妊娠水平孕酮体外对HIV-1感染和复制的影响

目的：研究相当于妊娠后外周血和胎盘局部浓度的孕酮（P4）体外对HIV-1感染及复制的影响。方法：检测终浓度为10^-6mol／L（相当于妊娠妇女外周血水平）和10^-5mol／L（相当于母胎界面局部浓度）的P4对HIV-1介导细胞融合的影响；ELISA法检测感染HIV的MT-2细胞培养上清p24抗原含量来探讨对HIV-1复制的影响；流式细胞术检测对淋巴细胞活化后CD69表达的影响和^1H-TdR掺入法检测对淋巴细胞增殖的影响。结果：10^-5mol／L的P4可以明显抑制HIV-1介导的细胞融合，并且降低感染细胞培养上清中p24抗原含量，两种浓度对淋巴细胞的活化和增殖都有抑制作用。结论：相当于妊娠后母胎界面局部浓度的P4体外可以抑制HIV-1感染和复制，其机制与抑制淋巴细胞活化和增殖有关。     

表达GFP基因的溶瘤腺病毒对大肠癌细胞的体外杀伤作用

目的： 研究在端粒酶启动子驱动下表达绿色荧光蛋白（GFP）及 5型腺病毒早期基因（E1A）基因的腺病毒Ad/hTERT-GFP-E1对大肠癌细胞的杀伤作用及其可能的机制。方法:将不同滴度Ad/hTERT-GFP-E1感染大肠癌及正常细胞,以巨细胞病毒（CMV）启动子驱动下表达GFP基因的腺病毒Ad/CMV-GFP作为载体对照,通过半数组织培养感染量（TCID50）法测定病毒滴度及体外复制能力,然后用MTT法及细胞克隆形成试验评价该病毒在体外对肿瘤细胞及正常细胞的杀伤作用,并对病毒感染后的细胞进行原位凋亡检测。结果:Ad/hTERT-GFP-E1能够在DLD1细胞内持续复制,GFP的表达能够对病毒的感染和复制起到监测作用;MTT结果显示该病毒对于结直肠肿瘤细胞有显著杀伤作用,而对于正常细胞没有明显的杀伤作用;对病毒感染后的DLD1进行细胞凋亡检测发现Ad/hTERT-GFP-E1所引起的凋亡率显著高于对照组(P＜0.01)。结论:溶瘤腺病毒Ad/hTERT-GFP-E1能够选择性地在肿瘤细胞内复制并杀伤肿瘤细胞,其机制与细胞凋亡的途径有关。     

不同嗜性HIV复制与树突状细胞成熟状态关系的研究

目的 探讨不同嗜性HIV-1在人树突状细胞（dendriticcell，DC）内复制与DC成熟状态的关系。方法 采用MACS磁珠分选法纯化出CD14^＋细胞，加入粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4体外培养7d，得到未成熟DC；再加入肿瘤坏死因子-α继续培养3d，得到成熟DC。感染不同嗜性HIV-1病毒株后，检测培养上清p24含量，观察HIV-1复制能力。结果 受T嗜性HIV-1攻击后，未成熟DC及成熟DC培养上清p24抗原含量随培养时间延长无明显增加。受M嗜性HIV-1攻击后，未成熟DC培养上清p24抗原含量随培养时间延长明显增加，成熟DC培养上清p24抗原含量不随培养时间延长明显增加。结论 T嗜性HIV-1不能在未成熟DC和成熟DC内复制，M嗜性HIV-1能在未成熟DC内复制但不能在成熟DC内复制，提示HIV-1能否在DC内复制与HIV-1嗜性和DC成熟状态有关。     

反基因锁核酸体外抑制乙肝病毒前S1基因表达

 目的 探讨针对乙肝病毒前S1基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用。方法 针对乙肝病毒前S1基因同聚嘌呤区,利用RNAstructure软件分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术（FQ-PCR）、时间分辨免疫荧光技术（TRFIA）和酶联免疫法（ELISA）分别监测1、3、5和7 d细胞培养上清液中HBV DNA、HBsAg和前S1抗原的含量;四甲基偶氮唑蓝（MTT）法检测锁核酸对细胞代谢的影响。结果 加入锁核酸后,对HBV DNA复制、HBsAg和前S1抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7 d后抑制率分别达64.32%、67.51%和63.88%。LNA对细胞代谢无明显影响。结论 针对乙肝病毒前S1基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据。     

蚯蚓体腔液体外抗呼吸道合胞病毒的实验研究

目的研究蚯蚓体腔液（Earthworm coelomic fluid,ECF）体外抗呼吸道合胞病毒（RSV）的作用。方法以Hep-2细胞为宿主细胞,利巴韦林作为阳性对照药物,通过观察细胞病变效应（CPE）和MTT染色法来研究蚯蚓体腔液体外抗RSV的作用。结果ECF和利巴韦林对Hep-2细胞的CC50分别为3．11mg／ml和1．35mg／ml。在直接杀灭作用实验中,IC50为184．1μg／ml,SI值为16．87;在抑制病毒复制实验中,IC50为1555．8μg／ml,SI值为1．99;在阻断病毒侵入实验中,没有观察到抗病毒的作用。结论蚯蚓体腔液具有直接杀灭和抑制RSV复制的作用,其中直接杀灭作用的抗病毒效果较明显,但不具有阻断RSV入侵的作用。     

早、中、晚孕期胎盘因子体外抗HIV-1的研究

目的 探讨早、中、晚孕期胎盘因子(PF)体外抗人免疫缺陷病毒-1(HIV-1)及在HIV-1垂直传播中的作用.方法 荧光染料Calcien-AM标记的H9/HIV-1ⅢB分别与早、中、晚孕期不同稀释浓度的PF作用后,与MT2细胞培养,荧光显微镜下观察合胞休的形成;用HIV-1ⅢB感染MT2细胞,并分别与早、中、晚孕期不同稀释浓度的PF作用后,用MTT法检测HIV-1感染细胞的存活率,用ELISA方法检测细胞培养上清中p24抗原水平.结果 各孕期PF并不能抑制MT2和H9/HIV-1ⅢB细胞的融合,但可以增加HIV-1感染细胞的存活率及减少HIV-1 p24抗原的表达,且效应以早孕期PF最大,中孕期PF其次晚孕期PF最小,并与剂量呈正相关.结论 PF在体外具有抗HIV-I的作用,并呈现孕期和剂量相关性,可能在阻断HIV-1垂直传播中具有一定作用.     

金银花体外抗呼吸道合胞病毒的作用研究

目的了解金银花体外抑制呼吸道合胞病毒的效果和强度。方法采用细胞病变抑制实验．噻唑蓝（MTT）比色法检测细胞活性,观察金银花在人宫颈癌传代细胞（Hela）中对人呼吸道合胞病毒3型的抑制作用,以治疗指数（TT）为评价指标。结果金银花对Hela细胞半数中毒浓度（TC50）为5mg／ml,最大无毒浓度（TC50）为3．6mg／ml;对呼吸道合胞病毒有直接灭活作用,其半数抑制率（IC50）为0．16mg／ml,TI为31．2;在吸附阶段也有作用,其IC50为0．48mg／ml,TI为10．5;同时金银花有抑制呼吸道合胞病毒生物合成作用,其IC50为1．0mg／ml,TI为5;金银花不能阻止呼吸道合胞病毒侵入细胞。结论金银花在体外主要通过直接灭活、阻止病毒吸附和抑制生物合成3种方式发挥抗呼吸道合胞病毒作用。     

细胞ERK信号通路对单纯疱疹病毒Ⅱ型增殖复制的影响

目的以Raf-MEK-ERK通路关键激酶MEK1（MAPK／extraeellular signal-regulated kinasekinase-1）为研究重点，利用MEK1-siRNA（small interfering RNA for MEK1）阻断宿主Raf-MEK-ERK通路，阐述该通路对病毒复制的作用。方法用HSV-2感染预转染MEK1-siRNA的HEK293细胞，应用观察细胞病变效应、病毒滴度以及病毒蛋白表达等研究方法，揭示细胞Raf-MEK-ERK通路对HSV-2增殖复制作用的影响。结果MEK1-siRNA在特异性阻断MEKl表达的同时，能明显抑制HSV-2的复制。结论体外实验表明，Raf-MEK-ERK通路在HSV-2增殖复制过程中具有十分重要的作用，MEK1-siRNA具有药物开发的潜能。     

熊果酸对单核细胞白血病细胞作用机制的初步研究

目的 探讨熊果酸在体外对单核细胞白血病细胞株THP-1（human acute monocytic leukemia cell line）的增殖抑制和杀伤机制。方法 采用MTT法和流式细胞术检测熊果酸对THP-1细胞的增殖抑制和杀伤效应．hoechst33258荧光染色观察DNA片段化。结果 熊果酸对THP-1细胞具有增殖抑制和杀伤效应，并呈浓度和时间依赖性。在熊果酸作用下THP-1细胞DNA受损，出现凋亡征象，G1期细胞数减少，细胞主要阻滞在S期。结论 熊果酸在体外能诱导THP-1细胞凋亡。     

灭活HIV-1颗粒对人CD4+T细胞活化和全血Th1/Th2细胞因子分泌的影响

目的：体外研究AT-2灭活的HIV-1颗粒对人CD4+T细胞活化和全血(whole blood，WB) Th1/Th2细胞因子分泌的影响。方法:AT-2灭活HIV-1ⅢB型病毒颗粒，运用ELISA法测定所制备的灭活病毒中p24抗原的含量，按照1/500、1/50和1/5 (V/V)的浓度加入到WB中，以植物血凝素(phytohemagglutinin，PHA)组为阳性对照；24 h后，收集WB培养上清，运用流式微球分析法（cytometric bead array，CBA）检测WB分泌Th1 (IL-2、IFN-γ和TNF-α)和Th2 (IL-4、IL-6和IL-10)细胞因子水平；同时运用免疫荧光抗体染色技术结合流式细胞术检测WB中CD4+T细胞早期活化标记分子CD69的表达百分率。结果:我们所制备的灭活病毒中p24抗原的含量为85.5 μg/L；24 h后，空白对照组中，CD4+T细胞CD69的表达百分率为(1.62±0.63)%，PHA组为(38.82±6.00)%，HIV-1(1/500)组为(3.83±1.07)%，HIV-1(1/50)组为(5.94±0.85)%，HIV-1(1/5)组为(9.30±1.22)%；空白对照组WB培养上清中细胞因子主要为IL-6和TNF-α，PHA组中Th1和Th2细胞因子全部升高，3个浓度的HIV-1组中Th1和Th2细胞因子也全部升高。结论:AT-2灭活的HIV-1ⅢB颗粒能够明显引起WB中CD4＋T细胞活化，并上调WB培养上清中Th1和Th2细胞因子的水平，其机制可能是除了HIV-1病毒蛋白的作用外，HIV-1出胞时，许多宿主细胞来源的免疫分子整合到病毒颗粒包膜中，而模拟抗原提呈细胞，从而产生免疫调节作用。     

硫酸酯化箬叶多糖抗HIV-1机制的初步研究

目的 探讨硫酸脂化箬液多糖（Ｓ－ＩＴＰＳ）抗ＨＩＶ的机制,为进一步开发该药提供理论依据。方法 于病毒接种前,接种同时及接种后分别在培养细胞（ＭＴ－４）中加入硫酸脂化箬叶多糖（Ｓ－ＩＴＰＳ）,在Ｓ－ＩＴＰＳ存在或不存在的条件下培养１￣４周。终点以病毒半数感染量（ＴＣＩＤ５０法）,细胞病变（ＣＰＥ）,ＭＴＴ染色细胞保护率（ＭＴＴ法）及培养上清中的ｐ２４抗原滴度９ＥＬＩＳＡ法）作为评价指标,判断药效,分     

Analysis of HIV-1 in the cervicovaginal secretions and blood of pregnant and nonpregnant women.

OBJECTIVES: To detect HIV-1 in cellular and acellular fractions of cervicovaginal secretions obtained by cervicovaginal lavage (CVL) and evaluate viral genotypes in the HIV-1-positive CVL samples. STUDY DESIGN/METHODS: This study consists of 37 HIV-1-seropositive pregnant and nonpregnant women from the United States. A total of 63 paired CVL and blood samples were collected. HIV-1 DNA from cervical cells (CC) and virion RNA from cervical supernatant (CS) was detected by gag polymerase chain reaction (PCR) assays. The HIV-1 genotypes were determined by analyzing the nested PCR-amplified V3 region sequences of the HIV-1 gp120 envelope gene. RESULTS: Within this cohort, 95% of the women were on single or combination antiretroviral therapy. Of the pregnant women, 63% of samples had HIV-1 viral DNA in the CC, and 29% of samples were positive for viral RNA in the CS. Among nonpregnant women, 71% of samples were positive for HIV-1 DNA in CC, and 46% of samples tested positive for virion RNA in CS. Plasma viral load ranged between 10,000 and 100,000 copies/mL and showed significant correlation with the detection of HIV-1 RNA in the CVL; this relation was less apparent with viral DNA in CC. The viral blood and CVL specimens were further analyzed by evaluating the genotypes of HIV-1 variants. In most patients, a high degree of similarity was observed between the viral sequences derived from blood and CVL samples. Two patients demonstrated closely related but somewhat distinct genotypic variants in CVL and blood. One subject showed clear compartmentalization in which distinct viral genotypes were observed in CVL and blood. Based on V3 loop analyses of gp120, with one exception, the cervicovaginal secretions harbored viral populations with a macrophage (CCR5)-tropic phenotype. CONCLUSIONS: This study demonstrates the unique characteris tics of HIV-1 strains in the genital secretions of a relatively large cohort of HIV-1-infected women in the United States. These results are important for further analysis of HIV-1 transmission and pathogenesis in vivo and for rational vaccine design.     

Quality of human immunodeficiency virus viral load testing in Australia

This study determined the proficiencies of laboratories measuring human immunodeficiency virus type 1 (HIV-1) viral loads and the accuracies of two assays used for HIV-1 viral load measurement in Australia and investigated the variability of the new versions of these assays. Quality assessment program panels containing (i) dilutions of HIV-1 subtype B, (ii) replicates of identical samples of HIV-1 subtype B, and (iii) samples of subtype E and B were tested by laboratories. Total variability (within and between laboratories) was tested with quality control samples. The coefficients of variation (CVs) for the Roche AMPLICOR HIV-1 MONITOR version (v) 1.0 and Chiron Quantiplex bDNA 2.0 assays ranged from 53 to 87% and 22 to 31%, respectively. The widespread occurrence of invalid runs with the AMPLICOR HIV-1 MONITOR 1.0 assay was identified. The CVs of the new versions of the assays were 82 to 86% for the AMPLICOR HIV-1 MONITOR v 1.5 assay and 16 to 23% for the Quantiplex bDNA 3.0 assay. For virus dilution samples, all but 5 of 19 laboratories obtained results within 2 standard deviations of the mean. The Quantiplex bDNA 2.0 assay reported values lower than those reported by the AMPLICOR HIV-1 MONITOR version 1.0 assay for samples containing HIV-1 subtype B, whereas the reverse was true for subtype E. Identification and resolution of the problem of invalid runs markedly improved the quality of HIV-1 viral load testing. The variability observed between laboratories and between assays, even the most recent versions, dictates that monitoring of viral load in an individual should always be by the same laboratory and by the same assay. Results for an individual which differ by less than 0.5 log(10) HIV-1 RNA copy number/ml should not be considered clinically significant.     

Detection of HIV-1 RNA/DNA and CD4 mRNA in feces and urine from chronic HIV-1 infected subjects with and without anti-retroviral therapy

HIV-1 infects gut associated lymphoid tissues (GALT) very early after transmission by multiple routes. The infected GALT consequently serves as the major reservoir for HIV-1 infection and could constantly shed HIV-1 and CD4⁺ T cells into the intestinal lumen. To examine this hypothesis, we monitored HIV-1 RNA/DNA and CD4 mRNA in fecal samples of chronically infected subjects with and without antiretroviral therapy (ART). We compared this to levels of HIV-1 RNA/DNA in urine and blood from the same subjects. Our results show that HIV-1 DNA, RNA and CD4 mRNA were detected in 8%, 19% and 31% respectively, of feces samples from infected subjects with detectable plasma viral load, and were not detected in any of subjects on ART with undetectable plasma viral load. In urine samples, HIV-1 DNA was detected in 24% of infected subjects with detectable plasma viral load and 23% of subjects on ART with undetectable plasma viral load. Phylogenetic analysis of the envelope sequences of HIV-1 revealed distinct virus populations in concurrently collected serum, feces and urine samples from one subject. In addition, our study demonstrated for the first time the presence of CD4 mRNA in fecal specimens of HIV-1 infected subjects, which could be used to assess GALT pathogenesis in HIV-1 infection.     

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.     

Early markers of HIV-1 disease progression in a prospective cohort of seroconverters in Bangkok, Thailand: implications for vaccine trials

BACKGROUND: Some candidate HIV-1 vaccines may not prevent HIV-1 infection but may alter the course of disease. Surrogate endpoints based on early laboratory makers in HIV-1-infected persons who are antiretroviral therapy (ART)-naive will be useful for evaluating vaccine efficacy in slowing disease progression (VEp). We examined pretreatment HIV-1 viral loads and CD4 cell counts in recent HIV-1 seroconverters to inform selection of these endpoints. METHODS: We studied 130 newly HIV-1-infected injection drug users identified from a prospective cohort of initially uninfected persons in Bangkok during 1995 through 1998. We analyzed trends in HIV-1 viral loads and CD4 cell counts as well as progression to the surrogate endpoint, defined as 2 consecutive CD4 cell counts of fewer than 350 cells/mm, during 24 months after the first HIV-1 seropositive (FP) visit. RESULTS: Median HIV-1 RNA copies/mL with interquartile ranges were 43,693 (14,320-94,767) at the FP visit, 46,924 (16,273-104,314) at 6 months, 28,446 (11,292-54,325) at 12 months, and 18,080 (8713-54,059) at 18 months. HIV-1 viral loads at the FP visit and at 18 months were positively correlated (r = 0.53, P < 0.0001). Of 130 participants, 12% reached the surrogate endpoint by 6 months, 16% by 12 months, and 27% by 18 months. In Cox regression analyses, HIV-1 viral loads of more than 50,000 copies/mL at the FP visit (hazard ratio [HR] = 2.3, 95% confidence interval [CI]: 1.1-4.8) and first CD4 cell count of 500 or fewer cells/mm (HR = 7.6, 95% CI: 3.2-17.6) were independently associated with faster progression to the surrogate endpoint. CONCLUSIONS: Participants with high HIV-1 RNA levels and low CD4 cell counts close to the time of seroconversion were more likely to experience early immunologic progression. Approximately one quarter of seroconverters reached the surrogate immunologic endpoint within 18 months of their FP visit and before starting ART, suggesting the utility of this endpoint for analyses of VEp in some ongoing and planned HIV-1 vaccine efficacy trials.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

Measurement of HIV RNA in patients infected by subtype C by assays optimized for subtype B results in an underestimation of the viral load

Quantitation assays of HIV-1 RNA used currently were designed and optimized for subtype B viruses. However, infection with non-B HIV viruses has become more common worldwide. Unfortunately, little information is available regarding the suitability of these assays for measurement of viral load in specific non-B subtypes. The performance of two commercial HIV-1 RNA quantitation assays was evaluated in 82 HIV subtype C-infected patients and in 43 HIV-1 subtype B-infected patients. Blood samples were tested by the Amplicor HIV-1 Monitor Assay, Version 1.5, and by the nucleic acid sequence-based amplification HIV-1 assay (NucliSens). The results were compared by using a paired, two-tailed Student's t-test; the difference between the assays was found to be significant only for subtype C. Discordant results (>0.5 log difference) between the two assays were detected in 39% of subtype C samples, compared to 23.2% of subtype B samples. In all cases in which a discordant result was detected, the lower results were obtained by the NucliSens assay. Discordant results between CD4 and viral load (CD4 < 200 cells/ml with a viral load <5,000 copies/ml) were observed in eight of the subtype C-infected patients when a viral load was measured by NucliSens (9.7%), compared to three patients (3.6%) when measured by the Amplicor assay. In conclusion, in patients with HIV subtype C infection, measurement of HIV RNA by the NucliSens assay resulted in a significant underestimation of the viral load as compared to the Amplicor assay. As a consequence, such an underestimation may result in sub-optimal care of patients infected with HIV subtype C.