不同方法注射乙肝免疫球蛋白阻断HBsAg、HBeAg双阳性孕妇母婴传播的研究

目的研究不同方法注射乙肝免疫球蛋白(HBIG)对HBsAg伴HBeAg双阳性孕妇的乙肝病毒(HBV)宫内感染阻断作用。方法将137例双阳性孕妇分为4组:A组从孕16周起注射HBIG,B组从孕20周起注射HBIG,C组从孕28周起注射HBIG,A、B、C 3组孕妇每次均注射HBIG200IU,并间隔4周1次,直至临产;D组作为对照组,不注射HBIG;出生后4组所有新生儿均于16h内和生后2周注射HBIG200IU,满月起按1、2、7月龄分别接种乙肝疫苗,并定期随访。结果新生儿出生时外周血测得HBsAg,A组与B组无显著性差别(P>0.05),A、B 2组与C组、D组以及C组与D组均有显著性差别(P均<0.05);经随访A、B、C、D 4组HBV宫内感染率分别为5.6%、5.3%、19.4%和48.5%,母婴传播阻断率A组与B组无显著差异(P>0.05),A组和B组均显著高于C组和D组、C组显著高于D组(P均<0.05)。结论对HBsAg伴HBeAg双阳性孕妇,选择孕20周开始注射HBIG比较合适,阻断率最高,能有效减少HBV宫内感染的发生率。     

针对S基因的siRNA或shRNA表达框对HepG2.2.15细胞中HBV基因表达和病毒复制的抑制作用比较

目的 化学合成针对乙型肝炎病毒（HBV）S基因的siRNA，同时制备针对相同区段的shRNA表达框，并比较二者对HepG2．2．15细胞中HBV基因表达和病毒复制的抑制作用。方法 化学合成针对HBVS基因的带有FITC标记的siRNA，设计合成带有FTrc标记的引物，PCR扩增含有RNA聚合酶Ⅲ启动子H1序列和shRNA编码序列的表达框，并对扩增产物进行纯化，将等摩尔数siRNA和shRNA表达框分别用脂质体转染稳定表达HBV的HepG2．2．15细胞，转染1d后流式细胞仪检测转染效率，转染3d后RT-PCR检测靶基因mRNA的水平，用SDS-PAGE、Westem blot及间接免疫荧光染色检测HBsAg的表达。收集转染前和转染后1、3、5和7d的细胞培养上清，检测HBsAg水平，同时用荧光定量PCR方法检测HBV　DNA的含量。结果成功制备了针对HBVS基因的shRNA表达框，将其与等摩尔数siRNA分别转染HepG2．2．15细胞后，RT-PCR证实细胞中HBV　mRNA水平降低，SDS-PAGE、Westem blot及间接免疫荧光染色检测到HBsAg的表达受到抑制，细胞培养上清中HBsAg和HBV DNA含量下降。与siRNA相比，shRNA表达框对HBV的抑制作用虽然起效较慢，但持续时间更长。结论shRNA表达框和siRNA均可明显抑制HBV的转录和表达，与siRNA相比，shRNA表达框能够更持久地抑制靶基因的表达。     

Lack of PTEN expression in endometrial intraepithelial neoplasia is correlated with cancer progression

We tested the hypothesis that PTEN inactivation may stratify cancer progression risk among putative endometrial hyperplasias, classified prognostically by means of the morphometric D score (DS). The DS, calculated from 3 morphometric variables measured in routine hematoxylin-eosin-stained endometrial biopsy slides, is the most sensitive and specific method of endometrial cancer risk prediction currently available. Clinical outcomes of 103 women with endometrial hyperplasia on biopsy were tallied according to the DS. Seven (7/103; 7%) patients with carcinoma during follow-up were all distributed within the high-risk prognostic group (ie, DS <1 = endometrial intraepithelial neoplasia [EIN]) (7/21; 33% progression). None of the 82 cases with a DS higher than 1 progressed. All cases that progressed were PTEN null, indicating that this genotype is capable of further stratifying cancer progression risk in hyperplasias irrespective of histological categorization. However, only 16% of the PTEN-null cases progressed. When PTEN expression pattern was combined with EIN, the prognostic power was greatly increased (specificity from 63% for PTEN and 85% for EIN to 93% when combined; positive predictive value from 16% and 33% to 50%). We conclude that loss of PTEN expression is the first biomarker in EIN that increases the accuracy of the prognostic DS to predict cancer progression risk. Unless endometrial hyperplasias are stratified by histological morphometric D-Score, PTEN has a low positive predictive value.     

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.     

Oligomerization and polymerization of the filovirus matrix protein VP40

The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle.     

Genome analysis of dengue type-1 virus isolated between 1990 and 2001 in Brazil reveals a remarkable conservation of the structural proteins but amino acid differences in the non-structural proteins

We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.     

Prevalence of hepatitis B virus infection among women at reproductive age at a German university hospital

BACKGROUND: Mother to infant transmission of hepatitis B virus (HBV) represents a major factor in maintaining chronic infection and depends on the degree of maternal infectivity status. OBJECTIVES: To examine the seroprevalence of hepatitis B virus surface antigen (HBsAg) in women at reproductive age admitted to the Department of Gynaecology at a German university hospital. STUDY DESIGN: The seroprevalence of hepatitis B surface antigen (HBsAg) in 5518 women at reproductive age was examined, HBsAg-positive samples were tested for additional HBV markers to verify the infection status. RESULTS: Out of 5518 samples from women at reproductive age, 88 women (1.59%) were positive for HBsAg and 7 of these HBV-positive women (7.95%) were additionally positive for HBeAg. The majority of the study population were German citizens, however most HBV infected persons originated from countries with a high HBV prevalence. The HBV seroprevalence in our study group is about two times higher compared to the average seroprevalence in the German citizen adult population, thus probably resulting in an underestimation of the infection rate in a multinational setting. CONCLUSIONS: Screening for HBsAg during pregnancy is still necessary and important for reduction of perinatal HBV transmission even in countries with low HBV prevalence.     

52 例病毒性肝炎患者肝组织中丙型肝炎病毒 RNA 和 HCAg-NS3 的测定

应用地高辛标记探针原位杂交法和单克隆抗ＨＣＶ－ＮＳ３－ＨＲＰ建立直接酶标免疫组化法分别测定５２例肝炎患者肝组织ＨＣＶＲＮＡ和ＨＣＡｇ－ＮＳ３。结果抗ＨＣＶ阳性组ＨＣＶＲＮＡ检出率５７．１％（１６／２８），ＨＣＡｇ－ＮＳ３检出率５３．６％（１５／２８）；抗ＨＣＶ阴性组其两项检出率均为１２．５％（３／２４）。肝组织中ＨＣＶＲＮＡ阳性物呈蓝紫色细小颗粒存在于肝细胞核或胞浆内，其在肝小叶中的分布可分为３型，即弥漫型、局灶型、散在型。肝组织中ＨＣＡｇ－ＮＳ３阳性物呈棕黄色细小颗粒分布于肝细胞核或胞浆内，以单个或数个阳性细胞散布于肝小叶中。２３例ＨＣＶＲＮＡ或／和ＨＣＡｇ－ＮＳ３阳性病例以肝炎后肝硬化（ＬＣ）病例占多数（１４／２３），其次为慢性重型肝炎（ＣＳＨ）和中度慢性肝炎（ＣＡＨ）。此两种检测方法具有较高符合率（９０．４％，４７／５２），表明病毒核酸及其表达产物均存在于肝细胞内，与ＨＣＶ感染密切相关。这为ＨＣＶ感染诊断提供了直接依据，有利于研究ＨＣＶ感染中病毒复制、慢性化进程、抗病毒治疗监测及重叠感染时病毒相互关系。     

Tattooing as a risk of hepatitis C virus infection.

The association of hepatitis C virus (HCV) infection and tattooing was studied in 87 tattooed and 126 tattoo free healthy young men who did not engage in intravenous drug use or multiple sexual activity. Antibody against HCV (anti-HCV) was tested in serum specimens by enzyme immunoassay with C100-3, NS3, and core antigens; 11 of the 87 (12.6%) tattooed and 3 of the 126 (2.4%) tattoo free subjects were positive for anti-HCV (odds ratio = 5.9, 95% CI = 1.6-22.0). A relationship was demonstrated by an increased risk for HCV infection with an increasing number of tattooed site (P(trend) = 0.002). All but one of the 87 tattooed subjects had been infected by hepatitis B virus (HBV) and 25 were carriers of hepatitis B surface antigen (HBsAg). None of the 25 HBsAg carriers was positive for anti-HCV whereas 11 of the 62 HBsAg non-carriers had anti-HCV, suggesting a negative association between the HBsAg carriage and the long lasting anti-HCV (P = 0.02, Fisher's exact). The status of the tattooer was also an important determinant for HCV infection; the risk was higher if tattooing was done by a non-professional friend than by a professional tattooist. Tattooing, probably with improperly sterilized needles, can clearly pose an increased risk for HCV infection in Taiwan. This study indicates the need for legal standards for hygienic tattooing as part of preventive measures for the control of parenterally transmitted infections.     

Differential diagnosis of HTLV-I-associated myelopathy and multiple sclerosis in Iranian patients

Summary Two Iranian patients with chronic progressive spastic paraparesis and urinary dysfunction were referred to our hospital with the presumptive diagnosis of multiple sclerosis (MS). Routine CSF analysis and magnetic resonance imaging of the two patients were only partially characteristic of MS. Testing for antibodies to human T-cell leukemia virus type I [HTLV-1] in serum using a radioimmune precipitation assay revealed antibodies to HTLV-I in both patients. The infection with HTLV-I was confirmed by polymerase chain reaction (PCR) and liquid hybridization analysis using primers to the tax/rex region and a corresponding probe, demonstrating proviral DNA in peripheral blood mononuclear cells of both patients. On the basis of these findings demonstrating the presence of proviral HTLV-1 DNA in the two Iranian patients, the initial diagnosis of MS was corrected to that of HTLV-I-associated myelopathy (HAM). In contrast, several patients with definite MS (nine from Germany, two from Iran) with a relapsing and remitting form of the disease were tested for HTLV-1 infection by enzyme-linked immunosorbent assay and PCR, which yielded negative results. However, the mother of one HAM patient was found to be infected with HTLV-I. To support an association between HTLV-I infection and CNS disease in the two HAM patients, we analyzed the production of specific IgG antibodies within the CNS based on a simple enzyme immunoassay for viral IgG antibodies in CSF and serum. In the two HAM patients there was significant intrathecal antibody production directed against HTLV-I, but this was not found in any of the samples from MS patients. These findings demonstrate an immune reaction to HTLV-I in the CNS of HAM patients, thus confirming the association of infection and CNS disease. The demonstration of intrathecal HTLV-I antibody production also proved useful for the differential diagnosis of MS or HAM, especially in patients from areas endemic for HTLV-I.Abbreviations DTPA diethylenetriaminepentaacetic acid - ELISA enzyme-linked immunosorbent assay - HAM HTLV-I-associated myelopathy - HTLV-I human T-cell leukemia virus type I - MRI magnetic resonance imaging - MS multiple sclerosis - PBMC peripheral blood mononuclear cells - PCR polymerase chain reaction - RIPA radioimmune precipitation assay - SDS sodium dodecyl sulfate - TSP tropical spastic paraparesis