HIV-1感染者滤泡辅助性T细胞高表达CD32亚群的特点及临床意义

目的分析艾滋病病毒(HIV-1)感染者滤泡辅助性T细胞(Tfh)CD32高表达亚群(CD32~(hi)Tfh)的特点及其与疾病进展、病毒活跃复制的关系,以期探讨CD32~(hi)Tfh细胞亚群在艾滋病进展中的特点及临床意义。方法通过流式细胞术检测CD4~+T淋巴细胞(简称CD4细胞)及Tfh细胞亚群CD32~(hi)的比例,分析CD32~(hi)CD4细胞及CD32~(hi)Tfh细胞亚群百分比与免疫指标及外周单个核细胞(PBMCs)内病毒活跃复制指标的相关性。结果入组17例健康对照(HCs)和62例HIV-1感染者,其中未抗病毒治疗患者(TNPs)48例、抗病毒治疗完全应答患者(CRs)14例。(1)相较于HCs,TNPs组CD32~(hi)CD4细胞(平均值0.35%vs 0.12%,P0.000 1)及CD32~(hi)Tfh细胞亚群(平均值0.51%vs 0.21%,P=0.001 3)百分比显著增加;相较于TNPs,抗病毒治疗后,CRs组CD32~(hi)CD4细胞百分比显著增加(平均值0.35%vs 0.24%,P=0.030 7);(2)在TNPs中CD32~(hi)CD4细胞百分比与CD4细胞计数(r=-0.377 1,P=0.011 6)和CD4/CD8值(r=-0.328 6,P=0.029 4)呈负相关,与血浆HIV-1病毒载量(VL)呈正相关(r=0.367 4,P=0.014 9);TNPs患者CD32~(hi)Tfh细胞百分比与CD4细胞计数呈负相关(r=-0.376 3,P=0.023 7),与VL正相关(r=0.330 4,P=0.049 1);(3)TNPs患者CD32~(hi)CD4细胞及CD32~(hi)Tfh细胞亚群百分比与HIV脱氧核糖核酸呈正相关(r=0.534 4,P=0.042 4;r=0.575 0,P=0.027 4)。结论 HIV-1感染者体内CD32~(hi)Tfh细胞亚群增加可能与HIV-1活跃复制有关。     

慢性HIV-1感染者CD8细胞的铁死亡特点及其临床意义北大核心CSCD

目的 分析慢性HIV-1感染者外周血中CD8细胞的铁死亡水平,探讨CD8细胞铁死亡的临床意义。方法 利用流式细胞术检测HIV-1感染者和健康对照者外周血中CD8细胞的脂质过氧化水平,分析不同疾病状态下HIV-1感染者CD8细胞的不同记忆分化亚群及CXCR5+CD8细胞和CXCR3+CD8细胞的铁死亡情况,探究其变化特点及与疾病进展的关系。结果 纳入符合标准的30例未经ART的HIV-1感染者(TN),ART大于两年的免疫应答者(IR)17例,免疫无应答者(INR)9例与13例健康对照(HC),分析发现HC组和IR组相比TN组的CD8细胞的脂质过氧化水平显著升高(中位数TN vs. HC,0.068 85 vs. 0.041 16,P=0.000 8;TN vs. IR,0.068 85 vs. 0.045 06,P=0.001 6)。TN组CD8细胞EM亚群的脂质过氧化水平高于CM亚群、Naive亚群和EMRA亚群(中位数EM vs. CM vs. Na?ve vs.EMRA:0.083 14 vs. 0.061 19 vs. 0.071 27 vs. 0.063 09)。在TN组中,CXCR5+CD8细胞的脂质过氧化水平显著高于CXCR5-CD8细胞(中位数0.082 65 vs. 0.068 75, P<0.000 1),CXCR3+CD8细胞显著低于CXCR3-CD8细胞(中位数0.065 68 vs. 0.072 80,P<0.000 1)。TN组的CD8细胞脂质过氧化水平与CD4细胞计数具有显著负相关性(r=-0.470 1,P=0.008 8)。结论 在未经ART的HIV-1感染者中CD8细胞的铁死亡水平显著升高,其铁死亡水平与CD4细胞计数显著负相关,暗示了CD8细胞的铁死亡与HIV-1感染后的疾病进展程度有关。     

感染急性期HIV-1 DNA在CD_4~+T淋巴细胞亚群中的分布

目的阐明艾滋病病毒1型(HIV-1)感染急性期,HIV脱氧核糖核酸(DNA)在CD+4T淋巴细胞(简称CD4细胞)亚群中的分布特点。方法将来自北京佑安医院的男男性行为者(MSM)HIV感染急性期队列,根据病人感染后病情进展速度的不同,将病人分为两组:一组20例病人,病情进展迅速,CD4细胞计数在2年内降低到200个/μL以下,为CD4细胞低组;另外一组23例病人,病情进展较慢,CD4细胞计数一直维持在500个/μL,为CD4细胞高组。收集病人急性期外周血,分离CD4细胞亚群,实时聚合酶链反应(RT-PCR)检测亚群中HIV DNA。结果记忆性CD4细胞中的HIV DNA含量为(4199±317.4)拷贝/100万细胞,高于幼稚CD4细胞中的(75±12.1)拷贝/100万细胞(P=0.0117);幼稚CD4细胞中HIV-1DNA的含量,CD4细胞低组高于CD4细胞高组(P0.01)。结论 HIV-1急性感染期幼稚CD4细胞中HIV-1DNA可能和疾病进展有关。     

HIV感染者B细胞的特点及其对疾病进展的影响

艾滋病(AIDS)是由艾滋病病毒-1(HIV-1)感染后引起的一种严重危害人类健康的重大传染病。除了进行性的CD4+T淋巴细胞减少,HIV-1感染还造成多种免疫细胞的功能改变,这些功能改变极大地影响艾滋病的疾病进展。B细胞是人体免疫系统中重要的免疫细胞,其不但能介导体液免疫,还是重要的免疫调节细胞。然而HIV-1感染会在多个方面造成B细胞的功能缺陷。文章分析了HIV-1感染对B细胞免疫超活化、B细胞亚群组成以及调节性B细胞的影响及这些影响与艾滋病疾病进展的关系。     

抗病毒治疗HIV-1感染者外周血CD39~+ NK细胞的表达特点

目的通过分析接受长期抗病毒治疗(ART)的1型艾滋病病毒(HIV-1)感染者体内CD39~+NK细胞的频率和表型特征,探索其与主要临床指标的关系及临床意义。方法本研究入组10例健康对照(HCs)和28例长期接受ART的HIV-1感染者。利用流式细胞术检测NK细胞亚群分布以及NK细胞上CD39的表达情况,分析CD39+NK细胞亚群比例与CD4、CD8~+T淋巴细胞(简称CD4、CD8细胞)计数、以及CD4/CD8细胞比值的相关性。结果 28例长期接受ART的HIV-1感染者中,18例免疫重建成功患者(CRs)和10例免疫重建失败患者(INRs)。相较于HCs和CRs组,INRs组CD56dim亚群比例明显下降(平均值94.75%vs.82.65%,P 0.01;平均值93.20%vs.82.65%,P 0.001);INRs组CD39~+NK细胞的频率明显高于CRs组(平均值21.45%vs.7.79%,P 0.05)和HCs组(平均值21.45%vs.4.115%,P 0.001);长期接受ART的HIV-1感染者CD39+NK细胞占比与其CD4/CD8细胞比值呈负相关(r=-0.392 4,P=0.038 9)。结论免疫重建失败患者外周血中CD39+NK细胞表达频率明显高于免疫重建成功患者,HIV-1慢性感染者外周血CD39~+NK细胞表达增加伴随着CD4/CD8细胞比值的降低。     

艾滋病絮谈(三十七)

一、艾滋病毒存在许多亚型 HIV是一种逆转录病毒。已明确有两个型即HIV-1和HIV-2。由于这两种人类免疫缺陷病毒具有基因多变的特点和抗原漂移现象,近年分子生物学的研究不断发现一些新的病毒亚型:如HIV-1除早已确定的A～E5个亚型外,最近又报道了F～H和O亚型,至少可分作9个亚型;而HIV-2分为A和B两个亚型。1994年来又把HIV-1的A～H亚型统归为M亚群,而把O亚型称为O亚群,对此国际上正在     

HIV-1潜伏感染人Jurkat T细胞模型的建立

目的 建立Ⅰ型艾滋病病毒(HIV-1)潜伏感染人Jurkat T细胞的模型,为进一步研究HIV-1潜伏感染机制奠定基础.方法 利用一种表达增强型绿色荧光蛋白( EGFP)的HIV-1假病毒感染人Jurkat T细胞,采用流式细胞术分选出GFP -细胞群,之后经激活剂曲古抑菌素A(Trichostatin A,TSA...     

HIV感染者外周血单个核细胞中HIVDNA载量与HIVRNA及CD4~+T淋巴细胞间的关系

目的分析未经抗病毒治疗(ART)的1型艾滋病病毒(HIV-1)感染者外周血单个核细胞(PBMC)中HIV-1脱氧核糖核酸(DNA)载量与血浆HIV-1核糖核酸(RNA)及外周血CD4+T淋巴细胞(简称CD4细胞)计数间的关系。方法 50例未经ART的HIV-1感染者,采用套式反转录荧光定量聚合酶链反应(RT-PCR)技术检测血浆中HIV-1 RNA含量,使用磁珠分离法从外周血中分离PBMC并提取DNA采用PCR进行HIV-1 DNA检测,使用流式细胞仪进行T淋巴细胞亚群CD4细胞检测;使用SPSS 19.0软件对数据进行分析。结果 50例未经ART的HIV-1感染者血清中HIV-1 RNA高于检测限的为42/50,平均水平为(4.28±2.57)Lg IU/mL,HIV-1 DNA高于检测限的为47/50,平均水平为(2.63±0.81)Lg拷贝/106 PBMC,CD4细胞计数中位数198(95～328)个/μL。结论未经抗病毒治疗的HIV-1感染者外周血单个核细胞中HIV-1 DNA载量与外周血浆HIV-1 RNA载量呈正相关,与CD4细胞计数呈负相关,HIV-1 DNA载量检测可用来评估HIV-1感染者体内HIV的含量。     

HIV-1慢性感染者CD39~+PD-1~+CD4~+T淋巴细胞特点与抗病毒治疗疗效的关系

目的通过分析1型艾滋病病毒(HIV-1)感染者不同疾病阶段CD39~+PD-1~+CD4~+T淋巴细胞(简称CD39~+PD-1~+CD4细胞)的特点及其与潜伏病毒库形成的关系,探讨CD39~+PD-1~+CD4细胞的临床意义。方法通过流式细胞术检测CD4细胞上CD39及PD-1的表达情况,分析CD39~+PD-1~+CD4细胞与CD4细胞计数、病毒载量及CD4细胞内病毒指标的相关性。通过实时荧光定量聚合酶链反应(PCR)检测免疫重建成功患者(CRs)和免疫重建失败患者(INRs)的HIV库,分析CD39~+PD-1~+CD4细胞与HIV库的关系。结果入组11例健康对照(HCs)和69例HIV-1感染者,其中包括38例未抗病毒治疗(ART)者(TNs)、21例CRs、10例INRs。1)与HCs相比,TNs组患者CD39~+PD-1~+CD4细胞亚群占比显著升高(平均值0.99%vs. 2.50%);与TNs组相比,ART后,CRs组患者的CD39~+PD-1~+CD4细胞亚群占比显著降低(平均值2.50%vs. 0.86%);而INRs组患者该细胞亚群占比显著高于CRs组(平均值2.74%vs. 0.86%);2)在TNs组患者中CD39~+PD-1~+CD4细胞亚群占比与CD4细胞计数呈负相关(r=-0.359 6, P=0.026 6),与病毒载量呈正相关(r=0.451 1, P=0.004 5);3)ART两年以上患者CD39~+PD-1~+CD4细胞亚群占比与HIV脱氧核糖核酸(HIV DNA)正相关(r=0.565 9,P=0.047 3),与细胞相关的未剪接HIV核糖核酸(HIV us RNA)正相关(r=0.675 8,P=0.013 7)。结论 CD39~+PD-1~+CD4细胞与ART后免疫重建失败相关,其机制可能是CD39~+PD-1~+CD4细胞促进HIV建立潜伏病毒库。     

Cellular reservoirs of HIV-1 and their role in viral persistence

A major obstacle in human immunodeficiency virus type 1 (HIV-1) eradication is the ability of the virus to remain latent in a subpopulation of the cells it infects. Latently infected cells can escape the viral immune response and persist for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). Given the appropriate stimulus, latently infected cells can reactivate and start producing infectious virions. The susceptibility of these cell populations to HIV-1, their life span, their proliferative capacity, and their ability to periodically produce infectious virus subsequent to alterations in cellular physiology and/or immunologic controls are critical issues which determine the contribution of these cells to viral persistence. Memory CD4+ T cells due to the long life span, which may be several years, and their ability to reactivate upon encounter with their cognate antigen or other stimulation, are considered a critical reservoir for maintenance of latent HIV-1 proviral DNA. Cells of the monocyte-macrophage lineage, which originate in the bone marrow (BM), are of particular importance in HIV-1 persistence due to their ability to cross the blood-brain barrier (BBB) and spread HIV-1 infection in the immunoprivileged central nervous system (CNS). Hematopoietic progenitor cells (HPCs) are also a potential HIV-1 reservoir, as several studies have shown that CD34+ HPCs carrying proviral DNA can be found in vivo in a subpopulation of HIV-1-infected patients. The ability of HPCs to proliferate and potentially generate clonal populations of infected cells of the monocyte-macrophage lineage may be crucial in HIV-1 dissemination. The contribution of these and other cell populations in HIV-1 persistence, as well as the possible strategies to eliminate latently infected cells are critically examined in this review.     

A potent anti-HIV immunotoxin blocks spreading infection by primary HIV type 1 isolates in multiple cell types

Although several immunotoxins that selectively kill HIV-1-infected cells have been described, their clinical utility is limited by low potency against spreading viral infection. We show here that changing the carboxyterminal sequence of an anti-HIV-1 envelope immunotoxin to the consensus endoplasmic reticulum retention sequence KDEL substantially improves its ability to block infection of peripheral blood mononuclear cells by primary HIV-1 isolates without increasing nonspecific toxicity. Polychromatic flow cytometry of peripheral blood mononuclear cells (PBMC) infected with an HIV-1-GFP reporter virus demonstrated that the improved immunotoxin is active against a variety of primary cell types including memory T cells, NK-T cells, and monocyte/macrophages. The subnanomolar potency of this agent suggests that it could be clinically useful either as an adjuvant to highly active antiretroviral therapy (HAART) in drug-resistant patients or to reduce the reservoir of latently infected cells that is implicated in HIV-1 persistence.     

HAART-persistent HIV-1 latent reservoirs: their origin, mechanisms of stability and potential strategies for eradication

HIV-1 infection persists despite long-term administration of highly active antiretroviral therapy (HAART). The mechanism of this persistence appears to result primarily from viral infection of CD4+ T-lymphocytes that have the ability to duplicate and revert into a quiescent state. These infected resting cells are long-lived and evade immune surveillance or clearance. The inability to eradicate this class of cells, bearing the viral DNA, suggests life-long persistence of virus in HIV-1-infected individuals, even if HAART were administered for decades. This review discusses the origins and mechanisms accounting for stability of these latent HIV-1 cellular reservoirs. It further provides an overview of recent clinical trials aimed at their eradication. There have been a limited number of immune activation (IAT) trials directed at HAART-persistent, viral reservoir eradication. These trials have not resulted in purging of these highly stable viral reservoirs though results from such efforts suggest partial effects. The properties of novel compounds that might be included into IAT eradication protocols are continuing to be evaluated and their potential for inclusion into future IAT trials will be discussed.     

Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection.     

HIV type 1 persistence in CD4- /CD8- double negative T cells from patients on antiretroviral therapy

The establishment of reservoirs of latently infected cells is thought to contribute to the persistence of HIV-1 infection in the host. Studies so far have mainly focused on the long-lived reservoir of HIV-infected resting CD4+ T cells. A discrete population of HIV-infected CD4-/CD8- double negative (DN) T cells has recently been shown to exist and may also play a role in HIV-1 persistence. DN T cells are CD3 positive, either TCRalphabeta or TCRgammadelta positive, but lack both CD4 and CD8 surface markers. We developed a novel, magnetic bead column-based cell fractionation procedure for isolating >99% pure DN T cells. CD4+, CD8+, and DN T cells were purified from 23 samples of a cohort of 18 HIV-1-infected patients. Each cell fraction was analyzed for levels of total and integrated HIV-1 DNA. A correlation was observed between the presence of HIV-1 DNA in the DN T cell fraction and plasma viral load (VL). Using a micrococulture technique, we saw an initial release of virus from DN T cells of a patient with high VL. Analysis of env and nef sequence data suggested that the HIV-1 present in CD4+ and DN T cells originated from a common infecting strain. Different from the published literature, we have demonstrated the presence of HIV-1 DNA in DN T cells only in patients who are experiencing HAART failure. While these cells may have a limited role in viral persistence in high VL patients, our results suggest DN T cells are unlikely to be a major reservoir in patients on HAART with clinically undetectable plasma viral RNA.     

When integrated in a subepithelial mucosal layer equivalent, dendritic cells keep their immature stage and their ability to replicate type R5 HIV type 1 strains in the absence of T cell subsets

Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC). The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage. Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown. Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro. We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors. These cells were either integrated in SEMLE or maintained in suspension. Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)). Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media. Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo. Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets. Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE. These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation. In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.     

Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus

Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells. The human cytomegalovirus US28 gene encodes a chemokine receptor homolog that has been reported to function as an HIV-1 coreceptor. However, studies of US28 have given conflicting results regarding its ability to mediate HIV-1 entry. We examined the ability of US28 to function as an HIV-1 coreceptor in various cell lines and found that its coreceptor activity is highly cell dependent. US28 could function as a coreceptor for HIV-1 entry in HeLa and U87 cells but not in COS-1 and Cf2Th cells. In COS-1 cells, US28 was expressed on the cell surface and could mediate cell-cell fusion with HIV-1 Env-expressing cells, suggesting that the block to infection may result from a defect in virus internalization or postentry steps. In Cf2Th cells, US28 was expressed at high levels intracellularly but was not transported to the cell surface. The block in US28 coreceptor function in COS-1 and Cf2Th cells was coreceptor dependent, since CCR5, CXCR4, and other coreceptors can mediate HIV-1 entry in these cell lines. HIV-1 viruses pseudotyped with the MuLV or VSV Env entered and replicated at similar efficiency in COS-1 and U87 cells in single-cycle infections, suggesting that postentry and other early events in the HIV-1 life cycle are not intrinsically inefficient in COS-1 cells. These results identify two distinct mechanisms that can restrict the HIV-1 coreceptor activity of US28 in a cell- and coreceptor-dependent manner, and help to explain the existing controversy regarding the ability of US28 to mediate HIV-1 entry.     

Reservoirs for HIV-1

The success of antiretroviral therapy for HIV-1 infection has generated interest in mechanisms by which the virus can persist in the body despite the presence of drugs that effectively inhibit key steps in the virus life cycle. There are several potential cellular and anatomic reservoirs for HIV-1. Among the most worrisome is a reservoir consisting of latently infected resting CD4+ T cells. Recent studies suggest that these cells can potentially provide a mechanism for life-long persistence of replication-competent forms of HIV-1, rendering unrealistic hopes of virus eradication with current antiretroviral regimens.     

Cervical and prostate primary epithelial cells are not productively infected but sequester human immunodeficiency virus type 1

Primary prostate and cervical epithelial cells and epithelial cell lines were examined for human immunodeficiency virus type 1 (HIV-1) infection or transmission to peripheral blood mononuclear cells (PBMC). Neither cell-free nor cell-associated HIV-1 infected primary epithelial cells or cell lines. Pretreatment of HIV-1 to enhance CD4-independent entry did not augment infection. Cell surface expression was detected for galactosyl ceramide but not for CC-chemokine receptor 5, CXC-chemokine receptor 4, or CD4. The ability to transfer HIV-1 to resting or activated PBMC was tested by culturing with rinsed or trypsinized and replated HIV-1-exposed epithelial cells. Virus was not recovered from the rinsed or replated cocultures with resting PBMC; however, activated PBMC recovered HIV-1 from rinsed epithelial cells and rarely from replated epithelial cells. Although urogenital epithelial cells are not infected, these data suggest that they can transfer virus to activated immune cells and have implications for sexual transmission of HIV-1.     

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.