Identification of FX-activating molecules of convulsion-inducing lipoprotein purified from egg granulomas in Schistosoma mansoni-infected mice and of its inducing mechanism

本研究对曼氏血吸虫感染小鼠肝虫卵肉芽肿中提取的惊厥诱导脂蛋白（CILIP）的X因子（FX）激活分子进行了鉴定，探讨了巨噬细胞和树突状细胞对CILIP的诱导机制，CILIP的诱导与信号转导途径的研究：1.体外实验表明，巨噬细胞和树突状细胞可以产生CILIP当用Toll样受体的几种配体如LPS、LTA、肽聚糖、聚IC和CpGDNA刺激时，用SWAP抗原致敏的脾DX 5 + NK细胞和/或CD 4 + T细胞，或与细胞因子如IFNγ和GM-CSF联合使用。SWAP致敏淋巴细胞激活PCA需要RNA和蛋白质的重新合成以及PTK和PKC的激活，而不是MAPK激活、钙调蛋白或细胞内Ca++浓度的增加。此外，巨噬细胞FX激活PCA的诱导可被胞内cAMP的增加所下调。3.制备了两种抗CILIP的单克隆抗体。大鼠单克隆抗体对天然CILIP具有高度的特异性和敏感性，并呈剂量依赖性地抑制CILIP的FX激活活性，但不与经去污剂处理的变性CILIP反应。建立了两种抗CILIP单克隆抗体的夹心ELISA检测方法，并将其应用于CILIP的定量检测。4.虽然利用抗组织因子单克隆抗体和抗各种凝血因子特异性抗体进行了各种生化分析，以鉴定CILIP的EX激活分子，但尚未获得确凿的证据。

Biochemical studies has been performed to identify the factor-X(FX)-activating molecules of convulsion-inducing lipoprotein (CILIP) purified from hepatic egg granulomas in Schistosoma mansoni-infected mice, the inducing mechanism of CILIP by macrophages and dendritic cells, and the signal transduction pathway involved in CILIP induction.1.In vitro analysis demonstrated that macrophages and dendritic cells could produce CILIP(≒FX-activating PCA) when stimulated with several ligands for Toll-like receptors such as LPS, LTA, peptidoglycan, poly IC and CpGDNA, with splenic DX5+ NK cells and/or CD4+ T cell sensitized with SWAP antigen, or with cytokines such as IFNγ and GM-CSF.2.The induction of macrophage FX-activating PCA by stimulation with SWAP sensitized lymphocytes required de novo RNA and protein synthesis and activation of PTK and PKC, but not MAPK activation, calmodulin or increase of intracellular Ca++ concentration. Moreover, the induction of macrophage FX-activating PCAwas down-regulated by increment of intracellular cAMP.3.Two kinds of monoclonal antibodies against CILIP were prepared from BALB/c mice and Wister rats. Rat's MoAb appeared to be highly specific and sensitive to native CILIP, and inhibited dose-dependently FX-activating activity of CILIP, but did not react with denatured CILIP treated with detergents. Sandwich ELISA system using two kinds of MoAbs against CILIP was developed and confirmed its usefulness when applied for quantitative determination of CILIP.4.Although various biochemical analysis using MoAb against tissue factor and specific antibodies against various coagulation factors have been conducted to identify the EX-activating molecules of CILIP, conclusive evidence has not been obtained yet.