IDENT TOLL LIKE RECEPTORS AGONIST INDUCED PBMC & CD8+TCELLS ANTIHIV MECHANISM

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 现在人们普遍认为，仅使用高效抗逆转录病毒疗法（HAART）来恢复免疫功能是不完全的。由于HIV-1耐药株的出现，需要治疗性免疫策略来加强HAART治疗HIV疾病。据报道，Toll样受体激动剂7/8（R-848）诱导抗HIV-1活性。 Toll样受体（TLR）是一类进化上保守的病原体受体，在先天性和适应性免疫系统中发挥重要作用。TLR具有诱导产生细胞因子和B-趋化因子的抗病毒因子并触发IFN（IFN-α和IFN-β）产生的能力。我们建议测试TLR激动剂7/8诱导的抗病毒活性可以通过蛋白质组学方法进行差异测定的假设。 具体目标 1.为了确定PBMC培养液中的差异抗HIV 1活性，用TLR激动剂R-848处理CD8 + T细胞和CD8 + T细胞耗尽的PBMC 2.利用蛋白质组学鉴定和比较具有和不具有抗病毒活性的培养液中差异表达的蛋白质 3.利用重组蛋白表达技术验证TLR激动剂R-848刺激的所鉴定因子的抗病毒活性 我们目前正在进行剂量反应测定，以确定最佳TLR激动剂浓度。TLR激动剂暴露9天后，收集培养液用于定量真实的时间PCR和蛋白质组学分析。初步结果表明，5 μ g/mL是TLR激动剂的最佳浓度。一式三份进行来自5 μ g/mL和1 μ g/mL培养液的二维凝胶电泳（2D-GE）实验，并与对照相比评价差异表达的蛋白质。我们相信，所确定的蛋白质将产生TLR激动剂-抗HIV-1反应的蛋白质组学特征。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease. It has been reported that Toll like receptor agonist 7/8 (R-848) induces anti-HIV-1 activity. Toll like receptors (TLR) are evolutionary conserved pathogen receptors that play a key role in innate and adaptive immune system. The TLR have the ability to induce antiviral factors producing cytokines and B-chemokines and triggering IFN (IFN-alpha and Beta) production. We propose to test the hypothesis that TLR agonist 7/8 induced antiviral activity can be differentially determined by a proteomic approach. Specific Aims 1. To determine the differential anti-HIV1 activity in culture fluids from PBMC, CD8+ T cells and CD8+ T cells depleted PBMC treated with TLR agonist R-848 2. To identify and compare differentially expressed proteins in culture fluids with and without antiviral activity using proteomics 3. To validate the antiviral activity of the identified factors stimulated by TLR agonist R-848 using recombinant protein expression technique We are currently performing dose-response assays to determine the optimal TLR agonist concentration. After 9 days of TLR agonist exposure, culture fluids were collected for Quantitative Real Time PCR and proteomics analysis. Preliminary results reveal that 5 ug/mL is the optimal TLR agonist concentration. Two Dimensional Gel Electrophoresis (2D-GE) experiments from culture fluids at 5 ug/mL and 1 ug/mL were performed in triplicates and differentially expressed proteins were evaluated as compared to the control. We believe that the identified proteins will generate a proteomic signature of the TLR agonist-anti HIV-1 response.