Mechanisms of placental dysfunction in pregnancy malaria

 描述（由申请人提供）：在疟疾流行地区，由恶性疟原虫引起的妊娠期疟疾（PM）每年导致约150，000名儿童和10，000名母亲死亡。PM中的婴儿死亡率主要是由于出生体重低（LBW）;然而PM也会导致死胎和流产。LBW与胎盘中寄生的红细胞（PE）隔离和随后的炎症相关;然而，PM中母体-胎儿相互作用的精确分子细节和导致胎儿生长受限/LBW的胎盘变化仍然知之甚少。该项目的长期目标是澄清这些过程的分子细节。本项目将重点研究胎盘合体滋养细胞高表达的megalin相关的多配体清除和信号转导系统的作用。该系统可以介导和调节大量的母体胎儿交换和维生素、脂质、激素、Ca 2+、基质蛋白酶和抑制剂以及其他载体蛋白（包括受体cubilin及其配体）的体内平衡。巨蛋白 megalin系统在胚胎发育中起着极其重要的作用，megalin或其细胞内接头蛋白Dab 2的小鼠敲除导致98%的围产期死亡率。我们假设PM干扰巨蛋白在合胞体滋养层中的功能/表达/分布，这反过来又可能对胎盘病理学有显著贡献，这是由于该受体处理的分子对于正常胎儿生长/发育的高度重要性。我们的初步结果强烈支持这一假设。为了解megalin系统在PM时母胎相互作用和胎盘病理学中的作用，我们提出以下具体目的：1）利用megalin、cubilin和Dab 2抗体，免疫荧光显微镜分析（IFA）和逆转录-PCR方法，分析疟疾流行区胎盘切片中megalin系统蛋白的丰度，并将其与PM病理学联系起来; 2）研究寄生红细胞（PE）与绒毛滋养层细胞BeWo细胞粘附对megalin系统蛋白表达、转运、配体转运和信号转导的影响。我们将使用真实的时间荧光显微镜，IFA，RT-PCR，生化和细胞生物学方法。我们还将测试在BeWo细胞表面结合的PE中加入人巨噬细胞是否会影响这些过程，从而在最易受PM病理影响的孕妇中发展适应性反应之前模拟先天免疫反应的初始步骤。这些实验将检验我们关于巨蛋白转运/信号传导系统的蛋白质在营养物和调节分子的胎盘转移中的潜在作用以及它们的丰度/分布与PM和/或LBW的关联的假设。这反过来又将为未来扩大研究母胎交换、病理变化和与PM中巨蛋白功能相关的胎儿生长受限/LBW机制奠定基础，PM是疟疾流行地区婴儿死亡率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.