胞活性无影响 (P >0 .0 5 )。结论HBVX翻译起始区是ASON抗HBV复制表达的有效靶向位点     

Baclofen promotes alcohol abstinence in alcohol dependent cirrhotic patients with hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) and alcoholic liver disease (ALD), either alone or in combination, count for more than two thirds of all liver diseases in the Western world. There is no safe level of drinking in HCV-infected patients and the most effective goal for these patients is total abstinence. Baclofen, a GABA(B) receptor agonist, represents a promising pharmacotherapy for alcohol dependence (AD). Previously, we performed a randomized clinical trial (RCT), which demonstrated the safety and efficacy of baclofen in patients affected by AD and cirrhosis. The goal of this post-hoc analysis was to explore baclofen's effect in a subgroup of alcohol-dependent HCV-infected cirrhotic patients. Any patient with HCV infection was selected for this analysis. Among the 84 subjects randomized in the main trial, 24 alcohol-dependent cirrhotic patients had a HCV infection; 12 received baclofen 10mg t.i.d. and 12 received placebo for 12-weeks. With respect to the placebo group (3/12, 25.0%), a significantly higher number of patients who achieved and maintained total alcohol abstinence was found in the baclofen group (10/12, 83.3%; p=0.0123). Furthermore, in the baclofen group, compared to placebo, there was a significantly higher increase in albumin values from baseline (p=0.0132) and a trend toward a significant reduction in INR levels from baseline (p=0.0716). In conclusion, baclofen was safe and significantly more effective than placebo in promoting alcohol abstinence, and improving some Liver Function Tests (LFTs) (i.e. albumin, INR) in alcohol-dependent HCV-infected cirrhotic patients. Baclofen may represent a clinically relevant alcohol pharmacotherapy for these patients.     

程序性死亡受体-1在慢性病毒性肝炎中的研究进展

摘要：乙型肝炎病毒（HBV）和丙型肝炎病毒(HCV)感染是导致慢性活动性肝炎、肝硬化和肝细胞癌的主要原因。近年来研究发现程序性死亡受体（programmed death-1 PD-1）负性调节病毒特异反应性T细胞的功能,被认为是HBV和HCV肝炎病毒持续感染的决定性因素,在慢性病毒性(HBV和HCV)性肝炎发病中扮演着重要角色。本文就程序性死亡受体（PD-1）在慢性病毒性肝炎中的研究进展进行综述。     

丙型肝炎患者血清透明质酸、生长抑素水平测定的临床意义

目的　探讨血清透明质酸 (HA)、生长抑素 (SS)在丙型肝炎患者中的变化。方法　应用放射免疫分析法测定 4 2例丙型肝炎患者血清中透明质酸和生长抑素含量 ,并与 30名正常健康人作对照。结果　丙型肝炎患者血清中 HA、SS含量显著地高于正常人组 (P<0 .0 1) ,且 HA、SS两组呈明显的正相关 (r=0 .6 2 78,P<0 .0 1)。结论　血清 HA、SS含量的增高与丙型肝炎相关     

Versatility of peptide nucleic acids (PNAs): role in chemical biology,drug discovery,and origins of life

This review briefly discussed nomenclature, synthesis, chemistry, and biophysical properties of a plethora of PNA derivatives reported since the discovery of aegPNA. Different synthetic methods and structural analogs of PNA synthesized till date were also discussed. An insight was gained into various chemical, physical, and biological properties of PNA which make it preferable over all other classes of modified nucleic acid analogs. Thereafter, various approaches with special attention to the practical constraints, characteristics, and inherent drawbacks leading to the delay in the development of PNA as gene therapeutic drug were outlined. An explicit account of the successful application of PNA in different areas of research such as antisense and antigene strategies, diagnostics, molecular probes, and so forth was described along with the current status of PNA as gene therapeutic drug. Further, the plausibility of the existence of PNA and its role in primordial chemistry, that is, origin of life was explored in an endeavor to comprehend the mystery and open up its deepest secrets ever engaging and challenging the human intellect. We finally concluded it with a discussion on the future prospects of PNA technology in the field of therapeutics, diagnostics, and origin of life.     

Inhibition of bovine viral diarrhea virus in vitro by xanthohumol: Comparisons with ribavirin and interferon-α and implications for the development of anti-hepatitis C virus agents

Xanthohumol (XN) is a natural compound with potential antiviral activity. In this study, the ability of XN to inhibit bovine viral diarrhea virus (BVDV), a surrogate model of hepatitis C virus (HCV), was investigated. The antiviral activity of XN was compared with that of ribavirin (RBV) and interferon (IFN)-α. The results showed that XN could inhibit BVDV induced cytopathic effects (CPE). At 1000 TCID₅₀ and 100 TCID₅₀, the values of 50% effective concentration (EC₅₀) were 3.24 ± 0.02 mg/l and 2.77 ± 0.19 mg/l, respectively, and the therapeutic indices were >7.72 and >9.03, respectively. XN inhibited BVDV E2 expression and viral RNA levels in a dose-dependent manner. At 6.25 mg/l, XN decreased the viral RNA from released virus by 3.83 log 10 at 1000 TCID₅₀ and to an undetectable level at 100 TCID₅₀, and decreased the viral RNA level in whole cell culture by 3.36 log 10 and 2.88 log 10 at 1000 TCID₅₀ and 100 TCID₅₀, respectively. The inhibitory activity of XN on CPE, BVDV E2 expression and viral RNA levels was stronger than that of RBV and weaker than that of IFN-α. These results indicate the need to investigate the anti-HCV potential of XN.     

The SATE pronucleotide approach applied to acyclovir: part II. Effects of bis(SATE)phosphotriester derivatives of acyclovir on duck hepatitis B virus replication in vitro and in vivo

The in vitro and in vivo antiviral activities of two mononucleoside phosphotriester derivatives of acyclovir (ACV) incorporating S-acyl-2-thioethyl (SATE) groups are reported using the duck model of hepatitis B (DHBV). In primary duck hepatocyte cultures, the described phosphotriesters significantly inhibited the replication of DHBV at submicromolar concentrations. They were found to be more potent than the parent nucleoside. This result was in agreement with our data concerning the anti-HBV activity of these pronucleotides in HepG2.2.15 cells (previous paper). In vivo, the studied SATE pronucleotide was also found to be more efficient than ACV in infected ducklings upon short-term oral therapy, while intraperitoneal treatment showed high anti-DHBV activity with both ACV and its SATE pronucleotide in this animal model. These findings demonstrate the potential of SATE pronucleotides of ACV as anti-HBV agents.     

Rational drug discovery: Ellagic acid as a potent dual‐target inhibitor against hepatitis C virus genotype 3 (HCV G3) NS3 enzymes

Structure‐based virtual screening (SBVS) has served as a popular strategy for rational drug discovery. In this study, we aimed to discover novel benzopyran‐based inhibitors that targeted the NS3 enzymes (NS3/4A protease and NS3 helicase) of HCV G3 using a combination of in silico and in vitro approaches. With the aid of SBVS, six novel compounds were discovered to inhibit HCV G3 NS3/4A protease and two phytochemicals (ellagic acid and myricetin) were identified as dual‐target inhibitors that inhibited both NS3/4A protease and NS3 helicase in vitro (IC₅₀ = 40.37 ± 5.47 nm and 6.58 ± 0.99 µm , respectively). Inhibitory activities against the replication of HCV G3 replicons were further assessed in a cell‐based system with four compounds showed dose‐dependent inhibition. Compound P8 was determined to be the most potent compound from the cell‐based assay with an EC₅₀ of 19.05 µm . The dual‐target inhibitor, ellagic acid, was determined as the second most potent (EC₅₀ = 32.37 µm ) and the most selective in its inhibitory activity against the replication of HCV replicons, without severely affecting the viability of the host cells (selectivity index > 6.18).     

丙型肝炎病毒抗体酶联免疫诊断试剂中酶结合物稀释液对酶结合物稳定性影响的研究

目的通过对不同酶结合物稀释液的丙型肝炎病毒(HCV)抗体酶联免疫诊断试剂的稳定性对比试验,优选出最佳的酶结合物稀释液用于HCV抗体诊断试剂的生产。方法用不同酶结合物稀释液的HCV抗体酶联免疫诊断试剂,对丙型肝炎质控血清进行检测,检测出4℃试剂盒实验结果各样品S/CO值和37℃3d试剂盒实验结果各样品S/CO值,将实验结果进行对比。结果以中性PBST(加有Tween-20的磷酸盐缓冲液)为缓冲液,以BSA(牛血清白蛋白)和Casein(酪蛋白)无关蛋白作稳定剂的酶结合物稀释液的试剂的实验结果明显优于其余试剂。结论以PBST为缓冲液,以BSA和Casein无关蛋白作稳定剂组成酶结合物稀释液适用于HCV抗体酶联免疫诊断试剂的生产。     

Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-kappaB and STAT-3

Human hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. The HCV RNA genome codes for at least ten proteins. The HCV non-structural protein 5A (NS5A) has generated considerable interest due to its effect on interferon sensitivity via binding and inactivating the cellular protein kinase, PKR. It has been shown that NS5A engages in the endoplasmic reticulum (ER)-nucleus signal transduction pathway. The expression of NS5A in the ER induces an ER stress ultimately leading to the activation of STAT-3 and NF-kappaB. This pathway is sensitive to inhibitors of Ca(2+) uptake in the mitochondria (ruthenium red), Ca(2+) chelators (TMB-8, EGTA-AM), and antioxidants (PDTC, NAC, Mn-SOD). The inhibitory effect of protein tyrosine kinase (PTK) inhibitors indicates the involvement of PTK in NF-kappaB activation by NS5A. This implicates an alternate pathway of NF-kappaB activation by NS5A. The actions of NS5A have also been studied in the context of an HCV subgenomic replicon inducing a similar intracellular event. Thus, activation of NF-kappaB leads to the induction of cellular genes, which are largely antiapoptotic in function. These studies suggest a potential function of NS5A in inducing chronic liver disease and hepatocellular carcinoma associated with HCV infection.