细胞因子及抗-CD3单抗对外周血单个核细胞活化作用

探讨联合应用ＩＦＮ γ、ＩＬ 2及抗 ＣＤ3单抗对分离自健康供血者的外周血单个核细胞 (ＰＢＭＣ)在体外的诱导活化作用 ,并以单独应用ＩＬ 2作比较 ,分析了细胞活化的特征性标志。同时通过动态测定这些标志 ,认识了免疫细胞活化的动态过程。现将研究结果报告如下 :材料与方法试剂 :基因重组人ＩＦＮ γ ,上海克隆生物高技术有限公司产品 ;基因重组人ＩＬ 2 ,中国军事医学科学院基础所及北京中化生物技术研究所联合研制产品 ;ＣＤ3单抗 ,古巴分子免疫中心产品 ;ＭＴＴ及ＤＭＳＯ均为美国Ｓｉｇｍａ公司产品 ;ＦＩＴＣ或ＰＥ标记鼠抗人ＣＤ3…     

强直性脊柱炎TH1/TH2相关细胞因子的检测

TH1/TH2相关细胞因子在强直性脊柱炎(anky losingspondylitis ,AS)发病机制中起着重要作用。本研究采用半定量RT PCR方法检测了AS患者外周血单个核细胞(peripheralbloodmononuclearcell ,PBMC)TH1/TH2相关细胞因子mRNA的表达,观察AS患者体内TH1/TH2相关细胞因子的平衡状态。材料和方法随机选择19例初诊腰痛后诊为AS患者,其中男性17例,女性2例。年龄为12～31岁,平均2 1.2 1±5 .6 2岁,病程为3个月～14年。诊断采用1984年纽约修订标准。19例正常对照为健康志愿者,其中男性16例,女性3例。年龄为14～4 7岁,平均2 2 .4 7±5 .2 1岁…     

多发性肌炎/皮肌炎外周血单个核细胞HLA-DR抗原的表达

有关多发性肌炎(PM)／皮肌炎(DM)外周血淋巴细胞亚群的研究仅见早期报道，且尚无统一结论。为探讨PM／DM外周血单个核细胞(PBMC)HLA-DR的表达及其意义，我们应用双荧光抗体标记、流式细胞仪(FACS)进行了检测。     

内皮细胞在感染性休克炎症性细胞因子释放中的作用

目的 以感染性休克患者血清刺激人原代内皮细胞(HPAEC)和外周血单个核细胞(PBMC),观察内皮细胞在感染性休克细胞因子释放中的作用.方法 采用密度梯度离心法分离健康人PBMC;RT-PCR及免疫印迹检测HPAEC细胞表面标志CD144和血管假性血友病因子(von Will-ebrand factor,vWF)的表达;ELISA检测感染性休克患者和健康对照者血清中的IL-6、TNF-α、MCP-1的水平;两种血清及LPS刺激HPAEC和PBMC后,ELISA检测培养上清中IL-6、TNF-α、MCP-1的水平;流式细胞技术检测休克患者血清和LPS刺激后HPAEC膜表面ICAM-1的表达;S1P1受体激动剂CYM-5442预处理后,休克血清刺激HPAEC培养上清中IL-6、TNF-α、MCP-1的水平.结果 HPAEC表达内皮细胞标志性分子CD144和vWF;感染性休克患者血清中炎症因子IL-6、TNF-α、MCP-1水平明显高于健康对照(P＜0.01);休克血清和LPS处理PBMC和HPAEC后,相对于对照组,均有明显升高(P＜0.01);PBMC的休克组炎症因子与PBMC的LPS组均无明显差异(P＞0.05),而HPAEC的休克组炎症因子相对于HPAEC的LPS组均明显升高(P＜0.01);同样地,LPS刺激两种细胞后,HPAEC的IL-6、TNF-α明显低于PBMC(P＜0.01),MCP-1的水平无明显变化(P＞0.05),但是休克血清刺激后,HPAEC的IL-6、TNF-α与PBMC无明显变化(P＞0.05),MCP-1则明显升高(P＜0.01);休克患者血清刺激S1P1受体特异性激动剂CYM-5442预处理的HPAEC后,培养上清中炎症因子IL-6、TNF-α、MCP-1水平明显降低(P＜0.01).结论 内皮细胞在感染性休克细胞因子释放中起着关键作用.     

卡介苗(BCG)通过诱导IL-12产生和受体表达促进人NK细胞功能

目的:研究卡介苗(BCG)对人自然杀伤细胞(NK)功能的作用及其机制.方法:分离抗结核抗体阴性志愿者外周血PBMC、纯化NK细胞, 分别与BCG、 IL-12、 BCG+IL-12、 BCG+抗IL-12Rβ1 mAb(2B10)培养.利用ELISA方法检测培养上清液IFN-γ、 IL-12p40含量;利用ELISpot方法检测IFN-γ、颗粒酶B产生细胞的频率;利用四甲基偶氮唑盐(MTT)比色法测定杀伤功能.利用流式细胞术检测NK细胞IL-12Rβ1的表达.结果:BCG呈剂量依赖的方式诱导PBMC产生IFN-γ.在BCG刺激条件下, PBMC颗粒酶B分泌细胞数明显高于不加任何刺激剂组(P<0.05).BCG增强PBMC杀伤活性.BCG不能诱导纯化NK细胞产生IFN-γ, 但与IL-12同时刺激则表现出协同作用.纯化NK细胞经BCG刺激后杀伤活性与未刺激相比差异无统计学意义.BCG呈剂量依赖方式诱导PBMC产生IL-12、并促进NK细胞不同亚群表达IL-12Rβ1.2B10抗体抑制BCG对PBMC产生IFN-γ和分泌颗粒酶B的诱导作用.结论:BCG间接地促进NK细胞的生物学活性, 其部分机制是通过诱导单核细胞产生内源性IL-12、并上调NK细胞表达IL-12R.     

低剂量γ射线辐照外周血单个核细胞对细胞因子表达的影响

目的:探讨低剂量辐射对外周血单个核细胞细胞因子表达的刺激效应.方法:人外周血单个核细胞采用1 Gyγ射线照射,吸收剂量率为17 Gy/min,并在培养过程中不同时间采用ELISA方法检测上清液中IL-2、TNFα及IFN-γ含量.结果:经γ射线照射24 h后培养上清中IL-2、TNFα及IFN-γ含量明显升高,与对照组比较有明显差异(P<0.05),并随培养时间延长,各细胞因子含量会进一步升高.结论:低剂量辐射对外周血单个核细胞中IL-2、TNFα及IFN-γ表达具有刺激效应.     

融合蛋白hCGβ-hC3d3增强人免疫活性细胞对hCGβ抗原的免疫原性

目的:探讨分子佐剂hC3d3与hCGβ的融合蛋白hCGβ-hC3d3作用于人外周血单个核细胞(PBMC)对hCGβ抗原的免疫原性的影响.方法:对人PBMC进行细胞分离纯化,分别用1、10、100 nmol/L的hCGβ、hCGβ-hC3d3及PWM等抗原体外作用于B细胞、B T细胞、PBMC和Raji细胞8～12天,用3H-TdR掺入法了解不同抗原刺激后各组细胞增殖情况;用ELISA检测培养上清中总免疫球蛋白(Ig)水平;而ELISPOT可测定各种抗原刺激后Ig分泌细胞数量.结果:用不同浓度hCGβ、hCGβ-hC3d3及PWM作用于B细胞、B T细胞、PBMC、Raji细胞8～12天后,3H-TdR掺入法分析显示:与hCGβ处理组相比,融合蛋白hCGβ-hC3d3能使各组细胞增殖明显升高,且呈浓度依赖性;100 nmol/L hCGβ-hC3d3与前3组细胞共培养上清中总Ig的含量分别为hCGβ刺激组的4倍、10倍和10.85倍,且呈浓度依赖性.ELISPOT结果与培养上清液检测结果类似,经与hCGβ-hC3d3共培养后Ig生成细胞较hCGβ组明显增加.结论:融合蛋白hCGβ-hC3d3明显增强人免疫活性细胞抗hCGβ的免疫原性.     

慢性丙型肝炎患者抗病毒治疗前PBMC细胞因子的研究

目的 研究准备抗病毒治疗的慢性丙型肝炎患者的免疫特点.方法 将30例慢性丙型肝炎患者和10例正常对照外周血单个核细胞(PBMC)体外培养72 h后,用ELISA法检测培养上清中细胞因子IL-2、IL-4、IL-10、IL-12、IFN-γ和TNF-α的浓度.结果 (1)慢性丙型肝炎患者PBMC培养上清中IFN-γ、IL-10和TNF-α的水平明显升高(P＜0.05),没有检测到IL-2、IL-4、IL-12的基础分泌.(2)不同病情患者间的细胞因子的分泌水平差异无统计学意义(P＞0.05).结论 慢性丙型肝炎患者体内TH2型细胞因子的分泌占优势.提示通过调整TH1/TH2失衡可能达到抗病毒治疗目的.     

卡介苗对hPBMC的TLR4表达调节及其免疫活化作用的研究

目的:了解卡介苗对人外周血单个核细胞( hPB-MC) TLR4的表达调节,以及BCG介导免疫细胞活化效应的新机制.方法:研究BCG对hPBMC的TLR4表达的调节,以及对表达TLR4的淋巴细胞的免疫活化效应,以(hPBMC+PBS)作为空白对照组,应用流式细胞仪对TLR4进行检测,ELISA方法测定BCG刺激组与对照组的IFN-γ和TNF-α的表达.结果:经过BCG刺激后,TLR4表达大大增加(P＜0.01),且随时间增加而增强,在72 h时,BCG组的TLR4表达率为(44.73±0.0066)％,而对照组的表达率仅为(1.02±0.0024)％.BCG可以促进淋巴细胞增殖,且这种增强作用也存在一定时间依赖性,在BCG与hPBMC共同孵育24h、48h和72h后,BCG组IFN-γ和TNF-α的表达量显著高于对照组(PBS组),差异有统计学意义(P＜0.05),且这种增强作用存在一定的时间依赖性.结论:BCG对hPBMC的TLR4表达有正调节作用,并加强表达TLR4的hPBMC的免疫活化作用.     

肾综合征出血热病毒对人外周血单个核细胞产生细胞因子的影响

目的研究肾综合征出血热(HFRS)病毒对人外周血单个核细胞(PMBC)产生细胞因子的影响。方法用HFRS病毒A69株及脂多糖(LPS)单独或联合作用于PMBC,于作用后12、24、48、72、96h分别收集培养上清液,用ELISA法检测上清液中细胞因子TNF-α、IL-6和IFN-γ的含量,实验数据用方差分析处理。结果病毒感染和LPS能使PBMC分泌TNF-α、IL-6和IFN-γ能力增强。感染后12h,TNF-α和IFN-γ开始升高,于感染后24h达高峰,然后开始下降;而IL-6水平于感染后72h达高峰,然后开始下降。HFRS病毒与LPS联合作用显著提高病毒诱导PBMC分泌TNF-α、IL-6和IFN-γ的能力。结论HFRS病毒能提高PBMC分泌TNF-α、IL-6和IFN-γ的能力,LPS可增强病毒诱导PBMC分泌细胞因子的能力,在HFRS病毒感染过程中合并细菌感染可能调节细胞因子介导的疾病进程,细胞因子在HFRS病毒的致病与免疫过程中可能起重要作用。     

Aberrant Expression of CC and CXC Chemokines and Their Receptors in Patients with Asthma

This study further elucidates the roles of selected chemokines (IP-10, MIG, and RANTES) and their receptors (CCR3, CCR5, and CXCR3) in asthma. We compared their profiles in six groups of participants—atopic cohort and nonatopic cohort (each including controls and asthmatic patients with or without steroid therapy). Plasma concentration of IP-10 was significantly lower while that of RANTES and the expression of CCR3 were higher in asthmatic patients (all p < 0.05). Plasma RANTES correlated positively with the GINA severity score in all asthmatic patients (r=0.27, p < 0.05), and with IL-13 in nonatopic asthmatic patients (r=0.46, p < 0.05). In asthmatic patients, the ex vivo release of IP-10 and MIG was attenuated in PBMC activated with allergen, mitogens and IL-18 (p < 0.05). In conclusion, plasma RANTES may be a surrogate marker for asthma and the diminished Th1 related CXC chemokine production may contribute to Th2 predominance in asthma.     

In vitro marker gene expression analyses in human peripheral blood mononuclear cells: A tool to assess safety of influenza vaccines in humans

Vaccines are inoculated in healthy individuals from children to the elderly, and thus high levels of safety and consistency of vaccine quality in each lot must meet the required specifications by using preclinical and lot release testing. Because vaccines are inoculated into humans, recapitulation of biological reactions in humans should be considered for test methods. We have developed a new method to evaluate the safety of influenza vaccines using biomarker gene expression in mouse and rat models. Some biomarker genes are already known to be expressed in human lymphocytes, macrophages and dendritic cells; therefore, we considered some of these genes might be common biomarkers for human and mice to evaluate influenza vaccine safety. In this study, we used human peripheral blood mononuclear cells (PBMC) as a primary assessment tool to confirm the usefulness of potential marker genes in humans. Analysis of marker gene expression in PBMC revealed biomarker gene expressions were dose-relatedly increased in toxic reference influenza vaccine (RE)-stimulated PBMC. Although some marker genes showed increased expression in hemagglutinin split vaccine-stimulated PBMC, their expression levels were lower than that of RE in PBMC from two different donors. Many marker gene expressions correlated with chemokine production. Marker genes such as IRF7 were associated with other Type 1 interferon (IFN)-associated signals and were highly expressed in the CD304⁺ plasmacytoid dendritic cell (pDC) population. These results suggest PBMC and their marker genes may be useful for vaccine safety evaluation in humans.     

Chlamydia pneumoniae infection enhances cellular proliferation and reduces steroid responsiveness of human peripheral blood mononuclear cells via a tumor necrosis factor-α-dependent pathway

BACKGROUND: Although epidemiological studies have found an association between Chlamydia pneumoniae infection and severe asthma, the causality and underlying mechanism are largely unknown. We hypothesized that C. pneumoniae infection increases the proliferation and enhances the survival of immune and inflammatory cells, resulting in reduced responsiveness to corticosteroids and suggesting that the underlying mechanism is related to a TNF-alpha-dependent pathway. METHODS: Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence or absence of C. pneumoniae infection. Responsiveness to corticosteroids was assayed by adding dexamethasone, and the underlying mechanism was investigated by treating cells with infliximab that is a chimeric anti-TNF-alpha monoclonal antibody. Cellular proliferation and apoptosis was assessed by thymidine uptake and counting apoptotic cells using flow cytometry. RESULTS: Cellular proliferation was significantly higher in C. pneumoniae-infected PBMCs than in uninfected PBMCs, which is more prominent in Th2-dominant microenvironment. The anti-proliferative and pro-apoptotic effect of corticosteroid were significantly reduced in C. pneumoniae-infected PBMCs compared with uninfected PBMCs. The proliferative effect of C. pneumoniae infection and the reduced response to corticosteroid were effectively reversed by blocking the TNF-alpha pathway at least partially. CONCLUSION: C. pneumoniae infection enhanced the proliferation and survival of immune and inflammatory cells, resulting in steroid resistance. The reversal of these phenomena by the TNF-alpha inhibitor suggests that TNF-alpha may play an important role in the induction of steroid dependence or resistance. A TNF-alpha inhibitor may therefore be a candidate agent for managing steroid-dependent or -resistant severe asthma.     

Expression and Functional Analysis of Toll-Like Receptors of Peripheral Blood Cells in Asthmatic Patients: Implication for Immunopathological Mechanism in Asthma

Background  We investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry. Discussion  Ex vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P < 0.05). Expression of TLR4 and TLR5 was also found to be significantly lower in asthmatic patients when compared to that of control subjects (all P < 0.05). Therefore, the decreased activation of TLR4 and TLR5 in asthmatic patients might contribute to the immunopathological mechanisms of asthma by reducing the release of Th1 and anti-inflammatory cytokines. Samantha W. M. Lun and C. K. Wong contributed equally to this study.     

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.     

The interferon-alpha and interleukin-10 responses in neonates differ from adults, and their production remains partial throughout the first 18 months of life

Previous studies have suggested that the susceptibility of newborns to infections is linked to the immaturity of their immune system, but very few data are available on the early stages of maturation of the immune response. Therefore, we decided to investigate the evolution of the interferon (IFN)-α and interleukin (IL)-10 responses in neonatal mononuclear cells. To this end, mononuclear cells isolated from cord blood and peripheral blood of 2-, 6- and 18-month-old children and adults were stimulated with unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) 2216 (IFN-α response) or lipopolysaccharide (LPS) (IL-10 response) for 24 h. The production of IFN-α and IL-10 was then measured in culture supernatants using enzyme-linked immunosorbent assay (ELISA) or a 6-plex cytokine array, respectively. Compared to adults, we found a significant impairment in both the IFN-α and IL-10 responses of neonatal mononuclear cells. Interestingly, both responses had increased significantly after 2 months, but remained lower than the adult responses throughout the first 18 months of life. This study shows that although the immune response of neonates tends to mature fairly quickly, it remains different when compared to the adult immune response throughout the first 18 months of life. This could have important consequences on children's ability to mount an appropriate immune response to various challenges and to establish tolerance and immune homeostasis.     

The prospective relationship between subjective aging and inflammation: Evidence from the health and retirement study

This study tested the prospective associations and potential mediators between subjective aging, indexed by subjective age and self-perceptions of aging (SPA), and a range of inflammatory markers, including C-reactive proteins (CRP) and pro- and anti-inflammatory cytokines among older adults. Participants (N = 6099, 59% women, age range = 50 to 94, Mean Age = 65.32, SD = 8.85) were drawn from the Health and Retirement Study. Subjective age, SPA, and demographic factors were assessed in 2008/2010. Assessments of soluble transformation growth factor-beta 1 (sTGF-β1), interleukin 10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), interleukin 6 (IL-6), soluble tumor necrosis factor receptors (sTNFR1), and high sensitivity CRP (hsCRP) were measured in 2016. Potential mediators (body mass index, disease burden, physical inactivity, and depressive symptoms) were asssessed at baseline and in 2012/2014. Linear regression analyses indicated that an older subjective age and negative SPA were related to higher level of IL-10, IL-1Ra, IL-6, sTNFR1 and hsCRP. These associations were mediated by higher disease burden and physical inactivity. Negative SPA (but not subjective age) was associated with lower sTGF-β1. The link between subjective aging and inflammatory markers was relatively independent from chronological age. The present study provides new evidence that subjective aging is prospectively associated with inflammation, including systemic inflammation and pro-and anti-inflammatory cytokines.     

川芎嗪和地塞米松对狼疮肾炎患者PBMC IL-12表达及合成Ig的影响

应用细胞培养技术 ,采用半定量RT PCR法和ELISA检测等方法 ,以正常人为对照 ,观察川芎嗪 (33μg/ml)和地塞米松 (10 5mol/L )对培养的狼疮肾炎 (LN )活动期、静止期外周血单个核细胞 (PBMC )表达IL 12和产生免疫球蛋白 (Ig)的影响。结果表明 ,LN活动期、静止期较正常对照组IL 12蛋白、基因水平明显增高 (P <0 0 1) ,川芎嗪仅对活动期狼疮肾炎PBMCIL 12表达明显抑制 ,而地塞米松明显抑制狼疮肾炎PBMCIL 12表达 (P <0 0 1) ,两种药物对正常人对照IL 12表达及合成Ig无明显抑制作用 (P >0 0 5 ) ,川芎嗪和地塞米松均明显抑制狼疮肾炎活动期及静止期Ig合成 (P <0 0 1或 <0 0 5 )。提示 :狼疮肾炎患者有高水平的IL 12 ,川芎嗪、地塞米松通过下调狼疮肾炎IL 12表达及Ig的合成 ,减轻狼疮肾炎的发生和发展。     

Identification of human cell responses to hexavalent chromium

Hexavalent chromium [Cr(VI)] is a recognized environmental toxin with ubiquitous distribution in industrialized societies. Its concentration in ambient air derives from several sources including but not limited to chemical processes, the burning of fossil fuels and the production of cement. It is a food contaminant because of its deposition into bodies of water. The majority of published studies on the effects of Cr(VI) concern animal models and these studies have shown that it can induce a variety of cytotoxic and genotoxic reactions that affect the immune system. In order to identify the specific cellular impact of Cr(VI) on humans, we studied its effect on protein production and gene expression in human peripheral blood mononuclear cells (PBMC) obtained from both men and women of each major ethnic group including Caucasians, Hispanics, Asians and African-Americans. High-throughput protein profiling using bead-based protein arrays showed a concentration-dependent biphasic effect of Cr(VI) on the expression of many cytokines and chemokines by activated PBMC. High-density oligonucleotide microarray analysis identified several functional families of genes including those involved in immune response, intracellular signaling, cell cycle, apoptosis, RNA transport and binding, organelle organization and biogenesis that were strongly affected by Cr(VI). Cr(VI) suppressed many cellular receptor genes involved in immune response and activated many cell cycle-related and proapoptotic genes. These results defined responses that were unique to Cr(VI). This methodology defined an effective manner for identifying injurious/toxic human exposures to Cr(VI) at the cellular level that may facilitate the identification and monitoring of efficacious treatments for Cr(VI)-related maladies.