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

受体激动剂诱导的 PBMC 识别损伤

• 批准号: 8357103
• 负责人: NAWAL BOUKLI
• 金额: $ 7.91万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8357103
• 关键词: Agonist; Antiviral Agents; Biological Assay; Biomedical Research; CD8B1 gene; Disease; Dose; Drug resistance; Funding; Grant; HIV; HIV-1; Highly Active Antiretroviral Therapy; Immune system; Immunization; Interferon-alpha; Interferons; Liquid substance; National Center for Research Resources; Peripheral Blood Mononuclear Cell; Play; Principal Investigator; Production; Proteins; Proteomics; Recombinant Proteins; Reporting; Research; Research Infrastructure; Resources; Source; T-Lymphocyte; Techniques; Testing; Therapeutic; Time; Toll-like receptors; Two-Dimensional Gel Electrophoresis; United States National Institutes of Health; chemokine; cost; cytokine; immune function; pathogen; protein expression; receptor; research study; resistant strain; response; restoration; two-dimensional

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 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耐药菌株的出现，需要治疗性免疫策略来加强HAART治疗HIV疾病的效果。据报道，Toll 样受体激动剂 7/8 (R-848) 可诱导抗 HIV-1 活性。 Toll 样受体 (TLR) 是进化保守的病原体受体，在先天性和适应性免疫系统中发挥关键作用。 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 IFN（IFN-α 和 β）产生。我们建议测试以下假设：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确定。 具体目标 1. 确定用 TLR 激动剂 R-848 处理的 PBMC、CD8+ T 细胞和 CD8+ T 细胞耗尽的 PBMC 培养液中抗 HIV1 活性的差异 2. 使用蛋白质组学来识别和比较具有和不具有抗病毒活性的培养液中的差异表达蛋白质 3. 使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实时 PCR 和蛋白质组学分析。初步结果表明，5 ug/mL 是最佳 TLR 激动剂浓度。对 5 ug/mL 和 1 ug/mL 培养液进行二维凝胶电泳 (2D-GE) 实验，一式三份，并与对照相比评估差异表达的蛋白质。我们相信，所鉴定的蛋白质将产生 TLR 激动剂抗 HIV-1 反应的蛋白质组学特征。