Analysis of pathogen recognition by TLR4 and RP105 and their application to immunothrapy

TLR4和RP105病原体识别分析及其在免疫治疗中的应用

基本信息

批准号：
13470073
负责人：
KIMOTO Masao
金额：
$ 8.7万
依托单位：
Saga University (2004)佐賀医科大学 (2001-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

1.Analysis of the TLR and RP105 molecular structure : We obtained highly purified form of recombinant bacterial products of MD-2,CD14 and LBP. Using these purified proteins, we analyzed thier critical structures for binding to LPS. We also revealed the differential binding of CD14 and MD-2 to LPS.2.Analysis of TLR4 and RP105 ligands : We found that MD-2 directly binds to LPS. On the other hand, MD-1 was found not to bind LPS directly, although LPS can signal through RP105/MD-1 molceule. We compared the LPS binding pattern between MD-1 and MD-2 using their site-specific mutant molecules.3.Role of TLR4 and RP105 in immunological diseases : We found increased frequency of RP105-negative B cells in SLE patients. Culture of RP105-negative B cells resulted in the secretion of IgG and IgM, the production of these were augmented by SAC or IL-6 stimulation. RP105-negative B cells from SLE patients produced dsDNA antibodies. These results suggest that RP105-negative B cells are in an activated state and possibly involved in the production of auto-antibodies.4.Manipulation of TLR4 and RP105 signal transduction : We found alternative spliced forms of MD-2 by RT-PCR. Such alternative form of MD-2 molecules may potentially function as dominant negative molecules for TLRA/MD-2 signaling, and may function as a potential tool for the manipulation of LPS signaling. We also obtained monoclonal antibodies against TLRA/MD-2. This mAb was found to transmit activation signals through TLRA/MD-2 and resulted in the secretion of pro-inflammatory cytokines.

1. TLR和RP105分子结构的分析：我们获得了MD-2，CD14和LBP的重组细菌产物的高度纯化形式。使用这些纯化的蛋白质，我们分析了其与LPS结合的关键结构。我们还揭示了CD14和MD-2与LPS的差异结合。2。TLR4和RP105配体的分析：我们发现MD-2直接与LPS结合。另一方面，发现MD-1不直接结合LP，尽管LPS可以通过RP105/MD-1 molceule发出信号。我们使用其位点特异性突变体分子比较了MD-1和MD-2之间的LPS结合模式。3。免疫疾病中TLR4和RP105的role：我们发现SLE患者RP105阴性B细胞的频率增加。 RP105阴性B细胞的培养导致IgG和IgM的分泌，SAC或IL-6刺激增强了它们的产生。来自SLE患者的RP105阴性B细胞产生DSDNA抗体。这些结果表明，RP105阴性B细胞处于活化状态，可能参与自身抗体的产生。4。TLR4和RP105信号转导的操作：我们发现RT-PCR的MD-2的替代剪接形式。 MD-2分子的这种替代形式可能会作为TLRA/MD-2信号传导的主要负分子，并且可能是操纵LPS信号传导的潜在工具。我们还获得了针对TLRA/MD-2的单克隆抗体。发现该mAb通过TLRA/MD-2传输激活信号，并导致促炎性细胞因子的分泌。