单核细胞TLR7/8表达与HIV-1感染疾病进程关系的研究

目的 对HIV-1感染者单核细胞TLR7/8表达水平及与疾病进展的相关性进行研究,探讨单核细胞TLR7/8在HIV-1疾病进程中的作用.方法 选取63名HIV-1感染者及18名健康对照,应用MACS磁珠分选法纯化CD14+单核细胞,用2.5μg/ml R848刺激单核细胞TLR7/8,QIAGEN公司的RNA提取试剂盒提取单核细胞总RNA,实时定量RT-PCR测定TLR7/8的mRNA表达.结果 HIV-1感染者单核细胞TLR7和TLR8的表达水平与CIM+T细胞显著正相关(r=0.614,P＜0.01;r=0.419,P＜0.01).TLR7 mRNA表达:缓慢进展组明显高于HIV感染组、AIDS组和健康对照组(P＜0.05),HIV感染组明显高于AIDS组(P＜0.05);TLR8 mRNA表达:缓慢进展组明显高于AIDS组(P＜0.05).体外用R848刺激11LR7后表达显著下调,而TLR8的表达稳定.结论 AIDS疾病进程中HIV-1感染者单核细胞TLR7/8表达水平明显下降,TLR7的表达水平下降更显著.     

中国HIV/AIDS患者NK细胞及NKT细胞变化的检测

目的　探讨HIV感染后机体NK(naturalkillercells)及NKT细胞的变化情况。方法　取外周血细胞 ,用标记荧光的抗体进行染色 ,流式细胞仪分析HIV AIDS患者NK和NKT细胞的变化。结果　HIV AIDS患者NK、NKT细胞和CD4 + T细胞显著低于正常对照 ;CD8+ T细胞显著高于正常对照。HIV AIDS患者NK百分数显著低于正常对照 ,与CD4 + T细胞数量成正比 ,r=0 .2 89,P <0 .0 1 ;NKT细胞数量与CD4 + T细胞数量成正比 ,r =0 .378,P <0 .0 1 ;与CD8+ T细胞数量成正比 ,r =0 .340 ,P <0 .0 1 ;长期不进展组NKT、NK细胞数量与正常对照组差异无显著性。结论　HIV感染可明显降低HIV AIDS患者NK和NKT细胞数量 ,NK和NKT细胞变化与疾病进展密切相关。     

HIV/AIDS患者外周血Th9细胞及其转录因子PU.1与病程关系的分析

目的研究HIV/AIDS患者外周血Th9细胞及其转录因子PU.1的表达量,探讨其与艾滋病疾病进展的相关性。方法40例HIV/AIDS患者根据CD4~+T淋巴细胞分为A、B 2组(A组CD4~+T淋巴细胞350,B组CD4~+T淋巴细胞350),同时,选取20例健康人为对照组,采用流式细胞技术及RT-PCR技术检测其外周血CD4~+T淋巴细胞亚群(Th1、Th2、Th9)及其相关转录因子(T-bet、GATA3、PU.1)的表达量,通过比较3组间Th9细胞及其转录因子PU.1表达量的差异,分析Th9细胞及其转录因子与艾滋病疾病进展的相关性。结果与健康人群相比,HIV/AIDS患者CD4~+T细胞绝对计数与百分比明显降低,CD8~+T细胞的绝对计数与百分比明显升高;3组组间比较显示,Th1细胞百分比A组(6.71±3.05)和B组(5.82±2.52)低于健康对照组(10.89±6.82)差异有统计学意义(P0.05);Th2细胞百分比3组间差异无统计学意义;Th9细胞百分比A组(2.0±0.69)、B组(0.66±0.23)和健康对照组(0.40±0.16)之间差异有统计学意义(P0.05);转录因子PU.1相对表达量A组(3.30±0.78)和B组(1.59±0.44)之间差异有统计学意义(P0.05)。Th9细胞及其转录因子PU.1相对表达量与CD4~+T细胞成负相关(r=-0.637,r=-0.746,P=0.000)。结论 HIV/AIDS患者外周血Th9细胞及其转录因子PU.1的表达量随着疾病进展逐渐升高,两者与艾滋病疾病进展密切相关,可作为判断疾病进展的指标。     

中国HIV感染长期不进展者CD4+CD25+Foxp3+调节性T细胞变化研究

目的 对中国HIV感染长期不进展者(LTNP)CD4+CD25+Foxp3+调节性T细胞水平及其与疾病进展相关性进行研究,探讨CD4+CD25+Foxp3+ 调节性T细胞在LTNP保护机制中发挥的作用.方法 选取74名HIV-1感染者(LTNP、典型进展HIV组、AIDS组)及16名健康对照,应用流式细胞仪胞内染色技术在单细胞水平检测CD4+CD25+Foxp3+调节性T细胞表达水平,分析其与CD4+ T细胞数量、病毒载量、淋巴细胞活化、凋亡水平的相关性.结果 中国HIV感染LTNP CD4+CD25+Foxp3+ T细胞百分率明显低于典型进展HIV、AIDS组及健康对照组(P<0.05).HIV/AIDS患者CD4+CD25+Foxp3+ T细胞百分率与CD4+ T细胞显著负相关(r=-0.509,P<0.001),与病毒载量明显正相关(r=0.414,P<0.01),与CD4、CD8+ T细胞表面CD38、CD95表达水平明显正相关(P<0.05),与CD4、CD8+ T细胞表面HLA-DR表达无显著相关性.结论 中国HIV感染LTNP CD4+CD25+Foxp3+ 调节性T细胞百分率明显低于典型进展者,提示调节性T细胞与LTNP保护机制相关.     

HIV/AIDS患者B7-H1的表达与合并机会性感染关系的研究

目的:探讨HIV/AIDS患者外周血髓样树突细胞(mDC)上B7-H1表达水平与合并机会性感染的关系。方法:采集2009年1月至2012年5月住院的42例未经抗逆转录病毒治疗(ART)的HIV/AIDS患者及42名健康对照组抗凝全血,用流式细胞仪检测外周血mDC上B7-H1表达水平,并分析其与疾病进展的相关性。结果:HIV/AIDS患者合并机会性感染B7-H1表达水平显著高于未合并机会性感染组(P<0.05),未合并机会性感染组显著高于健康对照组(P<0.05);B7-H1表达水平>30%合并机会性感染率显著高于B7-H1表达水平<10%的HIV/AIDS患者(P<0.05);HIV/AIDS患者mDC上B7-H1表达水平与CD4+T细胞数量显著负相关(r=-0.665,P<0.05),而与病毒载量显著正相关(r=0.604,P<0.05)。结论:HIV/AIDS患者mDC上B7-H1表达水平与合并机会性感染密切相关,是疾病进展的指标。     

HIV/AIDS患者疾病进程与CD4+CD25hi调节性T细胞之间相关性研究

目的 通过分析不同阶段HIV感染者外周血CD4+CD25hi调节性T细胞(CD4+CD25hiregulatory T cells,Treg cells)与外周血免疫状态和病毒载量的相关性,探讨Treg细胞对HIV/AIDS发病进程的影响.方法 采集116例HIV感染者和21例正常人对照外周血,用4色流式细胞术进行CD4+和CD8+T细胞绝对数计数;用3色流式细胞术进行Treg细胞测定;用荧光定量PCR法进行HIVRNA载晕测定.实验数据用回归统计学方法和T检验方法进行分析.结果 HIV感染者外周血Treg细胞频率在HIV感染初期显著下降,之后随着疾病的进程逐渐升高.在CD4+T细胞大于300/μl的患者低于正常对照组,在CD4+T细胞小于100/μl的患者高于正常对照组,差异具有统计学意义.Treg细胞频率与CD4+T淋巴细胞绝对数和CD4+/CD8+之间均呈负相关.其相关系数r和P值各为r=-0.564,P＜0.001和r=-0.377,P＜0.001;Treg细胞频率与血浆HIV病毒载量呈正相关,其相关系数r=0.514.P＜0.001.结论 CD4+CD25hi Treg细胞可能是参与艾滋病免疫发病机理的重要细胞,在HIV感染发病进程的不同阶段具有不同的意义,其确切机制有待进行进一步研究.     

免疫调控受体PD-1和TIGIT在HIV感染者/HIV患者CD3~+CD4~-T细胞表达研究

深入了解免疫调控受体程序性死亡分子1(programmed death 1,PD-1)和T细胞免疫球蛋白及免疫酪氨酸样抑制基序(T cell immunoglobulin and ITIM domain,TIGIT)在人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者/HIV患者外周血CD3~+CD4~-T细胞的表达情况及其与疾病进展的关系。用流式细胞仪检测24例未治疗的HIV感染者/HIV患者外周血CD3~+CD4~-T细胞表面PD-1和TIGIT的表达百分比、CD4+T细胞绝对值计数,实时荧光定量PCR检测HIV病毒载量,并与20例HIV抗体阴性者表达情况比较(HIV-negative normal control,NC)。结果发现HIV感染者/HIV患者CD3~+CD4~-T细胞表面TIGIT受体表达百分数较健康对照组明显增高(P0.000 1),与CD4+T细胞绝对计数呈负相关(r=-0.450 4,P=0.027 1),与病毒载量呈正相关(r=0.621 4,P=0.001 2);HIV感染者CD3~+CD4~-T细胞PD-1受体表达百分数较健康对照组明显增高(P0.000 1),与CD4+T细胞绝对计数呈负相关(r=-0.455,P=0.0255);PD-1和TIGIT共表达的CD3~+CD4~-T细胞百分数在HIV感染者/HIV患者中表达明显增高(P0.000 1)。HIV感染机体后,CD3~+CD4~-T细胞PD-1受体和TIGIT受体表达百分数均明显增高,且与HIV疾病进展密切相关。     

HIV/AIDS患者外周血CD4~+T细胞亚群及中性粒细胞CD64的变化及临床意义

目的探讨HIV/AIDS患者外周血中CD4+T细胞亚群及中性粒细胞CD64的变化及临床意义。方法筛选2014年5月至9月河南中医学院第一附属医院艾滋病临床研究中心收治的47例HIV/AIDS患者,根据CD4+T淋巴细胞数将其分为A组(CD4≤350 cell/μl)23人,B组(CD4350 cell/μl)24人,同期选取健康对照组20人。用流式细胞仪(FCM)检测3组人员外周血中CD4+T细胞亚群(Th1、Th2、Th17、Treg)及中性粒细胞表面CD64表达水平,用SPSS17.0软件分析数据。所有数据用中位数表示,2组间比较采用Mann-Whitney检验,相关性分析采用Spearman秩相关。结果 A组和健康对照组Th1细胞(CD4+IFN-γ+)百分比均高于B组(P=0.040,P=0.028);A组Th2细胞(CD4+IL4+)百分比高于B组(P=0.015);A和B组Treg细胞(CD4+CD25+FOXP3+)百分比均高于健康对照组(P均0.05),A组Treg细胞高于B组(P=0.001);Th17细胞以及中性粒细胞表面CD64的表达量3组比较差异无统计学意义;Treg细胞与CD4+T细胞呈负相关(r=-0.734,P=0.000),Treg细胞与Th1/Th2比值呈负相关(r=-0.300,P=0.015)。结论 HIV/AIDS患者CD4+T细胞亚群之间的平衡失调,调节性T细胞处于优势状态,CD4+T细胞亚群变化可作为HIV/AIDS患者疾病进展的动态监测指标。CD64是机体急性炎症感染的一个重要参考指标,但在HIV/AIDS患者中变化不明显。     

中国HIV/AIDS患者外周血单核细胞上TLR4表达和血浆TNF-α水平

目的 观察中国HIV/AIDS患者外周血单核细胞上Toll样受体4(TLR4)表达水平和血浆TNF-α浓度,初步探讨TLR4在HIV感染中的作用.方法 分别运用流式细胞术、ELISA法测定46例HIV/AIDS患者和30例健康对照者的外周血单核细胞上TLR4的表达及血浆TNF-α浓度,采用t检验和相关分析进行数据分析.结果 HIV/AIDS患者外周血单核细胞TLR4的表达率、血浆TNF-α浓度分别是52.19%±4.37%和(35.79±5.08)pg/ml,均显著高于健康对照组;单核细胞上TLR4的表达率与血浆TNF-α浓度、HIV-1病毒载量的相关系数分别是0.645和0.708,呈正相关.结论 中国HIV/AIDS患者外周血单核细胞TLR4表达上调,并且与患者体内HIV复制存在一定的相关性.     

HIV感染后CD4~+ T细胞表面NK相关受体表达的变化

目的研究人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染后CD4+T细胞表面NK相关受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV感染者、11例AIDS患者、15例进行高效抗逆转录病毒治疗(highly activeantiretroviral therapy,HAART)者和13例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血CD4+T细胞表面NKG2D、NKG2A和KIR3DL1的表达。结果 AIDS患者CD4+T细胞表面NKG2D表达的百分比显著高于其他各组,且NKG2D表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.352,P<0.05),与HIV病毒载量呈正相关(R=0.426,P<0.05)。AIDS患者CD4+T表面NKG2A表达的百分比显著高于其他各组,NKG2A+NKG2D-表达的百分比显著高于其他各组,且NKG2A表达的百分比与CD4+T细胞的绝对数量呈负相关(R=-0.432,P<0.01)。结论 HIV感染机体后,CD4+T细胞NK相关受体表达变化与疾病进展相关。     

HIV 感染者调节性T 细胞表面B、T 淋巴细胞衰减因子的表达研究

目的:对HIV 感染者调节性T 细胞(Regulatory T cell,Treg)上B 、T 淋巴细胞衰减因子(B and T lymphocyte at-tenuator,BTLA)的表达水平进行检测,并探讨其在HIV 感染进程中的作用。方法:选取24 例感染在一年之内的HIV 早期感染者(Early HIV infected patients,EHI 组)、14 例感染超过一年的HIV 慢性感染者(CD4+ T 计数>200 cells/ μl,HIV 组)、6 例AIDS患者(CD4+ T 计数<200 cells/ μl,AIDS 组)和9 例健康人作为对照,应用流式细胞仪检测不同时期感染者及健康对照者Treg 细胞BTLA 的表达水平,分析其与疾病进展及免疫活化的相关性。结果:随着HIV 疾病进展,EHI 组、HIV 组及AIDS 组Treg 细胞BTLA 表达水平依次升高,其中HIV 组与AIDS 组Treg 细胞BTLA 表达水平显著高于EHI 组(P<0.05 及P<0.01),AIDS 组Treg 细胞BTLA 的表达水平高于健康对照(P<0.05);Treg 细胞BTLA 表达水平与CD4+ T 淋巴细胞计数呈负相关(P<0.001),与病毒载量呈正相关(P<0.01);Treg 细胞BTLA 表达水平与活化CD4+ CD38+ T 淋巴细胞及CD4+ HLA-DR+ T 淋巴细胞呈正相关(P<0.001,P<0.001)。结论:HIV 感染者Treg 细胞BTLA 表达升高,与疾病进展显著相关,提示其可能通过加强Treg 细胞的抑制功能加速疾病进展,并为未来HIV 感染的干预提供信息。     

Alterations of natural killer cell and T-lymphocyte counts in adults infected with human immunodeficiency virus through blood and plasma sold in the past in China and in whom infection has progressed slowly over a long period

Natural killer (NK) cells, natural killer T (NKT) cells, and T lymphocytes were analyzed by using a flow cytometer in 225 human immunodeficiency virus (HIV)-positive individuals infected through the past sale of blood and plasma without receiving antiretroviral therapy in the People's Republic of China. According to CD4 T-cell counts these HIV-infected adults were stratified into three groups: long-term slow progressors, HIV-infected subjects, and AIDS patients. NK cell counts in long-term slow progressors were higher compared to HIV infection and AIDS patients (P < 0.05) and lower compared to normal controls (P < 0.05), whereas NKT cell counts in slow progressors and the HIV infection group were not different from those of normal controls. NK cell counts in HIV-seropositive subjects were positively correlated with CD4 T-cell counts (P < 0.05), and NKT cell counts were positively correlated with CD4 T-cell and CD8 T-cell counts (P < 0.05). The CD8 T-cell counts were higher in slow progressors compared to those with HIV infection, AIDS patients, and normal controls. These results indicated that HIV infection causes alterations of NK cells and T cells in slow progressors, HIV-infected subjects, and AIDS patient groups, but no difference was found in NKT cell counts and percentages in slow progressors and the HIV-infected group compared to normal controls.     

Peripheral B cells with intracytoplasmic mu chains in HIV infection.

Besides the major alteration of T lymphocytes, B-cell anomalies have been reported in HIV infection, related to late stages of B-cell maturation, and considered to result from the dysregulation of T/B interactions. Because T cells are also involved in the control of lymphopoiesis and/or because of specific alterations of the B lineage, anomalies of B-cell maturation could occur in HIV-infected patients. We investigated the presence of immature pre-B lymphocytes, characterized by cytoplasmic mu chains, in 35 peripheral blood samples from healthy controls, 82 from HIV-positive/non-AIDS patients, and 45 from AIDS patients. Significant numbers of such cells were observed in 48% of HIV-seropositive patients and in 40% of the patients with AIDS disease. The presence of pre-B cells correlated with higher numbers of CD8+ and/or CD57+ cells and of peripheral lymphocytes. These data suggest that B-cell dysregulation in HIV infection may lead to the abnormal release of immature B cells in the peripheral blood. This observation may be interpreted as a sign of bone marrow activity.     

Increased plasma levels of matrix metalloproteinase-9 are associated with the severity of chronic urticaria

BACKGROUND: Matrix metalloproteinase (MMP)-9 is produced by many inflammatory cells such as macrophages, neutrophils, mast cells, eosinophils and T lymphocytes. Activated T cells are capable, through cell-cell contact, of inducing MMP-9 expression in human mast cells. OBJECTIVE: To investigate the activation status of peripheral CD4+ T cells and the level of MMP-9 in the plasma of patients with chronic urticaria (CU), and whether MMP-9 levels are in association with CU severity. METHODS: Study subjects included 29 patients with CU and 30 healthy control subjects. At the time of assessment, patients were divided into subgroups according to urticarial severity. Plasma levels of total MMP-9 (free pro-MMP-9 and free MMP-9) were determined by ELISA. CD4+ lymphocytes were positively selected with magnetic microbeads. After 48 h of activation, CD4+ T cells were assayed for both nuclear factor-kappa B (NF-kappa B) expression and proliferation. RESULTS: Plasma levels of MMP-9 were found to be significantly higher in 29 CU patients compared with 18 healthy controls (186 +/- 174 vs. 31 +/- 21 ng/mL, P<0.0001). We also found a significant correlation between MMP-9 levels and urticarial severity (r = 0.92, P<0.001). In addition, CD4+ T cells from CU patients expressed higher levels of NF-kappa B than CD4+ T cells from healthy controls (82 +/- 30 vs. 69 +/- 20 optical density, P = 0.007). Finally, as compared with seven healthy individuals, DNA synthesis in CD4+ T cells from seven CU patients was found to be significantly elevated (1000 +/- 240 vs. 751 +/- 166 counts per minute, P = 0.01). CONCLUSION: Increased levels of MMP-9 are found in CU patients, and particularly among those with severe disease. We also demonstrated that CD4+ T cells from such patients are highly activated.     

HIV感染者NKT样细胞基线功能对疾病进程的影响

目的本实验通过检测HIV感染者NKT样细胞基线功能的变化,研究NKT样细胞对HIV感染疾病进程的影响。方法应用流式细胞术直接对HIV感染者以及健康对照外周血NKT样细胞IFN-γ分泌和CD107a表达进行研究。结果 NKT样细胞分泌IFN-γ百分比高者HIV疾病进展慢(P<0.008,P<0.001),与CD4+T细胞计数呈显著正相关(r=0.402,P=0.027),而与病毒载量呈显著负相关(r=-0.472,P=0.037)。结论 NKT样细胞功能较强,具有免疫保护作用,是延缓HIV病程的重要因素之一,可作为监测HIV疾病进展的指标。     

Alterations of Natural Killer Cell and T-Lymphocyte Counts in Adults Infected with Human Immunodeficiency Virus through Blood and Plasma Sold in the Past in China and in Whom Infection Has Progressed Slowly over a Long Period

Natural killer (NK) cells, natural killer T (NKT) cells, and T lymphocytes were analyzed by using a flow cytometer in 225 human immunodeficiency virus (HIV)-positive individuals infected through the past sale of blood and plasma without receiving antiretroviral therapy in the People’s Republic of China. According to CD4 T-cell counts these HIV-infected adults were stratified into three groups: long-term slow progressors, HIV-infected subjects, and AIDS patients. NK cell counts in long-term slow progressors were higher compared to HIV infection and AIDS patients (P < 0.05) and lower compared to normal controls (P < 0.05), whereas NKT cell counts in slow progressors and the HIV infection group were not different from those of normal controls. NK cell counts in HIV-seropositive subjects were positively correlated with CD4 T-cell counts (P < 0.05), and NKT cell counts were positively correlated with CD4 T-cell and CD8 T-cell counts (P < 0.05). The CD8 T-cell counts were higher in slow progressors compared to those with HIV infection, AIDS patients, and normal controls. These results indicated that HIV infection causes alterations of NK cells and T cells in slow progressors, HIV-infected subjects, and AIDS patient groups, but no difference was found in NKT cell counts and percentages in slow progressors and the HIV-infected group compared to normal controls.     

高效抗逆转录病毒治疗对中国HIV/AIDS患者淋巴细胞活化及第二受体表达的影响

目的　了解高效抗逆转录病毒治疗后中国HIV/ AIDS患者淋巴细胞活化及CCR5、CXCR4表达的变化,探讨HIV感染者对于抗病毒治疗的免疫应答。方法　10例HIV /AIDS患者给予高效抗逆转录病毒治疗(HAART),用流式细胞仪检测治疗前和治疗第3、6 个月T淋巴细胞活化(HLA DR、CD38表达)及第二受体CCR5、CXCR4表达情况,比较HIV/ AIDS患者治疗前后淋巴细胞活化、第二受体表达的变化。结果　治疗前,10例HIV /AIDS患者CD4+、CD8+ T淋巴细胞活化水平均明显高于健康对照,CD8+ T淋巴细胞表面CCR5的表达明显高于健康对照,CXCR4 的表达明显低于健康对照(P<0.05);HAART治疗后,患者淋巴细胞活化水平随治疗时间明显下降(P<0.05),CD8+ T淋巴细胞表面CCR5表达水平显著降低(P< 0. 01), CXCR4 的表达升高;治疗6 个月时, CD38 CD4、HLA DRCD38 CD4、CCR5 CD8、CXCR4 CD8表达水平恢复至健康人水平;HIV AIDS淋巴细胞活化水平及第二受体CCR5的表达降低与HAART治疗后CD4+T淋巴细胞数量的升高具有显著的相关性。结论HAART能够降低中国HIV /AIDS患者淋巴细胞活化水平,使第二受体表达水平趋于正常,促进免疫功能的恢复。     

用流式细胞仪检测HIV感染者和AIDS患者的T细胞亚群

目的用流式细胞仪（ｆｌｏｗｃｙｔｏｍｅｔｅｒ,ＦＣＭ）检测周围血中ＣＤ４＋、ＣＤ８＋淋巴细胞,结合临床情况对ＨＩＶ感染者和ＡＩＤＳ患者的免疫状况进行评价。方法将抗凝全血进行白细胞分类计数,用双色荧光标记的单克隆抗体染色,经溶血和固定后,用ＦＣＭ计数,从而得出ＣＤ４＋、ＣＤ８＋淋巴细胞数。结果ＨＩＶ感染者和ＡＩＤＳ患者的ＣＤ４＋淋巴细胞数都比正常人低,特别是ＡＩＤＳ患者,他们的ＣＤ４＋淋巴细胞数都低于２００个／ｍｍ３,临床表现也很差。结论ＦＣＭ检测结果与临床评价高度一致,而且ＦＣＭ比一般人工计数法更准确、方便、迅速,同时也证明ＦＣＭ是监测ＨＩＶ感染者和ＡＩＤＳ患者的免疫状况的最佳方法。