HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects na(i)ve to antiretroviral therapy

Background The frequencies of regulatory T cells (Tregs) increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs. Methods Tregs were defined as CD4+CD25+CD127lo/-T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer. ResultsCompared to HIV-1 seronegative donors, the levels of HLA-DR on CD4+CD25+CD127lo/- Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads (r=0.3163,P=0.004) and negatively with CD4 T-cell counts (r=-0.4153, P<0.0001). In addition, significant associations between HLA-DR expression on CD4+CD25+CD127lo/-Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4+ and CD8+ T cells were also identified. Conclusion HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.     

Activation and coreceptor expression of T lymphocytes induced by highly active antiretroviral therapy in Chinese HIV/AIDS patients

Background At the end of 2005, 650 000 people lived with human immunodeficiency virus type-1 (HIV-1) in China, of whom 75 000 were AIDS patients. Many AIDS patients received highly active antiretroviral therapy (HAART) supported by the “China CARES” program but the immune responses of HAART were seldom reported. This study investigated the effect of HAART on the activation and coreceptor expression of T lymphocytes in Chinese HIV/AIDS patients and evaluated its effect on immune reconstitution. Methods Seventeen HIV/AIDS patients were enrolled and three-color-flow cytometry was used to detect the activation of HLA-DR CD38 and the coreceptor CCR5, CXCR4 expression on T lymphocytes in whole blood samples taken from the patients before and after 3- or 6-month HAART.Results The activation percents of CD4(+), CD8(+) T lymphocytes were significantly higher before therapy than the normal controls (HLA-DR/CD4: 40.47±18.85 vs 11.54±4.10; CD38/CD4: 81.34±10.86 vs 53.34±11.44; HLA-DR/CD8: 63.94±12.71 vs 25.67±9.18; CD38/CD8: 86.56±11.41 vs 58.84±6.16, all P&lt;0.01). After 6-month combined antiretroviral treatment, the activation of T lymphocytes in HIV/AIDS patients was significantly decreased (HLA-DR/CD4: 28.31±13.48; CD38/CD4: 69.88±12.64; HLA-DR/CD8: 46.56±18.64; CD38/CD8: 70.17±14.54, all P&lt;0.01 compared with the pre-treatment values). Before the treatment, CCR5 expression on CD8(+) T lymphocytes was up-regulated while CXCR4 expression on CD8(+) T lymphocytes downregulated in HIV/AIDS patients compared with the normal controls (CD8/CCR5: 70.91±10.03 vs 52.70±7.68; CD8/CXCR4: 24.14±11.08 vs 50.05±11.68, all P&lt;0.01). After 6-month HAART, CCR5 expression on CD8(+) T lymphocytes significantly decreased (56.35±12.96, P&lt;0.01), while CXCR4 expression on CD8(+) T lymphocytes increased (36.95±9.96, P&lt;0.05) compared with the pre-treatment and the normal controls. A significant statistical relationship was observed between the expression of activation markers, CCR5 and the CD4(+) T lymphocyte counts after HAART (P&lt;0.05).Conclusions Reduced activation of T lymphocytes and a normalization of coreceptor expression were observed in Chinese HIV/AIDS patients after HAART. Immunity can be restored in HIV/AIDS patients receiving HAART.     

ART治疗对HIV感染者CD4+FoxP3+T细胞水平的影响

目的:探索抗逆转录病毒疗法(ART)治疗在HIV-1疾病进程中对调节性T细胞(Treg细胞)的影响,并探讨Treg细胞频率在HIV-1疾病进程中的作用.方法:抽取114例(男96例、女18例)HIV-1阳性患者及17例健康对照者外周血,应用流式细胞术检测Treg细胞,并分析其表达水平(频率和绝对数)在 HIV-1疾病进程中的变化趋势及其与CD4+细胞绝对数之间的相关性.结果:随着HIV-1感染者病情进展,患者外周血中Treg细胞绝对数趋向下降并且与CD4+T细胞绝对数呈正相关,而Treg细胞频率趋向升高并且与CD4+T细胞绝对数呈负相关.Treg细胞频率及绝对数在ART治疗无症状HIV-1阳性感染者中显著降低,而在AIDS患者中却显著升高.结论:Treg细胞参与艾滋病免疫发病过程,并且在HIV-1感染的不同阶段,ART治疗对Treg细胞水平具有一定的影响,提示通过控制Treg细胞的水平可能有助于HIV-1感染疾病的临床控制.     

慢性HIV-1感染者经HAART治疗后T细胞CD38、HLA-DR和Ki67表达的变化

目的观察慢性人类免疫缺陷病-毒1(HIV-1)感染者经高效抗逆转录病毒治疗(HAART)后CD4+、CD8+T淋巴细胞中CD38、HLA-DR及Ki67水平的变化特点。方法纳入20例慢性HIV-1感染者,采用AmplicorHIV-1 monitor超灵敏技术测定病毒载量,用流式细胞术测定CD4+、CD8+T淋巴细胞及其治疗前(基线)、治疗2、4、8、12周CD38、HLA-DR及Ki67的表达,并用SPSS 16.0分析其变化特点。结果 HAART治疗后(72周)慢性HIV-1感染者的CD4+T淋巴细胞计数明显升高(P<0.01),而CD8+T淋巴细胞计数明显下降(P<0.01)。HIV-1感染者治疗前后CD4+T淋巴细胞的激活和增殖无显著变化;CD8+T淋巴细胞激活显著下降(HLA-DR:79.9%vs71.2%,P<0.01;CD38:84.9%vs75.2%,P<0.01),而增殖显著升高(Ki67:3.90%vs4.63%,P<0.05)。2周、4周、8周、12周HLA-DR/CD4百分比与72周CD4+T淋巴细胞计数均呈负相关,其r值分别为-0.623(P=0.008)、-0.598(P=0.00...     

Immune activation markers in individuals with HIV-1 disease and their correlation with HIV-1 RNA levels in individuals on antiretroviral therapy

BackgroundCurrently CD4+ T lymphocyte counts and HIV-1 RNA levels are being utilized to predict outcome of human immunodeficiency virus (HIV) disease. Recently, the role of immune activation in HIV disease progression and response to treatment is being investigated. This study focused on the expression of CD38 and HLA-DR on lymphocyte subsets in various groups of HIV-infected individuals and to determine their association with HIV-1 disease progression.MethodsNinety-eight cases of patients with HIV/AIDS in different disease stages and twenty-four healthy HIV-negative individuals were included in the cross-sectional study. Their immune function and abnormal immune activation markers (CD38 & HLA-DR) were detected using a flowcytometer, and HIV-1 RNA levels in individuals receiving antiretroviral drugs were estimated.ResultsThe immune activation marker levels were significantly different between patients with different disease stages (P < 0.001). A significant negative correlation was observed between peripheral blood CD4+ T cell counts and immune activation markers. Also, a significant positive correlation was observed between HIV-1 RNA levels and CD38+CD8+ T lymphocyte.ConclusionImmune activation markers (CD38 & HLA-DR) increase with disease progression. CD38+ on CD8+ T lymphocyte correlates well with HIV1 RNA levels in individuals failing on antiretroviral therapy.     

感染HIV-1儿童经抗病毒治疗后乳酸浓度检测及其相关因素研究

目的研究中国感染艾滋病毒(human immunodeficiency virus type 1,HIV-1)儿童经抗病毒治疗后血浆乳酸浓度及相关影响因素。方法选择31例抗病毒治疗的感染HIV-1儿童与30例正常对照儿童,检测血浆乳酸浓度,结合儿童的基本信息、治疗方案及实验室检测结果进行综合分析。结果感染HIV-1患儿血浆乳酸浓度(2.32±0.60)mmol/L显著高于对照组儿童(1.60±0.88)mmol/L(P<0.01);d4T治疗组和AZT治疗组间血浆乳酸浓度、基线CD4+T淋巴细胞计数、基线病毒载量、性别、年龄以及HIV-1传播途径差异均无统计学意义(P>0.05)。结论外周血乳酸浓度可反映感染HIV-1儿童经抗病毒治疗后的线粒体毒性,有望成为线粒体毒性检测指标。     

Characteristics of HIV-1-specific CD8 T-cell responses and their role in loss of viremia in children chronically infected with HIV-1 undergoing highly active antiretroviral therapy

Background Few studies have examined the properties of human immunodeficiency virus type 1 （HIV-1） epitope-specific cytotoxic T lymphocyte （CTL） responses in children. To address this issue, we characterized epitope-specific CTL responses and analyzed the determinants that may affect CTL responses before and after highly active antiretroviral therapy （HAART） in children with HIV-1 infection. Methods A total of 22 HIV-1-infected children and 23 uninfected healthy children as control were enrolled in the study. Circulating CD4 T cells and HIV-1 RNA load in plasma were routinely measured. Peripheral HIV-l-specific CTL frequency and HIV-1 epitope-specific, interferon-y （IFN-y）-producing T lymphocytes were measured using tetramer staining and enzyme-linked immunospot （ELISPOT） assay, respectively. Circulating dendritic cell （DC） subsets were monitored with FACS analysis. Results More than 80% of the children with HIV-1 infection exhibited a positive HIV-1-epitope-specific CTL response at baseline, but HIV-specific CTLs and IFN-y-producing lymphocytes decreased in patients who responded to HAART in comparison with non-responders and HAART-naive children. The duration of virus suppression resulted from HAART was inversely correlated with CTL frequency. While in HAART-naive children, HIV-l-specific CTL frequency was positively correlated with myeloid DC （mDC） frequency, although the cause and effect relationship between the DCs and CTLs remains unknown. Conclusions HIV-l-epitope-specific CTL responses are dependent on antigenic stimulation. The impaired DC subsets in blood might result in a defect in DC-mediated T cell responses. These findings may provide insight into understanding the factors and related mechanisms that influence the outcome of HIV-1 carriers to HAART or future antiviral therapies.     

中国恒河猴感染SIV后T淋巴细胞免疫活化及其肠粘膜归巢受体的表达

目的 研究中国恒河猴感染SIV后T淋巴细胞免疫活化及其肠粘膜归巢受体表达的情况。方法 选用12只健康中国恒河猴,分为正常动物组(对照组)4只,模型感染组(模型组)8只,静脉接种SIVmac239病毒株,分别于感染前、感染后1周、2周、3周、4周、8周、11周、14周采用免疫组化技术检测猴模型回肠组织中的肠淋巴归巢相关受体(intestinal lymphatic homing receptor) a47、CCR9、aE7、CD62L及肠淋巴组织活化分子CD69的表达; real-time PCR检测病毒载量,流式细胞仪检测T淋巴细胞亚群、CD4T淋巴细胞活化水平、外周血肠淋巴归巢受体表达水平; ELISA检测血清CD62L。结果 中国恒河猴感染SIV两周后,模型组和对照组的血液学指标(WBC、 LYM%、LYM、RBC)均在正常范围值里,两组差异无统计学意义(P>0.05)。模型组CD4/CD8低于对照组,CD8计数高于对照组(P<0.01)。模型组病毒载量显著高于对照组(P<0.01)。AIDS猴感染模型在感染2周起,CD4百分比开始逐渐下降,于感染8周后下降明显(P<0.05)。感染11周后(亚急性期),CD4计数在下降明显(P<0.05)。AIDS猴感染模型在感染1周(急性期)开始,CD4⁺HLA-DR⁺出现一过性上升,随即下降,但感染14周时又骤然升高。CD4⁺CD45⁺HLA-DR⁺和CD3⁺HLA-DR⁺在感染1周时呈现波动上升趋势,随后逐渐下降。血浆中CD62L在感染1周时开始逐渐上升,并在感染到第8周左右到达一个高峰期,随后开始出现下降(P<0.01)。中国恒河猴感染SIV后,肠淋巴归巢受体 47、CCR9、E7的表达升高,L-选择素的表达降低,同时E7、CCR9在外周血CD4⁺T细胞上的表达及肠道淋巴组织免疫活化分子CD69的表达升高。结论 中国恒河猴感染SIV后,效应性T淋巴细胞的肠淋巴归巢增强,初始T淋巴细胞肠淋巴归巢受到抑制,肠粘膜免疫系统出现过度活化现象。     

Epidemiology, clinical and laboratory characteristics of currently alive HIV-1 infected former blood donors naive to antiretroviral therapy in Anhui Province, China

Background Unregulated commercial blood/plasma collection among farmers occurred between 1992 and 1995 in central China and caused the second major epidemic of human immunodeficiency virus type 1 （HIV-1） infection in China. It is important to characterize HIV-l-infected former blood donors and to study characteristics associated with disease progression for future clinical intervention and vaccine development. Methods A cross-sectional study was performed on HIV-l-infected former blood donors （FBDs） and age-matched HIV-seronegative local residents. Demographic, epidemiologic, clinical and key laboratory data were collected from all study participants. Both unadjusted and adjusted multivariate linear regressions were employed to analyze the association of the decrease of CD4^＋ T-cell counts with other characteristics. Results Two hundred and ninety-four HIV-l-infected FBDs and 59 age-matched HIV-seronegative local residents were enrolled in this study. The unregulated blood/plasma collection occurred more than a decade （10.8 --12.8 years） ago, which caused the rapid spread of HIV-1 infection and the high prevalence of co-infection with hepatitis C virus （HCV, 89.5%）, hepatitis B virus （HBV） co-infection was observed in only 11 HIV＋participants （3.7%）. Deterioration in both clinical manifestation and laboratory parameters and increase of viral loads were observed in parallel with the decrease of CD4^＋ T-cell counts. The decrease of total lymphocyte counts （P〈0.001） and hemoglobin levels （P〈0.001） and the appearance of dermatosis （P=0.03） were observed in parallel with the decrease of CD4^＋ T-cell counts whereas viral loads （P〈0.001） and CD8^＋ T-cell counts （P=0.01） were inversely associated with CD4^＋ T-cell counts. Conclusions Co-infection with HCV but not HBV is highly prevalent among HIV-l-infected FBDs. CD4^＋ T-cell counts is a reliable indicator for disease progression among FBDs. Total lymphocyte counts, hemoglobin level and appearance of dermatosis were positively, whereas CD8^＋ T-cell counts and viral loads were inversely associated with the decreased CD4^＋ T-cell counts.     

HIV-I编码病毒蛋白R基因在真核细胞中的表达及其表达蛋白对KSHV溶解性周期复制的影响

目的:构建含人类免疫缺陷病毒1型(HIV-1)编码病毒蛋白R(Vpr)基因的重组真核表达质粒并初步探索Vpr基因编码蛋白对卡波济肉瘤相关疱疹病毒(KSHV)溶解性周期复制的影响.方法:构建pVpr-Flag重组质粒并进行酶切鉴定和序列测定;将pVpr-Flag重组质粒临时转染BCBL-1和NIH3T3细胞,采用RT-PCR、IFA和Western blot分别从mRNA和蛋白水平检测Vpr基因的表达情况:提取临时转染pVpr-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测KSHV次要衣壳蛋白编码基因ORF26(仅在病毒裂解期表达)mRNA转录水平,初步探索HIV-1 Vpr基因编码蛋白对KSHV复制的影响.结果:克隆的Vpr基因序列与GenBank中已登记的Vpr序列100%同源.RT-PCR、IFA和Western blot结果显示Vpr基因mRNA及其编码蛋白在真核细胞中得到了转录和表达.RT-PCR结果进一步显示,Vpr基因编码蛋白能够降低KSHVORF26mRNA转录水平.结论:成功构建含Vpr基因序列的重组质粒并在真核细胞中获得正确表达:初步探索表明Vpr蛋白能够抑制KSHV溶解性周期复制.     

终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及调节性T细胞的变化

目的 观察香烟烟雾暴露和终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及支气管肺泡灌洗液(BALF)中调节性T细胞(Treg)的变化.方法 将50只健康清洁级雄性Wistar大鼠随机分为5组:12周正常对照组(简称12周对照组)、24周正常对照组(简称24周对照组)、12周香烟烟雾暴露组(简称12周暴露组)、24周香烟烟雾暴露组(简称24周暴露组)、终止香烟烟雾暴露组(简称终止暴露组).烟熏法复制大鼠气道炎症的动物模型.12周后,12周对照组、12周暴露组处置取材,24周暴露组继续烟熏12周,终止暴露组终止烟雾暴露12周,将后2组及24周对照组处置取材.HE染色观察小气道病理改变,进行气道评分;收集BALF进行细胞学计数和分类计数;酶联免疫吸附法(ELISA)法测BALF中4种细胞因子:Th1/Th2型细胞因子干扰素(IFN)γ、白细胞介素(IL)4及促炎因子IL-8、肿瘤坏死因子(TNF)α的浓度;流式细胞术检测各组大鼠BALF中Treg细胞的比例,RT-PCR检测各组大鼠BALF中Foxp3 mRNA的表达.结果 (1)12周暴露组、24周暴露组、终止暴露组气道炎症评分较12周对照组、24周对照组明显高(均P＜0.01).24周暴露组、终止暴露组气道炎症评分均较12周暴露组高(均P＜0.01).(2)与12周对照组、24周对照组相比,12周暴露组、24周暴露组、终止暴露组BALF中IFN-γ、TNF-α和IL-8高,IL-4低(均P＜0.01).与12周暴露组相比,终止暴露组IFN-γ、IL-4、TNF-α差异均无统计学意义(均P＞0.05),IL-8较高(P＜0.01),24周暴露组IFN-γ、TNF-α和IL-8明显高(均P＜0.01).(3)BALF中Treg细胞比例,12周暴露组(7.4%±0.8%)、24周暴露组(7.8%±1.7%)、终止暴露组(7.0%±1.4%)较12周对照组(4.8%±1.2%)、24周对照组(4.7%±1.2%)高(均P＜0.01),前3组之间BALF中Treg细胞比例差异均无统计学意义(均P＞0.05).(4)12周暴露组(0.22±0.02)、24周暴露组(0.23±0.03)、终止暴露组(0.20±0.04)BALF中Foxp3 mRNA表达较12周暴露组(0.13±0.01)、24周暴露组(0.11±0.02)高(均P＜0.01).前3者之间BALF中Foxp3 mRNA表达差异均无统计学意义(均P＞0.05).结论 香烟暴露致大鼠气道Th1/Tc1介导炎症伴Treg细胞表达增高,终止香烟烟雾暴露后其炎症及Treg细胞高表达仍持续存在,提示该免疫失衡可能是导致终止香烟暴露后Th1/Tc1气道炎症仍持续进展的原因之一.     

人类免疫缺陷病毒１型病毒复制负调控因子编码基因的克隆及在ＢＣＢＬ-１细胞和ＮＩＨ／３Ｔ３细胞中的表达

目的:分离克隆人类免疫缺陷病毒1型(HIV-1)编码的病毒复制负调控因子(Nagative factor,Nef)基因,并将其导入BCBL-I细胞和NIH/3T3细胞中进行表达.方法:根据GenBank登记的Nef基因核苷酸序列设计PCR引物,在其5'端分别引入EcoR I和Sal I酶切位点;以含有Nef基因的pCINL质粒为模板,扩增Nef基因,经双酶切后克隆进真核表达载体pCI-neo中.为了便于蛋白检测,在下游引物5'端引入Flag序列,构建重组真核表达质粒.重组质粒经酶切鉴定、核酸序列测定和分析后分别瞬时转染BCBL-I细胞和NIH/3T3细胞,用RT-PCR、Western blot分别从核酸和蛋白水平检测Nef基因的表达情况.结果:成功构建了含Nef基因的重组真核表达质粒,核酸序列分析显示,克隆的Nef基因序列全长651 bp,与GenBank中登记的Nef基因序列100%同源,引入的Flag序列完全正确;RT-PCR和Western blot都在Nef预期位置检测到特异性条带.结论:HIV-1 Nef基因在BCBL-1细胞和NIH/3T3细胞中获得了正确表达.     

抗HIV-1基因治疗新进展

虽然高效抗反转录病毒治疗（highly active anti-retroviral therapy,HAART）取得了显著的成果,但是抗人类免疫缺陷病毒（human immunodeficiency virus,HIV）药物治疗仍有其局限性（如引起毒素蓄积和病毒突变）.基因治疗在理论上具有较好的抗HIV能力,可以通过持续干扰病毒复制,提供了阻止HIV进行性感染的希望.本篇综述主要探讨当前多种基因治疗策略及其最新进展.     

Early changes of CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation

Background  Hemorrhagic shock induces immune dysfunction. Regulatory T cells (Tregs), T-helper (Th) cells, and cytotoxic T-lymphocytes (CTLs) can execute many crucial actions in immune and inflammatory responses. This study was conducted to investigate the early pathophysiological changes of CD4⁺CD25⁺Foxp3⁺ Treg and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation.

Methods  A rat model of controlled hemorrhagic shock with no fluid resuscitation was established. Peripheral blood samples were taken before and four hours after hemorrhagic shock with no fluid resuscitation. Three color flow cytometry was used to detect Tregs, Th1, Th2, Tc1 and Tc2 cells in the samples.

Results  In the peripheral blood of rats, the percentage of Tregs four hours after hemorrhagic shock was significantly lower than before hemorrhagic shock (P=0.001). The ratios of Th1/Th2 and Tc1/Tc2 were changed from (23.08±8.98)% to (23.91±15.36)%, and from (40.40±21.56)% to (65.48±23.88)%, respectively.

Conclusions  At an early stage, the advent of hemorrhagic shock is related to an early decrease of Tregs, and a mild shift in the Th1/Th2, Tc1/Tc2 balance toward Th1 and Tc1 dominance. These changes are part of a hyper-inflammatory state of the host, and will deteriorate the maintenance of immune balance. Further influences and detailed mechanisms need to be investigated.     

Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of ＂DNA prime plus protein boost＂

Background The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes. Methods Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48（AE）, GX79（AE）, NX22（BC）, GS22（BC）, HN24（Thai-B） were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of ＂DNA prime plus protein boost＂ and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains. Results Response of gp120-specific antibody was relatively low after DNA primes （mean titer=10^4.72）; however, the titer of gp120-specific antibody went up with 2 protein boosts （mean titer=10^6.81）. Above all, neutralizing antibody （Nab） titers induced by this combined approach were much better than those elicited by DNA or protein used alone （P 〈0.01）. Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses. Conclusion Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.     

T1DM患者外周血记忆性T细胞表面PD⁃1表达水平与其疾病发生发展的初步研究

目的：通过检测1型糖尿病（type 1 diabetes mellitus,T1DM）、2型糖尿病（type 2 diabetes mellitus,T2DM）患者以及健康对照外周血T细胞亚群免疫负调控分子程序性死亡蛋白1（programmed cell death protein 1,PD?1）mRNA相对表达水平和记忆性T细胞（memory T cells,Tm）表面PD?1表达情况,从分子生物学、细胞免疫学等多种角度研究记忆性T细胞PD?1表达异常与以胰岛β细胞破坏为主要机制的T1DM发生发展的相关性。方法：分离T1DM、T2DM患者以及健康对照组外周血单个核细胞（peripheral blood mononuclear cells,PBMCs）;从PBMCs中分离出CD4+T细胞和CD8+T细胞,使用荧光定量PCR分别检测其细胞内PD?1 mRNA相对表达水平;将PBMCs分别标记荧光抗体CD4?FITC、CD45RO?PE、CCR7?APC、CD8?FITC、PD?1?PerCp,应用流式细胞术分别检测CD4+CD45RO+CCR7+Tcm细胞、CD4+CD45RO+CCR7-Tem细胞、CD8+CD45RO+CCR7+Tcm细胞、CD8+CD45RO+CCR7-Tem细胞表面免疫负调控分子PD?1的表达水平。结果：①T1DM组患者外周血中CD4+T细胞内PD?1 mRNA相对表达水平低于T2DM组和正常对照组,差异有统计学意义;②T1DM组患者外周血中CD8+T细胞内PD?1 mRNA相对表达水平未见明显异常;③T1DM组患者外周血中CD4+CD45RO+CCR7-Tem细胞亚群与CD4+CD45RO+CCR7+Tcm细胞亚群PD?1表达水平均显著低于正常对照组和T2DM组患者,差异有统计学意义;④T1DM组患者外周血中CD8+CD45RO+CCR7-Tem细胞亚群与CD8+CD45RO+CCR7+Tcm细胞亚群PD?1表达水平均无明显异常。结论：胰岛β细胞可通过细胞表面PD?L1与CD4+Tm细胞表面PD?1结合从而负调节后者细胞免疫作用,因此当CD4+Tm细胞PD?1表达异常而失去负调控作用时,细胞效应则会进一步增强,最终可能通过破坏胰岛β细胞而导致T1DM的发生发展。     

PD-L1在结肠癌患者CD4＋CD25hiCD127low／-调节性 T 细胞中的表达及其临床意义

目的：探讨PD‐L1在结肠癌患者外周血CD4＋ CD25hi CD127low／‐T reg细胞上的表达及其与肿瘤病理分期之间的相关性。方法采用流式细胞术分析了47例结肠癌患者外周血中CD4＋CD25hi CD127low／‐T reg细胞的含量及其PD‐L1的表达。结果和健康人比较,结肠癌患者外周血中CD4＋CD25hi CD127low／‐T reg细胞的含量显著上升,并且PD‐L1的表达明显升高。进一步的分析表明,PD‐L1的表达和结肠癌的病理分期呈正相关,而术后患者CD4＋ CD25hi CD127low／‐T reg细胞 PD‐L1的表达则显著下降。结论 CD4＋CD25hi CD127low／‐T reg细胞数量及PD‐L1表达的升高可能导致了免疫抑制的增强,这可能是促进肿瘤免疫逃逸的重要因素。     

硫化氢对幽门螺杆菌感染GES-1细胞CSE,NF-κB及IL-8 mRNA表达的影响

目的: 观察硫化氢(hydrogen sulfide,H₂S)对幽门螺杆菌(H. pylori)感染的GES-1细胞CSE,NF-κB及IL-8 mRNA表达及其形态学的影响,探讨其对H. pylori所致胃黏膜细胞炎症的作用及机制。方法: 将GES-1细胞培养24 h后分为对照组(不加H. pylori及NaHS)、H. pylori组、NaHS组(又分为4个亚组,分别加入 50,100,200或400 μmol/L NaHS)和H. pylori+NaHS组(又分为4个亚组,分别加入H. pylori与50,100,200或400 μmol/L NaHS),每组分别培养3,6,及12 h,用RT-PCR法检测各组GES-1细胞CSE,NF-κB 及IL-8 mRNA表达,并分析其相关性。结果: H. pylori组CSE,NF-κB及IL-8 mRNA表达均较对照组增加(P<0.05),200 μmol/L NaHS组和400 μmol/L NaHS组CSE表达较对照组降低(P<0.05);而NaHS各组NF-κB和IL-8 mRNA表达与对照组比较差异无统计学意义(P>0.05);NaHS各组、H. pylori+200 μmol/L NaHS组及H. pylori+400 μmol/L NaHS组CSE,NF-κB及IL-8 mRNA表达均较H. pylori组降低(P<0.05);H. pylori组、H. pylori+200 μmol/L NaHS组及H. pylori+400 μmol/L NaHS组CSE,NF-κB及IL-8 mRNA表达之间均呈正相关(P<0.05)。结论: H. pylori诱导GES-1细胞NF-κB和IL-8 mRNA表达,并上调CSE mRNA表达;200和400 μmol/L NaHS能抑制H. pylori感染诱导的GES-1细胞NF-κB和IL-8 mRNA表达,改善H. pylori感染所致的细胞形态学变化,对细胞起保护作用。