Mechanisms of placental dysfunction in pregnancy malaria

 描述(申请人提供)：在疟疾流行地区，由恶性疟原虫引起的妊娠期疟疾每年造成约15万名儿童和10,000名母亲死亡。早产儿死亡在很大程度上是由于低出生体重(LBW)；然而早产儿也会导致死产和流产。LBW与寄生性红细胞(PE)隔离和随后的胎盘炎症有关；然而，PM中母胎相互作用的确切分子细节以及导致胎儿生长受限/LBW的胎盘变化仍然知之甚少。这个项目的长期目的是阐明这些过程的分子细节。本项目将重点研究在胎盘合体滋养层细胞中高表达的巨蛋白相关多配体清除和信号转导系统的作用。这个系统可以调节和调节大量的母胎交换和维生素、脂类、激素、钙离子、基质蛋白和抑制物，以及其他载体蛋白的动态平衡，包括受体Cubilin及其配体。《巨无霸》 系统在胚胎发育中起着极其重要的作用，敲除巨蛋白或其细胞内适配蛋白DAB2可导致98%的围产儿死亡。我们推测，PM干扰了合体滋养层细胞中巨蛋白的功能/表达/分布，这反过来可能对胎盘病理有重要作用，因为由该受体操纵的分子对正常胎儿的生长/发育具有高度的重要性。我们的初步结果有力地支持了这一假设。为了了解巨蛋白系统在母胎相互作用和PM期间胎盘病理中的作用，我们提出了以下具体目标：1)分析疟疾流行区胎盘切片中巨蛋白系统蛋白的丰度，并使用抗Meggalin、Cubilin和DAB2的抗体、免疫荧光显微镜分析(IFA)以及逆转录-PCR方法将其与PM病理相关联；2)研究寄生红细胞(PE)与绒毛细胞滋养层BeWo细胞的黏附对巨蛋白系统蛋白表达、运输、配基转移和信号转导的影响。我们将使用实时荧光显微镜、IFA、RT-PCR、生化和细胞生物学方法。我们还将测试将人巨噬细胞添加到结合在BeWo细胞表面的PE中是否会影响这些过程，从而模拟最容易受到PM病理影响的初孕妇女在发展适应性反应之前的先天免疫反应的初始步骤。这些实验将检验我们的假设，即巨蛋白运输/信号系统的蛋白质在胎盘营养物质和调节分子的转移中的潜在作用，以及它们的丰度/分布与PM和/或LBW的关联。这反过来将为未来扩大母胎交换、病理变化和胎儿生长受限/LBW机制的研究奠定基础，这些机制与PM中的巨蛋白功能有关，PM是导致疟疾流行地区婴儿死亡和发病率的重要因素。

 DESCRIPTION (provided by applicant): Pregnancy malaria (PM) caused by Plasmodium falciparum contributes to about 150,000 child and 10,000 mother deaths annually in malaria endemic regions. Infant mortality in PM is largely due to low birth weight (LBW); however PM also causes stillbirth and abortion. LBW is associated with parasitized erythrocyte (PE) sequestration and consequent inflammation in the placenta; however, the precise molecular details of maternal- fetal interactions in PM and placental changes leading to fetal growth restriction/LBW are still poorly understood. The long-term purpose of this project is to clarify molecular details of these processes. This project will focus on the role of megalin-associated multi-ligand scavenging and signal transduction system, which is highly expressed in placental syncytiotrophoblast. This system may mediate and regulate a substantial amount of maternofetal exchange and homeostasis of vitamins, lipids, hormones, Ca2+, matrix proteases and inhibitors, and other carrier proteins, including receptor cubilin and its ligands. The megalin system plays an extremely important role in embryonic development and murine knock-out of megalin or its intracellular adaptor protein Dab2 leads to 98% perinatal mortality. We hypothesize that PM disturbs megalin function/expression/distribution in syncytiotrophoblast which, in turn, may contribute significantly to the placental pathology due to the high importance of the molecules, handled by this receptor, for normal fetal growth/development. Our preliminary results strongly support this hypothesis. To understand the role of the megalin system in maternal-fetal interactions and in placental pathology during PM we suggest the following Specific Aims: 1) Analyze abundance of megalin system proteins in placental sections from malaria endemic regions and relate it to PM pathology using antibodies against megalin, cubilin, and Dab2 and immunofluorescence microscopy assay (IFA), as well as reverse transcription-PCR approach; 2) Study the effect of parasitized erythrocyte (PE) adhesion to the villous cytotrophoblast BeWo cells on megalin system protein expression, trafficking, ligand transfer, and signal transduction. We will use real time fluorescence microscopy, IFA, RT-PCR, biochemical and cell biological approaches. We will also test whether the addition of human macrophages to PE bound on the surface of BeWo cells may affect these processes thus modeling the initial steps of the innate immune response before the development of an adaptive response in primigravida women, most susceptible to PM pathology. These experiments will test our hypothesis about potential role of proteins of the megalin transporting/signaling system in the placental transfer of nutrients and regulatory molecules, and association of their abundance/distribution with PM and/or with LBW. This, in turn, will create a basis for future expanded studies of maternofetal exchange, pathological changes, and mechanisms of fetal growth restriction/LBW related to megalin functions in PM, a substantial contributor to infant mortality and morbidity in malaria endemic regions.