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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 现在人们承认，仅使用高效抗逆转录病毒疗法(HAART)恢复免疫功能是不完整的。由于HIV-1耐药毒株的出现，需要治疗性免疫战略来加强HAART在HIV疾病治疗中的作用。已有报道Toll样受体激动剂7/8(R-848)具有抗HIV-1活性。Toll样受体(Toll Like Receptor，TLR)是进化上保守的病原体受体，在先天免疫和获得性免疫系统中起着关键作用。TLR能够诱导抗病毒因子产生细胞因子和B-趋化因子，并触发干扰素(干扰素-α和β-干扰素)的产生。我们建议检验这样一种假设，即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来区别确定。 具体目标 1.检测TLR激动剂R-848处理的PBMC、CD8+T细胞和CD8+T细胞去除的PBMC的培养液中抗HIV1活性的差异。 2.用蛋白质组学方法鉴定和比较有抗病毒活性和无抗病毒活性的培养液中差异表达的蛋白质 3.用重组蛋白表达技术验证TLR激动剂R-848刺激的已鉴定因子的抗病毒活性 我们目前正在进行剂量反应分析，以确定TLR激动剂的最佳浓度。TLR激动剂作用9天后，收集培养液进行实时定量聚合酶链式反应和蛋白质组学分析。初步结果表明，TLR激动剂的最佳浓度为5 ug/m L。分别用5ug/mL和1ug/mL的培养液对三个样本进行双向凝胶电泳(2D-GE)实验，比较差异蛋白的表达。我们相信，识别的蛋白质将产生TLR激动剂-抗HIV-1反应的蛋白质组特征。

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.