Effects of Entecavir on Hepatitis B Virus Covalently Closed Circular DNA in Hepatitis B e Antigen-Positive Patients with Hepatitis B

The aim of this study was to assess the effect of 48-week entecavir therapy on serum and intrahepatic hepatitis B virus, covalently closed circular DNA (HBV cccDNA) levels in hepatitis B e antigen (HBeAg)-positive patients. A total of 120 patients with HBeAg-positive chronic hepatitis were treated with entecavir for 48 weeks. Serum HBV markers, total HBV DNA, and HBV cccDNA levels were measured at baseline and week 48. Biopsies from 20 patients were available for both intrahepatic total HBV DNA and cccDNA testing at these timepoints. HBV cccDNA levels were decreased from a median level of 5.1×10⁶ copies/mL at baseline to a median level of 2.4×10³ copies/mL at week 48. Reduction magnitudes of HBV cccDNA in patients with normalized alanine aminotransferase levels and those undergoing HBeAg seroconversion were significantly greater than those in alanine aminotransferase-abnormal and HBeAg positive patients. Intrahepatic HBV cccDNA was decreased significantly after 48 weeks of treatment, but could not be eradicated. In conclusion, treatment of HBeAg-positive hepatitis B patients with entecavir for 48 weeks decreased serum and intrahepatic HBV cccDNA significantly, and the magnitude of HBV cccDNA reduction was related to total HBV DNA decrease, alanine aminotransferase normalization, and HBeAg seroconversion.     

Quantitation of hepatitis B virus DNA in serum of patients with hepatoma

Hepatitis B virus (HBV) DNA in the serum of 31 patients with histologically confirmed primary hepatocellular carcinoma (PHC) from Malaysia and Indonesia was quantitated by densitometric scanning of autoradiograms obtained by Southern blot DNA hybridization, after electrophoresis using a 32P DNA cloned into plasmid pBR325 as a probe. This quantitation after electrophoresis is more informative than the usual spot hybridization technique. Five of the 31 sera were positive for HBV DNA. Levels ranged between 1.36 pq and 143.18 pq per ml of serum, and the levels of HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe in the serum were serologically determined. All five sera positive for HBV DNA were also positive for HBsAg. Three of the five positive for HBV DNA were positive for HBeAg and negative for anti-HBe. Two of the sera positive for HBV DNA were negative for HBeAg but positive for anti-HBe. All sera negative for HBV DNA were also negative for HBeAg. Many sera which were negative for HBV DNA and HBeAg were positive for HBsAg. Of the 31 sera from PHC patients, 23 had at least one HBV marker positive (74.2%).     

实时定量RT-PCR对乙型肝炎病毒全长RNA(fRNA)的定量检测

目的:建立一种简便的定量检测慢性乙型肝炎患者血清中终止于聚腺苷酸化位点的乙型肝炎病毒全长RNA(f RNA)的方法。方法:选取53例未治疗的乙型肝炎患者及22例HBs Ag阴性的健康者为研究对象,使用锚定oligo-d T的引物对其血清中f RNA进行实时定量反转录PCR检测,统计分析其与HBV DNA、HBcr Ag和HBe Ag的相关性。结果:对f RNA进行实时荧光定量RT-PCR检测的下线为2.3 log copies/ml,标准曲线的相关系数为0.99(P0.0001)。53例乙型肝炎患者中,29例(54.7%)可以检测到f RNA,22例正常对照中没有检测到f RNA。27例HBe Ag阳性和/或高水平HBV DNA的患者全部检测到f RNA,26例HBe Ag阴性并且低水平HBV DNA的乙型肝炎患者中有2例(7.7%)检测到f RNA(P0.0001)。HBe Ag阳性患者血清中f RNA水平高于HBe Ag阴性患者(5.0±0.3 vs.2.9±0.4 log copies/ml,P0.001)。f RNA与HBV DNA/HBcr Ag具有显著相关性(r=0.905、0.881,P0.0001)。Hayashi's定量分析法I显示f RNA与HBV DNA相关性强于其与HBcr Ag的相关性。结论:与HBV DNA和HBe Ag一样,f RNA可作为常规检测判断HBV的复制水平并指导用药。     

不同临床类型乙型病毒感染者外周血T细胞亚群与血清HBVDNA载量及HBeAg滴度相关性分析

目的：探讨慢性乙型肝炎病毒（HBV）感染患者外周血T细胞亚群与血清HBVDNA载量及HbeAg滴度的关系。方法：选取103名HBV感染患者和20名健康者为研究对象。流式细胞术检测外周血T细胞亚群,聚合酶链式反应及酶免疫分析法分别检测血清HBVDNA载量及HbeAg滴度。结果：慢性乙型肝炎患者和慢性HBV携带者外周血CD3可、CD4T淋巴细胞亚群百分数低于健康对照组,结果有统计学意义（P〈0．05或0．01;而CD8＋T细胞亚群则呈现相反趋势,结果亦有统计学意义（P〈0．05或0．01）。HBeAg阴性组中,HBVDNA水平与CD8T细胞亚群百分数呈正相关（r=0．567,P〈0．01）,与CD47CD8＋T细胞亚群百分数比值呈负相关（r=-0．601,P〈0．01）,而与CD3＋T、CD4＋T细胞亚群百分数无相关性。HBeAg阳性组中,HBVDNA水平及HbeAg滴度与cD3＋1r、cD41、CD8叮细胞百分数及CD47CD8＋T细胞百分数均无相关性（P〉0．05）。结论：不同临床类型的慢性乙型肝炎病毒感染患者外周血T细胞亚群存在不同程度细胞免疫功能降低和细胞免疫调节异常。HbeAg阴性的HBV感染患者,其血清HBVDNA水平与外周血T淋巴细胞免疫存在相关性。     

不同临床类型乙型病毒感染者外周血T 细胞亚群与血清HBV DNA 载量 及HBeAg 滴度相关性分析

目的:探讨慢性乙型肝炎病毒(HBV)感染患者外周血T细胞亚群与血清HBV DNA载量及HbeAg滴度的关系。方法:选取103名HBV感染患者和20名健康者为研究对象。流式细胞术检测外周血T细胞亚群,聚合酶链式反应及酶免疫分析法分别检测血清HBV DNA载量及HbeAg滴度。结果:慢性乙型肝炎患者和慢性HBV携带者外周血CD3+T、CD4+T淋巴细胞亚群百分数低于健康对照组,结果有统计学意义(P<0.05或0.01;而CD8+T细胞亚群则呈现相反趋势,结果亦有统计学意义(P<0.05或0.01)。HBeAg阴性组中,HBVDNA水平与CD8+T细胞亚群百分数呈正相关(r=0.567,P<0.01),与CD4+/CD8+T细胞亚群百分数比值呈负相关(r=-0.601,P<0.01),而与CD3+T、CD4+T细胞亚群百分数无相关性。HBeAg阳性组中,HBV DNA水平及HbeAg滴度与CD3+T、CD4+T、CD8+T细胞百分数及CD4+/CD8+T细胞百分数均无相关性(P>0.05)。结论:不同临床类型的慢性乙型肝炎病毒感染患者外周血T细胞亚群存在不同程度细胞免疫功能降低和细胞免疫调节异常。HbeAg阴性的HBV感染患者,其血清HBV DNA水平与外周血T淋巴细胞免疫存在相关性。     

229例低复制慢性乙型肝炎患者血清相关指标的分析

目的探讨血清HBV—DNA低复制的CHB患者相关指标的变化及意义。方法收集HBV—DNA低复制的CHB患者血液标本229例,ALT、乙肝标志物、mIL-2R、IL-10、miRNA-122等指标均采用全自动分析仪测定,对结果进行比较和线性相关分析。结果低复制CHB患者中e抗原阳性患者占38．00％,e抗原阴性患者占51．53％;229例患者ALT升高者占总数的66．81％,患者血浆miRNA-122的表达量的变化与ALT浓度呈正相关（r=0．841）。e抗原阳性与e抗原阴性组IL-10、mIL-2R浓度与对照组比较差异均有统计学意义（P〈0．05）。结论HBV—DNA低复制CHB患者ALT、mlL-2R、IL-10、miRNA-122等指标的变化证明此类患者部分病例存在肝细胞实质性的损害,临床应高度重视,尤其要与非活动性乙型肝炎病原携带状态相鉴别,不可一概而论。     

Long-term effects of peginterferon alfa-2a therapy in Japanese patients with chronic hepatitis B virus infection

Background

There is no information on the long-term effects of peginterferon (PEG-IFN) alfa-2a therapy for chronic hepatitis B (CHB) in Japan. This double-blind, randomized trial investigated the efficacy of PEG-IFN therapy.

Methods

We analyzed 22 Japanese patients with CHB (hepatitis B e antigen [HBeAg]-positive: 17, HBeAg-negative: 5) treated with PEG-IFN alfa-2a and followed-up posttreatment for 5 years. Responders represented patients who showed persistent normalization of alanine transferase (ALT) levels, HBeAg clearance, and low hepatitis B virus (HBV) DNA levels (HBeAg-positive patient; <5 log copies/mL, HBeAg-negative patient; <4.3 log copies/mL) at end of treatment, and at 1, 2, 3, 4 and 5 years posttreatment. In addition, baseline HBeAg-positive patients who showed sustained normalization of ALT level, HBeAg clearance, and low HBV DNA level for more than 6 months until at 1, 2, 3, 4, and 5 years after completion of PEG-IFN were also classified as “triple responders” and the proportion of triple responders relative to all patients was termed the “triple response rate”.

Results

The response rates among HBeAg-positive patients were 13 %, 25 %, 14 %, 21 % and 21 % at end of treatment, and at 1, 2, 3, 4, and 5 years, respectively. The response rate tended to be higher in patients treated for 48 than 24 weeks. The respective response rates among HBeAg-negative patients were 0 %, 20 %, 20 %, 20 % and 25 %. During the treatment period, hepatitis B surface antigen (HBsAg) clearance at 3.5 years was noted in one patient, who was 37-year-old, male, had genotype C and received PEG-IFN alfa-2a at 90 μg for 48 weeks.

Conclusion

At 5 years after completion of PEG-IFN, the triple response rate in HBeAg-positive patients and combined response rate in HBeAg-negative patients were 21 % (3/14) and 25 % (1/4), respectively. The triple response was seen in three patients who had all been treated with PEG-IFN for 48 weeks.

     

The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.     

Formation of covalently closed circular DNA in Hep38.7-Tet cells,a tetracycline inducible hepatitis B virus expression cell line

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection.     

Hepatitis B virus precore G1896A mutation in chronic liver disease patients with HBeAg negative serology from North India

Hepatitis B with precore stop codon mutation is related with severe liver damage in HBeAg negative patients. It is of utmost importance to screen the G1896A precore mutation. The study was designed to assess the impact of G1986A mutations in patients with different clinical spectra of the liver disease by PCR–LCR. 210 HBV positive patients with HBeAg negative serology of different kind of liver diseases (AVH = 72, FH = 21, CH = 79, Cirrhosis = 20 and HCC = 18) were screened. Patients were screened for the presence or absence of precore G1896A mutation by PCR–LCR. Direct nucleotide sequencing was done to confirm the results of LCR. Precore mutant in HCC was 94.4% (17/18), 85.7% (18/21) in FH, 60% (12/20) in liver cirrhosis, 48.1% (38/79) in chronic hepatitis and 27.7% (20/72) in AVH cases. The serum ALT level was statistically significant between HBeAg negative WT and G1896A mutants in chronic hepatitis cases. ALT level and HBV DNA level was slightly raised in the pre core mutant but and was not significant. Genotype D had a higher prevalence (79.5%) as compared to genotype A (20.5%). The mutations detected by PCR–LCR were in 100% concordance with direct sequencing. The exceptionally high prevalence of G1896A in FH and HCC demonstrates that the precore mutants are strongly associated with the progression of liver diseases in patients with HBeAg negative serology. The findings are also suggestive of screening HBV precore G1896A mutation particularly in HBeAg negative cases. The precore G1896A mutation increases proportionately in severe form of liver diseases. LCR can be a suitable tool for screening of G1896A mutations.     

Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B

The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.     

siRNA Combinations Mediate Greater Suppression of Hepatitis B virus Replication in Mice

Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.     

新疆维吾尔族与汉族乙型肝炎患者HBVDNA 及肝功能检测结果的比较

目的:探讨新疆乌鲁木齐地区伴有肝功能指标:丙氨酸氨基转移酶(ALT)浓度异常的维吾尔族(维族)及汉族HBeAg阳性乙型肝炎初次就诊患者,乙型肝炎病毒DNA复制载量及ALT浓度是否存在差异及其对患者诊断、预后的意义。方法:回顾性选取门诊伴有ALT浓度异常的汉族、维族初次就诊患者并筛选出HBeAg阳性患者汉族、维族共373例。采用实时荧光定量聚合酶链反应、生化测定及酶联免疫吸附试验法分别测定HBV DNA、ALT浓度及乙肝HBeAg。结果:(1)汉族HBV DNA组秩和8869,维族HBV DNA组秩和10359.36,经Mann-Whitney Test检验两组间尚不能肯定HBVDNA分布有统计学意义,即伴有肝功能损害的汉族、维族初次就诊HBeAg阳性患者HBV DNA复制程度没有差异。(2)汉族ALT组秩和26818.50,维族ALT组秩和22009.50,经Mann-Whitney Test检验两组间ALT分布有统计学意义,即伴有肝功能损害的初次就诊HBeAg阳性患者汉族肝功能损害程度高于维族。(3)HBVDNA低复制组(103-104copy/mL):汉族秩和3771.46,维族秩和4993.2;中复制组(104-106copy/mL):汉族秩和6412.4,维族秩和5088.2;高复制组(>106copy/mL):汉族秩和929.04,维族秩和666.96,经Mann-Whitney Test检验在低复制组两民族间ALT分布无统计学意义,在中、高复制组两民族间ALT具有统计学意义。即:伴有肝功能损害的初次就诊HBeAg阳性患者在HBV DNA低复制组两民族间肝功能损害程度无差异,但在中、高复制组汉族肝功能损害程度高于维族。结论:新疆乌鲁木齐地区伴有肝功能损害的初次就诊的HBeAg阳性的汉族与维族之间HBV DNA的病毒复制无统计学意义(P>0.05),但两民族间的ALT具有统计学意义,可能跟维族的民俗、饮食习惯及生存环境、免疫相关基因HLA基因频率分布差异等因素有关。     

Differential Plasma MicroRNA Profiles in HBeAg Positive and HBeAg Negative Children with Chronic Hepatitis B

Background and Aim

Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted. Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB.

Patients and Methods

MiRNA PCR panels were employed to screen plasma levels of 739 miRNAs in pooled samples from HBeAg positive, HBeAg negative, and healthy children. The three groups’ plasma miRNA profiles were compared, and aberrantly expressed miRNAs were identified. The identified miRNAs were then validated. Individual RT-qPCRs were performed on plasma from 34 HBeAg positive, 26 HBeAg negative, and 60 healthy children.

Results

A panel of 16 plasma miRNAs were identified as aberrantly expressed in HBeAg positive and HBeAg negative children (p<0.001). Levels of all of the miRNAs were upregulated in HBeAg positive children compared with in HBeAg negative children. A positive correlation was furthermore found between plasma levels of the identified miRNAs and HBV DNA (p<0.001).

Conclusion

We are the first to investigate the plasma miRNA profile of children chronically infected with HBV. Our data indicates the existence of a relationship between abundance of circulating miRNAs and immunological stages in the natural course of disease. Certain miRNAs may contribute to the establishment and maintenance of CHB in children. Further studies are warranted to advance understanding of miRNAs in the pathogenesis of CHB, hopefully leading to the identification of future therapeutic targets.