Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

重型病毒性肝炎中丁型肝炎病毒的检测

为了探讨丁型肝炎病毒（ＨＤＶ）感染在重型病毒性肝炎中的作用，对北京佑安医院１９８０年至１９８９年收治的５４例急性和亚急性重型肝炎和３８例急性乙肝患者血清，应用国产ＨＤＶＥＬＩＳＡ试剂测定抗－ＨＤ、抗－ＨＤＩｇＭ和ＨＤＡｇ，应用斑点杂交技术测定ＨＤＶＲＮＡ。结果发现重型肝炎组ＨＯＶ－Ｍ检出率明显高于急性乙肝组（２７．８％比５．３％．Ｐ＜０．０５）。单独ＨＢＶ感染和ＨＤＶ／ＨＢＶ混合感染的重型肝炎患者均有较高的病死率。提示ＨＤＶ感染是重型肝炎中重要的病原学因素之一，ＨＤＶ与ＨＢＶ具有协同作用加重肝损害，导致肝衰竭。     

乙型肝炎表面抗原对BALB/c小鼠细胞免疫应答的影响

目的研究不同来源乙肝表面抗原(HBsAg)对BALB/c小鼠的某些细胞免疫应答的影响.方法采用血源、CHO和酵母HBsAg分别免疫BALB/c小鼠,于免疫后不同时间制备脾脏单个核细胞(MNC),测定MNC对刀豆蛋白(ConA)和细菌脂多糖(LPS)的增殖反应性;采用绵羊红细胞(SRBC)作为致敏原,测定不同HBsAg免疫小鼠后迟发性超敏反应(DTH)的发生程度.结果不同来源HBsAg对ConA或LPS刺激的反应指数(SI)表现出不同的时间曲线,一般于免疫20?d和25?d时显著升高,其中血源HBsAg较高,酵母较低.H.M.-HBsAg在免疫5?d时即显出较高的SI值.CHO-HBsAg对SRBC诱导的DTH反应具有显著的抑制作用(P＜0.05).结论不同来源HBsAg诱导细胞免疫反应的类型和程度存在差异,CHO细胞来源的HBsAg对TH1细胞介导的DTH反应有抑制作用.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

pGenesil-siHBV X 对 HepG2.2.15 细胞 HBV 表达和复制的抑制效果研究

 目的 研究针对HBV X基因区设计的小干扰RNA（siRNA）表达载体质粒pGenesil-siHBV X 对HepG2.2.15细胞HBV表达和复制的抑制效果及特异性。方法 针对HBV X区设计siRNA表达载体质粒pGenesil- siHBV X。分别用培养液（空白对照）、脂质体Metafectene、pGenesil 空载体、pGenesil-siHK（阴性对照）、pGenesil-siAFP（特异性对照）、pGenesil-siHBV X处理或转染HepG2.2.15细胞各3次。于每次转染后24 h，取各组细胞培养上清液，用时间分辨荧光免疫测定技术检测上清液中HBsAg和HBeAg含量，用化学发光法检测AFP含量，用PCR荧光定量技术检测HBV-DNA复制水平。 结果 pGenesil-siHBV X转染能抑制HepG2.2.15细胞对HBV标志物的表达，且抑制作用随转染次数增加而增强。第3次转染后，pGenesil-siHBV X组细胞上清液中 HBsAg、 HBeAg和HBV-DNA检测结果分别为（6.26 ± 1.07）ng/ml 、（0.13 ± 0.05）Ncu/ml和（3.01 ± 0.40）×107拷贝/ml，与空白对照组的（22.50 ± 1.39）ng/ml、（1.12 ± 0.11）Ncu/ml和（12.33 ± 1.28）×107拷贝/ml比较，差异有统计学意义（t值分别为12.80、12.21、9.71，P < 0.05）；pGenesil-siHBV X 转染不影响细胞对AFP的表达（t = 0.18，P = 0.86）。结论 pGenesil-siHBV X可以有效和特异地抑制HepG 2.2. 15细胞HBV-DNA的复制及HBsAg和HBeAg表达。     

戊型肝炎病毒归科的形态学证据

采用超簿切片制样及负染色免疫电镜方法，对经细胞培养自戊型肝炎病人分离的８７Ａ株病毒进行了形态及形态发生的研究。结果显示，感染细胞胞浆局灶性空泡液泡病变区明显而普遍；病毒呈晶格样排列，在胞浆内装配成熟，与粗面内质网、病毒性包涵体、微丝等密切相关；病毒毒粒无囊膜，大小约３０ｎｍ呈圆形，表面形态不规整，电致密度不均一，偶见“凹杯形”、“刺突”等表面超微结构；感染细胞核呈现扭曲变形，异染色质趋边等显著受累及病变。其形态及形态发生特征表明其应为嵌杯病毒科新属—嗜肝ＲＮＡ病毒属的成员，而部分核苷酸序列分析也支持其为戊型肝炎病毒。     

小儿巨细胞病毒性肝炎治疗的临床对照研究

目的　探讨小儿巨细胞病毒 (HCMV)肝炎的有效治疗方法。方法　将诊断为小儿HCMV肝炎的 2 5例分为治疗组和对照组 ,对照组给予泼尼松、肝太乐、消炎利胆片及苯巴比妥等 ,治疗组在此基础上给予更昔洛韦 (GCV) 静脉用免疫球蛋白 (IVIG) ,分别在治疗前、出院时检查血常规、肝功能和HCMVDNA拷贝数 ,并在出院后 3个月、6个月、9个月随访 ,检查患儿的症状、体征和HCMV定量。结果　治疗组在 5个时点 (治疗前、出院时、3个月、6个月、9个月 )HCMVDNA拷贝数明显下降 ,治疗组和对照组之间用药前后比较 ,在住院天数、黄疸开始消退时间及黄疸完全消退时间的差异有显著意义 ,治疗组输血率明显减少 (P <0 0 5 )。结论　GCV联合IVIG治疗小儿HCMV肝炎能明显降低病毒量 ,缩短病程 ,减轻症状 ,又可纠正贫血 ,减少输血机会 ,未见明显副作用。     

IL-15真核表达质粒的构建及对乙肝疫苗诱导的体液免疫应答的影响

目的 研究两种不同的IL-15真核表达质粒对乙肝蛋白疫苗诱导的免疫应答的影响。方法：构建IL-15真核表达质粒(简称pIL-15)和含有IL-12信号肽的IL-15真核表达质粒(简称pIL-2s-15)，CTLL-2细胞增殖实验验证两种质粒真核表达产物的生物学活性。将这两种质粒分别与HBsAg共免疫BALB／C小鼠，用ELISA法检测小鼠血清抗-HBs IgG及IgGl、IgG2a亚类的效价。结果：与HBsAg蛋白疫苗共免疫时，pIL-15可使HBsAg诱导的抗-HBsIgG效价升高，显著高于载体pcDNA3．1与HB—sAg共免疫对照组，pIL-2s-15对HBsAg诱导抗-HBsIgC效价没有明显影响。与HBsAg pcDNA3．1组相比，HBsAg pIL-2s-15组和HBsAg pIL-15组诱生的抗HBsIgG2a亚类均升高，但前者IgG2a／IgG1比值最高，与HBsAg pcDNA3．1组相比差别有显著性；HBsAg pIL-15组IgG2a／IgG1比值与HBsAg pcDNA3．1组相比差别无显著性。结论 pIL-15真核表达质粒可增强蛋白疫苗诱导的体液免疫应答，pIL-2s-15真核表达质粒则主要使免疫应答趋向Th1型。     

Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen

The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNγ), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNγ-producing CTLs and their reaccumulation in the spleen. The data suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present.     

HLA-DRB1*07与慢性乙肝患者Th1/Th2因子表达水平的相关性

目的 :探讨广东地区汉族慢性乙型肝炎患者HLA DRB1 0 7与Th1 Th2因子表达水平的相关性。方法 :收集 12 0例广东地区汉族慢性乙肝患者新鲜抗凝血各 8ml,通过序列特异性引物套式PCR(PCR SSP)方法进行HLA DRB1 0 7检测 ,并同时用双抗体夹心法检测患者治疗前后外周血CD4 +T细胞分泌IFN γ、IL 2、IL 10和IL 4的水平。结果 :12 0例慢性乙肝患者HLA DRB1 0 7携带者 31例 ,携带率为 2 5 8% ,明显高于广东地区汉族人群的平均携带率 7 84 % ;HLA DRB1 0 7阳性患者IFN γ平均表达水平为 (1132 0 4± 75 36 )pg ml,IL 2平均表达水平为 (1184 0 6± 81 4 2 )pg ml,IL 4平均表达水平为 (876 79± 4 7 5 3)pg ml,IL 10平均表达水平为 (817 4 8± 2 4 4 0 )pg ml ;HLA DRB1 0 7阴性患者IFN γ平均表达水平为 (12 32 10± 198 13)pg ml,IL 2平均表达水平为 (12 0 8 17± 116 12 )pg ml,IL 4平均表达水平为 (6 81 99± 6 1 5 9)pg ml,IL 10平均表达水平为 (6 38 84± 76 17)pg ml。HLA DRB1 0 7阳性患者IL 4、IL 10表达水平高于阴性患者 (P <0 0 5 ) ,而IFN γ、IL 2表达水平与阴性患者差异无统计学意义 (P >0 0 5 )。结论 :HLA DRB1 0 7(+)慢性乙肝患者Th2因子表达水平高于HLA DRB1 0 7(- )慢性乙肝患者。