乙型肝炎病毒B与C基因型感染者临床和有关免疫细胞计数的比较

目的探讨乙型肝炎病毒B与C基因型感染者临床和有关免疫细胞计数的差别。方法在128例乙型肝炎患者,采用微板核酸杂交-ELISA技术进行HBV基因分型,采用流式细胞仪检测T细胞亚群、非特异性CTL、辅助性T（Th1）、Th2细胞、自然杀伤（NK）细胞,在64例人白细胞抗原（HLA）A2阳性的CHB患者检测HBV特异性CTL。结果在128例CHB患者中,B基因型感染者70例（54．69％）,C基因型57例（44．53％）,B／C混合型1例（0．78％）;C基因型感染者血清丙氨酸氨基转移酶和总胆红素水平高于B基因型感染者（P〈0．01和P〈0．05）;C基因型感染者HBVDNA水平（P＜0．01）、HBeAg阳性率（P〈0．01）、Th1细胞（P〈0．05）和非特异性CTL（P〈0．01）高于B基因型感染者,而HBV特异性CTL低于B基因型（P〈0．01）。结论C基因型感染者肝功能损害比B基因型重,可能与HBV特异性CTL低,导致HBV DNA水平和HBeAg阳性率高有关。     

慢性乙型肝炎患者树突状细胞诱导的HBV特异性细胞毒性T淋巴细胞PD-1的表达

目的 探讨慢性乙型肝炎(CHB)患者树突状细胞(DC)诱导的HBV特异性细胞毒性T细胞(CTL)表面程序性死亡受体1(PD-1)的表达情况及其与HBV DNA的关系.方法 采集30例CHB患者和10例健康人的抗凝外周静脉血,分离外周血单个核细胞(PBMC),在白细胞介素(IL)-4和粒-巨噬细胞集落刺激因子(GM-CSF)的作用下培养使DC增殖、成熟,培养第4d加入纯化的HBsAg进行冲击.采同一患者外周血,分离出自体T淋巴细胞,用含重组人白细胞介素(rhIL)-2的培养基维持T细胞的生长,培养第5d与HBsAg冲击的DC共培养.以流式细胞技术检测CTL的PD-1表达,并分析PD-1表达水平与HBV DNA的关系.结果 与健康对照组比较,CHB组DC诱导的HBV特异CTL的PD-1的表达明显升高(P=0.000).且HBeAg阳性组PD-1的表达率较HBeAg阴性组明显升高(P=0.000).CHB患者DC诱导的HBV特异性CTL的PD-1表达率与血清HBV DNA拷贝数的对数值呈正相关(r=0.53,P=0.008).结论 CHB患者DC诱导的HBV特异性CTL高表达PD-1分子,为HBV慢性感染过程中CTL功能低下,病毒难以清除提供了另一条重要线索.     

T淋巴细胞亚群及HBV特异性细胞毒性T淋巴细胞的变化在慢性乙型肝炎重症化中的意义

我们观察了209例慢性乙型肝炎(CHB)住院患者进展为慢性重型乙型肝炎前、后的HBV特异性细胞毒性T淋巴细胞(CTL)、非特异性CTL、T淋巴细胞(简称T细胞)亚群的变化,为预防或阻止CHB患者进展为重型肝炎提供依据.     

乙型肝炎病毒前C/BCP区基因变异对特异性细胞毒性T淋巴细胞免疫应答的影响

目的 研究HBV前C/BCP区基因变异对慢性乙型肝炎(CHB)患者特异性细胞毒性T淋巴细胞(CTL)免疫应答的影响.方法 采用HBV核心抗原表位肽core18-27,流式细胞术胞内细胞因子(CFC)分析法检测CHB患者外周血单个核细胞(PBMC)中的特异性CTL,对扩增产物进行测序分析.结果 54例CHB患者中G1896A突变毒株21例,占38.9%;1762/1764位核苷酸联合突变26例,占48.1%;3位点同时突变毒株13例,占24.1%.3种变异株体外HBV核心抗原表位肽core18-27刺激后,特异性CTL水平[(0.41±0.09)%、(0.36±0.08)%、(0.48±0.08)%]显著高于野毒株[(0.11±0.06)%,P＜0.05].结论 G1896A变异及1762/1764位核苷酸联合突变能显著增强特异性CTL水平,在CHB患者病情活动过程中起重要作用.     

乙型肝炎病毒基因型与滤泡辅助性T淋巴细胞的关系及其意义

我国的HBV基因型以B基因型和C基因型为主.C基因型感染者血清HBV DNA水平较高,肝组织学活动度较高,ALT反复或持续波动,其机制目前还不十分清楚.本研究通过比较慢性乙型肝炎(CHB)患者HBV B和C基因型感染者之间滤泡辅助性T淋巴细胞(Tfh)、IL-21、HBV特异性CTL、非特异性CTL、HBV DNA和ALT水平,探讨不同基因型对血清HBV DNA和ALT水平影响的差异的可能机制.     

乙型肝炎患者外周血病毒特异性CD8阳性细胞毒性T淋巴细胞的数量和功能分析

目的 分析急性乙型肝炎(AHB)和慢性乙型肝炎(CHB)患者外周血中乙型肝炎病毒(HBV)特异性细胞毒性T淋巴细胞( CTL)的数量及功能.方法 36例HLA-A2阳性的乙型肝炎患者,其中AHB 12例,CHB 24例.分别用含HBV抗原C、S和P区的c18-27、s183-191和p575-583三个肽段的四聚体[Tetramer (Tc 18-27、Ts183-191、Tp 575-583)]检测患者外周血单核淋巴细胞(PBMCs)中HBV特异性CD8阳性CTL细胞的数量,同时用酶联免疫斑点法(EHSPOT)检测其分泌IFN-γ的功能.用SPSS 13.0进行统计学分析.结果 AHB和CHB患者外周血中Tc18-27特异性的CTL数量无明显不同;而Ts 183-191特异性CTL的平均值分别为0.24％±0.39％和0.03％±0.02％,阳性率分别为75％和33.3％;Tp575-583特异性CTL平均值分别为0.08％±0.09％和0.02％±0.01％,阳性率分别为50％和16.7％,AHB较CHB显著升高(P＜0.05).此外,AHB患者平均Tetramer阳性的个数为1.58个,而CHB患者平均为0.67个,AHB较CHB显著增多(P＜0.01).在9例AHB患者中,其外周血Ts183-191特异性的CTL细胞的数量为139～21 735个/106 PBMCs,用ELISPOT方法检测相对应的分泌IFN-γ的斑点形成细胞(SFC)为0～252个/106 PBMCs,AHB患者Tetramer细胞数和ELISPOT检测的IFN-γ斑点数有明显相关性(P＜0.01),而CHB患者则无此相关性.结论 AHB与CHB患者外周血中HBV特异性CTL的数量和分泌IFN-γ的差异提示CTL可能在清除病毒方面发挥着至关重要的作用,是今后慢性乙型肝炎免疫治疗的重要研究方向之一.     

阿德福韦酯对慢性乙型肝炎患者外周血H B c A g特异性CTL的影响

目的: 探讨阿德福韦酯(ADV)对慢性乙型肝炎(CHB)患者外周血中HBcAg特异性CTL数量的影响.方法: 选择应用ADV治疗48 wk的HLA-A2阳性CHB患者11例作为研究对象, 应用Tetramer流式细胞技术检测治疗前后P BMC中的HBcAg特异性CTL细胞频率.结果: HBcAg特异性CTL为0.074%-0.937%.CHB患者体内的特异性CTL频率远低于急性乙型肝炎. 经ADV治疗CHB患者48 wk后, 其BcAg特异性CTL较治疗前无明显变化;亚组分析也表明无论治疗48 wk后HBV DNA是否转阴、ALT是否恢复正常, 对特异性CTL均无影响.结论: 应用ADV治疗48 wk, 对CHB患者体内HBcAg特异性CTL细胞频率无明显影响.     

急性乙型肝炎患者特异性核心抗原细胞毒性T淋巴细胞频率的变化及其意义

目的 动态观察急性乙型肝炎(AHB)患者外周血HBcAg18-27表位特异性细胞毒性T淋巴细胞(CTL)、血清ALT、HBV DNA、HBsAg和淋巴细胞亚群的变化,探讨HBV特异性CTL频率的消长在病毒清除以及肝脏损伤中的作用.方法 分别选取AHB、慢性乙型肝炎(CHB)患者外周血,根据人类白细胞抗原(HLA)-A0201结果分为两组:HLA-A0201阳性患者作为HBV特异性CTL检测组、HLA-A0201阴性患者作为特异性抗原表位对照组.用HLA-A0201限制性表位HBcAg18-27五聚体复合物通过流式细胞技术,动态定量检测外周血中HBV特异性CTL频率和T、B淋巴细胞与自然杀伤细胞(NK)和NKT淋巴细胞;以速率法检测血清ALT水平;荧光定量PCR检测HBV DNA水平; Abbott微粒子化学发光技术检测HBV血清学标志物.计量资料采用均数±标准差((x)±s)表示或中位数(P25-P75)描述,组间比较采用方差分析或非参数检验(KruskalWallis检验和Mann-Whitney U检验);两种计量指标的关系采用Pearson相关分析.结果 AHB患者入院第1、2、3周外周血HBcAg表位特异性CTL频率分别为2.11%(0.20%～3.64%)、3.56%(1.05%～5.91%)、2.03%(0.33%～3.58%),高于入院第4、5、6周的0.99%(0.12%～2.16%)、0.29%(0.05%～0.76%)、0.39%(0.05%～0.46%),也显著高于CHB的0.11%(0.06%～0.29%),z值分别为-3.258,-4.041,-3.259,P值均＜0.01.AHB患者HBcAg表位特异性CTL峰值延迟于血清HBV DNA、HBsAg和ALT等指标的峰值;在AHB患者中,HBcAg表位特异性CTL高频率患者的血清HBsAg消失时间早于CTL频率较低的患者[(1.75±1.04)周与(4.33±3.51)周,t=-2.018,P＜0.05].CD3+CD8+T淋巴细胞频率的峰值出现在入院后第2周,并与HBcAg表位特异性CTL峰值相重叠,两者动态变化规律呈相关性(r=0.420,P＜0.01).AHB患者早期外周血NK、NKT淋巴细胞数量显著低于正常对照组和CHB患者,但随着病情好转而逐渐恢复,AHB患者外周血NK细胞数量变化与HBcAg特异性CTL动态变化呈负相关(r=-0.435,P＜0.01).结论 急性HBV感染者高频率的HBcAg表位特异性CTL与HBsAg的更早消失有密切关系,动态监测外周血中HBcAg特异性CTL频率变化,可以作为预测HBV感染后临床转归的参考指标;AHB患者外周血CD8+T淋巴细胞数量的变化,可以间接反应AHB患者体内HBcAg特异性CTL频率的改变.

Abstract：

Objective This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Methods Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group.The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. Results The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBVspecific CTL was earlier than that in patients with low frequencies (t = 2.018, P ＜ 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P ＜ 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P ＜ 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P ＜ 0.01). Conclusion The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencie of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL. during acute HBV infection.     

乙型肝炎病毒基因型与滤泡辅助性T淋巴细胞的关系及其意义北大核心CSCD

我国的HBV基因型以B基因型和C基因型为主。C基因型感染者血清HBVDNA水平较高,肝组织学活动度较高,ALT反复或持续波动,其机制目前还不十分清楚。本研究通过比较慢性乙型肝炎（CHB）患者HBVB和C基因型感染者之间滤泡辅助性T淋巴细胞（Tfh）、IL-21、HBV特异性CTL、非特异性CTL、HBV DNA和ALT水平,探讨不同基因型对血清HBV DNA和ALT水平影响的差异的可能机制。     

乙肝病毒基因分型与慢性乙型肝炎患者临床和病理的相关性研究

目的:了解本地区慢性乙型肝炎(CHB)患者乙型肝炎病毒(HBV)基因型分布的特点,探讨基因型与临床及病理之间的关系。方法:采用荧光定量PCR方法检测120例CHB患者的HBV基因型,同时检测血清HBVDNA、HBV-M、肝功能等,并进行肝组织病理检查。结果:120例CHB患者B基因型43例,占35.83%,C基因型77例,占64.17%;在年龄、性别、肝功能及病情程度之间,B、C基因型差异均无显著性意义(P0.05);C基因型患者的HBVDNA水平、HBeAg阳性率均显著高于B基因型(P0.05),其肝组织炎症评分和纤维化评分也均明显高于B基因型(P0.05)。结论:本地区CHB患者HBV基因型以C型为主,C型血清HBVDNA水平及HBeAg阳性率高于B型,C型引起的肝组织病变较B型严重。HBV基因分型对判断肝脏病变的严重程度和指导临床抗病毒治疗具有重要意义。     

Recognition of HBV antigens and HBV DNA by dendritic cells

BACKGROUND:Hepatitis B virus(HBV)is a hepatotropic, noncytopathic,DNA virus which can cause acute and chronic infection.Viral persistence is associated with a weak or absent specific immune responses to HBV,particularly the cellular immune response.Dendritic cells(DCs)are professional antigen-presenting cells with a unique T cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection.An impaired function of DCs was suggested by recent studies to account for the T and B cell hyporesponsiveness in chronic HBV infection.This review summarizes recent insights into the recognition of HBV antigens by DCs. DATA SOURCES:Studies were identified by searching MEDLINE and/or PubMed for articles using the key words"hepatitis B virus (HBV)","dendritic cells","C-type lectins","mannose receptor", "toll-like receptor",and"dendritic cell-specific intercellular-adhesion-molecule-3 grabbing nonintegrin(DC-SIGN)"up to December 2009.Additional papers were identified by a manual search of the references from the key articles. RESULTS:DCs play an important role in the progress of hepatitis B,especially in the recognition of HBV.There are three main ways of recognition of HBV antigens by DCs. First,HBV DNA can be recognized by DCs through toll-like receptor 9(TLR9)which activates the NF-κB signal pathway and p38 MAPK to up-regulate the expression of interferon (IFN)regulatory factor 7(IRF-7)in a manner independent of type I IFN signaling,resulting in secretion of type I IFN and inflammatory cytokines,and induction of DC maturation and the adaptive immune response.Second,HBc/HBeAg cannot be recognized by DCs,but DNA or ssRNA encapsulated within HBcAg can be internalized by DCs through TLRs.Third,HBsAg can be internalized by DCs through the mannose receptor,which lacks the ability to induce DC maturation without the assistance of DC-SIGN.Meanwhile,there is some cross-talk among the three mechanisms,which induces an effective anti-viral response or HBV persistence. CONCLUSIONS:On the basis of these recognition processes, methods have been used to enhance the efficacy of DC-based vaccine against HBV and have been useful in the clinical application of HBV vaccine therapy.But the interactions between HBV antigens/HBV DNA and DCs are not clear, and cross-talk between TLRs and various ligands makes HBV antigen recognition by DCs more complicated.More efforts should be made to define the mechanisms and develop effective vaccines and therapies.     

乙型肝炎孕妇HBV血清标志物与HBVDNA载量及ALT的相关性分析

目的 分析乙型肝炎(乙肝)孕妇HBV血清标志物、HBVDNA载量及ALT检测结果,为HBV感染孕妇的诊治提供参考。方法 回顾性分析2016年11月—2017年11月在我区孕检的120例乙肝孕妇的临床资料,应用酶联免疫吸附法检测血清五项HBV标志物,同时采用荧光实时定量PCR技术检测HBVDNA水平,酶速率法检测ALT,并对检测结果进行统计分析。结果 120例孕妇血清中,感染模式Ⅰ(大三阳)HBsAg(+)、HBeAg(+)、HBcAb(+)58例,占48.33%;HBVDNA(+)49例,占84.48%,其中HBVDNA>106IU/ml42例,占72.41%;ALT增高39例,异常率为67.24%。感染模式Ⅱ(小三阳)HBsAg(+)、HBeAb(+)、HBcAb(+)45例,占37.50%;HBVDNA(+)27例,占60.00%,其中HBVDNA>106IU/ml15例,占33.33%;ALT增高20例,异常率为44.44%。感染模式Ⅰ孕妇HBVDNA阳性率、HBVDNA>106IU/ml率和ALT异常率最高,感染模式Ⅱ孕妇次之。结论 HBV血清标志物与HBVDNA高载量和ALT水平密切相关,三者相结合能为孕妇的临床诊断、围产期干预措施以及疗效观察提供参考依据。     

不同HBV标志物模式的乙型肝炎患者HBV表面大蛋白的检测及与HBVDNA水平的相关性

目的通过检测乙型肝炎病毒标志物（HBVM：HBsAg／抗-HBs、HBeAg／抗一HBe、抗一HBc）不同模式的乙型肝炎患者血清中乙型肝炎病毒外膜大蛋白（HBV—LP）、乙型肝炎病毒前s1抗原（HBVpreSl）、HBVDNA,探讨HBV—LP与HBVDNA及HBVM模式间的关系,研究HBV—LP用于乙型肝炎患者临床诊治的意义。方法采用酶联免疫吸附试验（ELISA）对HBV—LP、HBVpreS1和HBVM进行检测;采用荧光定量PCR方法对HBVDNA进行检测。结果乙型肝炎患者大蛋白检测结果与HBVDNA检测结果差异无统计学意义（X^2=1．97,P〉0．05）。HBV—LPA值随HBVDNA拷贝数的增加呈上升趋势,二者之间存在良好的相关性（r=0．565,P〈0．01）,在不同HBVDNA拷贝数组别间,HBV—LPA值差异有统计学意义（F=9．23,P〈0．01）。结论HBV—LP是反应HBV感染者体内病毒复制程度的良好的血清免疫学指标,血清中的HBV—LP与HBVDNA具有较好的相关性,特别是可作为HBeAg阴性患者体内监测病毒复制及预后判断的良好的血清学指标。     

Quantification of HBV core antibodies may help predict HBV reactivation in patients with lymphoma and resolved HBV infection

1. Download high-res image (221KB)
2. Download full-size image

     

Detection of HbsAg and HBV DNA in serum and saliva of HBV carriers

Serum and saliva samples from 23 patients known to be HBsAg-positive HBV carriers and 17 healthy control subjects were analyzed for hepatitis B virus (HBV) by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). All serum samples of the HBV carriers were positive for HBsAg, with 21 also positive for HBV DNA. In comparison, 22 saliva samples of HBV carriers were positive for HBsAg whereas only 11 of the 23 tested were positive for HBV DNA. Based on these results we have arrived at the conclusion that the saliva of HBV carriers might be potentially infectious and also that saliva testing could serve as an alternative technique for identifying HBV carriers.     

Distribution of HBV genotypes among HBV carriers in Benin： phylogenetic analysis and virological characteristics of HBV genotype E

AIM: To determine the distribution of Hepatitis B virus (HBV) genotypes in Benin, and to clarify the virological characteristics of the dominant genotype. METHODS: Among 500 blood donors in Benin, 21 HBsAg-positive donors were enrolled in the study. HBV genotypes were determined by enzyme immunoassay and restriction fragment length polymorphism. Complete genome sequences were determined by PCR and direct sequencing. RESULTS: HBV genotype E (HBV/E) was detected in 20/21 (95.2%), and HBV/A in 1/21 (4.8%). From the age-specific prevalence of HBeAg to anti-HBe seroconversion (SC) in 19 HBV/E subjects, SC was estimated to occur frequently in late teens in HBV/E. The comparison of four complete HBV/E genomes from HBeAg-positive subjects in this study and five HBV/E sequences recruited from the database revealed that HBV/E was distributed throughout West Africa with very low genetic diversity (nucleotide homology 96.7-99.2%). Based on the sequences in the basic core promoter (BCP) to precore region of the nine HBV/E isolates compared to those of the other genotypes, a nucleotide substitution in the BCP, G1757A, was observed in HBV/E. CONCLUSION: HBV/E is predominant in the Republic of Benin, and SC is estimated to occur in late teens in HBV/E. The specific nucleotide substitution G1757A in BCP, which might influence the virological characteristics, is observed in HBV/E.     

肝癌患者血清中乙型肝炎病毒基因分型及临床意义

目的:探讨血清中乙型肝炎病毒(HBV)基因型及HBV DNA水平与肝细胞癌的关系。方法:应用巢式聚合酶链反应扩增乙型肝炎病毒与基因,用末端标记方法对PCR产物标记并直接测序,测序结果和GenBank中登录的标准基因型序列相比较,应用荧光定量PCR法检测HBV DNA水平。对61例肝癌、65例慢性乙型肝炎、10例乙型肝炎病毒携带者进行了检测。结果:136例中B基因型59例(43.4%)、C基因型77例(56.6%),随着病情加重,C基因型比例逐渐增高;不同基因型HBV感染的肝癌患者间HBV DNA水平差异有显著意义,P<0.05;在慢性乙型肝炎患者中,HBV DNA水平差异无显著性意义。结论:本地区乙型肝炎病毒以B、C基因型为主,乙型肝炎病毒C基因型及高水平的HBV DNA感染与肝癌的发生相关。     

乙肝病毒基因型与患者临床预后关系的研究

研究不同HBV基因型感染者临床特征的异同。选取慢性HBV感染者297例,用特异性引物PCR法测定其HBV基因型,并比较不同基因型者在临床各方面表现有何异同。297例样本中B型占12．8％,C型占87.2％, 未发现其他基因型。B型者与C型者相比,年龄≤35岁者较多,血清ALT、AST水平较低,两组的血清HBVDNA水平无明显差异。B型在慢性HBsAg携带者、慢性肝炎、肝硬化及肝癌患者中所占比例逐步下降,而C型所占比例则逐步上升。B型者HBeAg阳性率低,HBeAg血清学转换率高。B型、年龄小者及女性者容易呈慢性HBsAg携带者状态。在17例死于肝病者中,B型者死亡时感染HBV时间较长。B型者对抗病毒治疗应答比C型者好。C型HBV 感染与严重肝脏疾病的发生有关,感染B型HBV者临床预后较好,对抗病毒治疗的应答较好。     

HBV and HCV therapy

One year of interferon therapy inhibits HBV replication in one third of the patients whereas long-term administration of oral nucleos(t)ide analogues is efficient in most of them, as long as early treatment adaptation in patients with partial virological response and resistance is provided. Following the demonstration of a more potent antiviral effect in terms of sustained virological response (SVR) rates, Pegylated-IFN coupled with Ribavirin has become the standard treatment for chronic hepatitis C, with nearly 65% of all treated patients achieving a SVR. Long-term suppression of HBV and eradication of HCV would halt the progression of chronic hepatitis to cirrhosis, hepatocellular carcinoma and liver decompensation.