基线极高HIV-1 RNA水平对初治HIV-1感染者外周血total HIV-1 DNA的影响

目的:观察基线HIV-1 RNA > 50万copies/mL的HIV-1感染者,在高效抗反转录病毒治疗（highly active antiretroviral therapy,HAART）前后外周血totalHIV-1 DNA的变化。方法:从国家十二五科技重大专项课题中选取基线HIV-1 RNA > 50万copi...     

γδT细胞与HIV-1感染相互作用机制的研究进展

γδT细胞是表达γ链和δ链的一群特殊T细胞,是参与天然免疫的重要成分,兼有天然免疫和获得性免疫特性。其在人外周血中数量较少,但功能强大,在机体的免疫调节、免疫保护、免疫监视、抗感染、细胞毒等生理功能方面具有重要作用。本文对γδΤ细胞的起源、发育与分布、生物学与免疫学功能及其与HIV-1感染的相互作用机制进行综述。     

HIV-1感染者病毒学指标与血浆中白细胞介素表达水平的相关性研究

目的：探究未治疗的人类免疫缺陷病毒（HIV-1）感染者不同HIV-1 RNA或HIV-1 DNA水平组患者血浆IL-1β、IL-6、IL-8、IL-10、IL-16和IL-18表达差异及其与HIV-1 RNA或HIV-1 DNA的相关性。方法：选取未治疗HIV-1感染者75例,检测其HIV-1 RNA、HIV-1 DNA水平、CD4+T细胞、CD8+T细胞计数及血浆IL-1β、IL-6、IL-8、IL-10、IL-16和IL-18表达。结果：不同组患者血浆IL-1β、IL-6、IL-10、IL-16和IL-18表达存在明显差异,且与HIV-1 RNA、HIV-1 DNA水平相关（P<0.05）。结论：高病毒载量的未治疗HIV-1感染者血浆IL-10、IL-18表达较高,IL-1β、IL-6和IL-16表达较低,且均与病毒载量相关;高HIV-1 DNA水平的未治疗HIV-1感染者血浆IL-10、IL-18表达较高,IL-6、IL-16表达较低,且均与HIV-1 DNA水平呈正相关,说明也可能与病毒储存库相关。     

HUT78淋巴细胞感染HIV-1病毒后基因表达变化的研究

人免疫缺陷病毒1型（HIV-1）攻击的靶细胞有CD4阳性的T淋巴细胞、单核细胞、巨噬细胞和骨髓树突状细胞.HIV-1感染对宿主细胞的形态、基因表达以及新陈代谢有明显影响,并且在mRNA水平和蛋白质水平上改变了宿主细胞的基因表达.本文应用基因芯片分析HUT78细胞在HIV-1感染后发生的基因表达变化.     

HIV-1 gp41融合多肽对CD3抗体激活的调节性T细胞的抑制作用

目的:观察HIV-1 gp41融合多肽(FP)能否影响CD3抗体激活的调节性T细胞(Treg).方法:用免疫磁珠分离小鼠脾脏CD4+ CD25+的Treg、CD4+ CD25-的效应性T细胞(Teff),丝裂霉素C处理小鼠脾细胞获得APC.通过CCK-8法和羧基荧光素双乙酸盐琥珀酰亚胺酯(CFSE)标记检测FP对CD3抗体刺激的Teff增殖效应分析其对Treg抑制功能的影响;用ELISA检测FP对Treg激活后IL-10分泌的影响;用激光共聚焦显微镜观察细胞表面FP与TCR的分布.结果:通过CCK-8法和流式细胞术分析发现,在CD3抗体刺激下Teff细胞有显著增殖效应;当Treg和Teff混合培养时,Treg能够明显抑制共培养的Teff增殖;当加入25 μg/mL FP后,Treg+ Teff组与Teff组细胞增殖没有明显变化;当FP浓度为5 μg/mL时,Treg+ Teff组比Teff组增殖显著降低.Treg未活化时IL-10分泌较低,经过CD3抗体刺激后其IL-10分泌显著增多,而当FP浓度为25μg/mL时IL-10显著下降,5μg/mL FP对IL-10分泌无显著影响.通过激光共聚焦显微镜检测发现Treg未活化时,T细胞受体(TCR)在细胞表面均匀分布,同时未见FP与TCR共分布;当Treg在CD3抗体刺激下,TCR活化形成半月形,且FP与活化的TCR在细胞表面共分布.结论:25μg/mL FP对Treg体外抑制功能具有显著抑制作用,而5 μg/mL FP不影响Treg的抑制功能,可能与其抑制Treg细胞IL-10分泌以及阻断APC在Treg细胞跨膜区域活化信号的递呈有关.     

HIV-1早期感染者γδT细胞对储藏库细胞的细胞毒作用

目的探讨HIV-1早期感染者γδT细胞是否具有清除HIV-1储藏库的能力。方法入组HIV-1早期感染者16例,分离外周血单核细胞,体外采用唑来膦酸(5μmol/L)加IL-2(1 000 IU/m L)扩增γδT细胞。乳酸脱氢酶(LDH)法检测γδT细胞对储藏库细胞(J-Lat Full Length Clone10.6)的细胞毒作用;流式细胞计量术检测扩增前后γδT细胞的表型和储藏库细胞中GFP的荧光强度。结果唑来膦酸加IL-2在体外刺激γδT细胞快速且大量扩增(P0.001)。γδT细胞对储藏库细胞具有较强的细胞毒作用,储藏库细胞中GFP的平均荧光强度显著减弱(P0.05)。扩增后γδT细胞表现出细胞毒性NK细胞样表型,HIV-1感染者CD56+Vδ2T细胞的比例较健康对照显著增加(P0.05)。结论γδT细胞具有清除HIV-1储藏库的能力,基于γδT细胞的自体或异体过继免疫治疗有望成为清除储藏库,治愈HIV-1感染的一种新策略。     

RNA干扰技术在抗HIV-1感染中的应用

艾滋病已成为危害人类健康的重要疾病 ,且至今没有行之有效的治疗及预防手段。本文主要介绍一种新型的方法 -RNA干扰技术在治疗和预防 HIV - 1感染方面的进展、取得的成就以及存在问题与展望 ,并证明 RNA干扰技术是抑制 HIV- 1感染及复制的有效工具     

病毒性肝炎患者外周血T细胞亚群的变化

<正> 应用APAAP桥联酶标法,检测50例急、慢性病毒性肝炎患者在不同病期时,其外周血T淋巴细胞亚群的变化,结果如下。 材料和方法 一、病例:急性黄疸型肝炎30例,其中甲型和乙型肝炎各半。患者男性21例,女性9例,平均年龄28.7±8.8岁,总胆红质(TBil)71.74±41.31μmol/L,谷丙转氨酶(ALT)均在150u以上(正常<40u)。慢性无黄疸型乙型肝炎20例,其中慢性迁延性肝炎12例,慢性活动性肝炎8例,ALT均在150u以上,男16例,女4例,平均年龄30.1±6.4岁。同时检测10例健康人T细胞亚群,以资对比。     

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay

A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10⁶ cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11–4.7 log copies/10⁶ cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21–58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus. J. Med. Virol. 54:54–59, 1998. © 1998 Wiley-Liss, Inc.     

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

A cell line (H2-5) producing defective doughnut-shaped particles of human immunodeficiency virus type 1 (HIV-1) was found to contain proviral DNA with a large deletion of 2558 bases, corresponding to the 3 half ofpol gene, the entirevif andvpr genes, and the 5 terminal of thetat gene.     

Interactive association of proviral load and IFN-gamma-secreting T cell responses in HIV-1C infection

We investigated the interactive relationship between proviral DNA load and virus-specific IFN-gamma-secreting T cell responses in HIV-1C infection. The presence or absence of correlation, and inverse or direct type of correlation, if any, were dependent on targeted viral gene product. Responses to Gag p24 or to Pol were associated with lower proviral DNA load. Associations between proviral DNA load and T cell responses did not necessarily mirror relationships between plasma RNA load and T cell responses. An interaction analysis showed a synergy in that lower proviral DNA and lower plasma RNA load were associated with high Gag p24-specific IFN-gamma-secreting T cell response (interaction test P = 0.0003). Our findings support the idea that HIV proteins have differential value for vaccine design, and suggest that, for HIV-1C, Gag p24 may be one of the most attractive regions to include in vaccine designs to control both plasma RNA load and cell-associated proviral DNA load.     

Transient increment of HTLV-2 proviral load in HIV-1-co-infected patients during treatment intensification with raltegravir

BackgroundNumerous studies have analyzed the effects of raltegravir intensification on HIV-1 viral replication in infected individuals receiving suppressive combined antiretroviral treatment (cART). Nevertheless, there are only two studies on the effect of raltegravir in HTLV-1 infection, and none in HTLV-2.ObjectiveTo study the effect of raltegravir on HTLV-2 infection in HIV-1-co-infected individuals.Study designThis retrospective longitudinal study included four HTLV-2-HIV-1-co-infected individuals who received raltegravir-based cART during 48 weeks and 11 HTLV-2-HIV-1-co-infected individuals under cART without raltegravir during 48 weeks. HTLV-2 proviral load, CD4 and CD8 count and frequency were analyzed.ResultsHTLV-2 proviral load significantly increased at week 24 compared to baseline among all the patients who received raltegravir (p = 0.003), while no significant increases were found in the control group. No significant variation in either CD8 or CD4 counts was found during the follow up in both groups.ConclusionsRaltegravir induced a transient increment on total HTLV-2 DNA proviral load in HTLV-2/HIV-1-coinfected individuals on suppressive cART after 24 weeks.     

Diversity of HIV-1 RNA and DNA in breast milk from HIV-1-infected mothers

We compared human immunodeficiency virus type 1 (HIV-1) RNA and DNA populations in the different fractions of breast milk (lactoserum, lipid layer, cell pellet) and between right and left breasts in four HIV-1-infected mothers by analyzing the hypervariable env C2-V5 region. Phylogenetic analyses of the viral quasispecies revealed that RNA populations and DNA populations were clearly distinct and that viral RNA sequences were similar in lipid layer and lactoserum in the milk of 3 out of 4 mothers. Comparison of viral DNA between milk from right and left breast showed a differential distribution of variants in three mothers. In contrast, RNA variants detected from milk of the two breasts were mixed in 3 out of 4 mothers. This study suggests that each mammary gland is subjected to microenvironmental pressure that may differ from the contralateral breast.     

HIV-1 proviral DNA copy number in peripheral blood leucocytes and bronchoalveolar lavage cells of AIDS patients.

In this study we have determined by polymerase chain reaction (PCR) the quantity of HIV-1 proviral DNA in cells obtained by bronchoalveolar lavage (BAL) from the lung of HIV-1+ individuals. This has been compared quantitatively with the proviral DNA in peripheral blood leucocytes (PBL) obtained simultaneously from the same patients. The mean HIV DNA copy number per 10(6) cells was 391 for PBL, with a range of 1-9000, and 2971 for BAL cells, with a range of < 1-70,000. The quantity of HIV DNA detected in BAL cells was higher than that detected in the corresponding PBL samples in 44 out of 78 (56%) individuals, whilst more HIV DNA was detected in the PBL compared with BAL cells in 14 out of 78 (18%) patients. In both BAL and PBL higher levels of HIV DNA were detected in the adherent (monocyte/macrophage) enriched cell populations compared with other non-adherent cells (leucocytes). A direct relationship between HIV DNA copy number and ability to recover infectious HIV progeny in vitro by co-cultivation with cord blood leucocytes was found for both PBL and BAL cells. Individuals known to be receiving azidothymidine treatment had a lower mean HIV DNA load in all cell fractions compared with those patients on no antiretroviral therapy.     

Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART

The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 +/- 2.76) than in DNA (2.88 +/- 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had >or=1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens.     

HIV-1感染者CD4+T细胞受体基因多样性特点及其与病毒载量的相关性

目的 分析HIV-1感染者CD4+T细胞受体(TCR)基因的多样性特征及其与病毒载量的相关性.方法 应用抗CD4单克隆抗体从25份HIV-1感染者和10份HIV-1阴性对照样本外周血单个核细胞(PBMC)中分离CD4+T细胞,提取细胞总RNA,然后通过逆转录及巢式多聚酶链反应(nestedPCR)对TCR 22个Vβ基因家族的互补决定区3(CDR3)进行扩增,利用ABI3700测序仪对扩增的PCR产物进行扫描,定最分析HIV-1感染者TCRCDR3区多样性变化特征及其与病毒载量的相关性.结果 HIV-1感染者CD4+T细胞TCR CDR3区平均D(distance)值显著高于正常对照组(P＜0 05),TCR Vβ基因各家族CDR3长度谱型成寡克隆分布,TCR CDR3区的紊乱与病毒载量呈正相关(r=0 494,P＜0 05);HIV-1感染引起TCR多样性的改变不仅表现在不同Vβ基因家族上,而且也表现在CDR3长度上,其中感染者Vβ8、Vβ22、Vβ23基因家族的变化与正常人差异有统计学意义.结论 HIV-1感染能引起CD4+T细胞TCR基因多样性的减少及高斯(Gaussian)分布的破坏,TCR CDR3区的紊乱与病毒载量呈正相关.

Abstract：

Objective To assess the impact of the virus on the complementary determining region 3 (CDR3) length diversity of T cell receptor(TCR) Vβ repertoires of CD4+ T lymphocytes and to explore its association with viral load in individuals with HIV-1 infection. Methods The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD4+ T cells in HIV infected and healthy adults. Separation of CD4+ T cells from peripheral blood mononuclear cells ( PBMCs) was carried out by using immunomagnetic beads coated with anti-CD4 antibody. Total RNAs from the purified CD4 + T lymphocytes were isolated and used to perform nested-PCR amplifications in CDR3 of 22 TCR gene families. CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed. Results An average diversity for all CDR3 profiles in CD4+ T cells from 25 HIV-infected individuals was significantly different as compared to 10 age-matched healthy donors (P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles. There was positive correlation between changes in TCR CDR3 diversity and viral load (r = 0. 494, P ＜ 0. 05). The changes in CDR3 length diversity of Vβ families in HIV-infected individuals, particular in Vβ8, Vβ22, Vβ23 were statistically different from the healthy controls. Conclusion HIV-1 infection might induce the loss of TCR Vp repertoire diversity and disrupt the CDR3 Gaussian distributions within CD4 + T cells. There should be positive correlation between changes in TCR CDR3 diversity and the viral load in HIV-1 infected patients.     

Studies on the cross-clade and cross-species conservation of HIV-1 Gag-specific CD8 and CD4 T cell responses elicited by a clade B DNA/MVA vaccine in macaques

Here, we evaluate the T cell responses raised by our HIV-1 clade B DNA/MVA vaccine for recognition of a HIV-1 circulating recombinant form (CRF) AG Gag sequence (CRF-02). The cross-clade activity for the AG sequence was better conserved for CD8 than CD4 T cells. CD8 T cells exhibited 75% conservation for height and 83% conservation for breadth, whereas CD4 responses exhibited 45% conservation for height and 50% conservation for breadth. Five CD8 epitopes and 8 CD4 epitopes were mapped. Three of the 5 CD8 epitopes and 2 of the 8 CD4 epitopes were conserved across multiple HIV-1 clades. Impressively, all of the CD8 epitopes and half of the CD4 epitopes have been reported for human infections. Our results demonstrate that the clade B DNA/MVA HIV vaccine elicits T cell responses against epitopes that are conserved in multiple clades and recognized by humans and macaques.     

Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection

The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 variants in plasma that are considered to be recently produced.Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals.Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.