Microparticles, a new player in cerebral malaria pathogenesis

描述（由申请人提供）：脑型疟疾（CM）的发病机制是由于血管、凝血和免疫系统以及寄生虫隔离之间的复杂相互作用。通过使用实验性脑型疟疾（eCM）模型，我们报告了泛硫乙胺治疗通过抑制微粒（MP）形成和脑血管渗漏保护所有受者免受eCM。ATP结合盒A1缺陷阻止MP形成，所有缺陷动物免于死亡。我们的初步数据表明，在eCM过程中MP形成显著增加，并与疾病的临床症状和组织学分析相关。通过使用我们的人血脑屏障（BBB）的体外模型，我们观察到MP是单核细胞的有效激活剂，并且通过粘附到pRBC或HBEC，促进恶性疟原虫寄生的RBC（Pf-pRBC）粘附到人脑内皮细胞（HBEC）的体外单层。我们随后证实了我们的结果与患者的2个模型;我们报告了CM患者的MP显着增加，血小板MP的数量与昏迷深度和血小板减少症相关。这一发现还表明，MP在人类CM凝血病中起作用，这也与患者中CM的发展相关，并强调了MP和凝血病在人类CM发病机制中的重要性。将这些发现转化为人类CM的连续治疗需要更好地理解MP作为生物标志物的意义，并确定其他MP特异性靶点进行干预。我们假设，在脑型疟疾期间引起微粒（MP）数量增加，并且MP通过以下方式在CM中起作用：（i）引起脉管系统中的促炎、促粘附和促凝血状态，以及（ii）促进血脑屏障（BBB）的破坏。为了解决这一假设，我们使用了两种公认的模型：体内模型：小鼠或eCM的伯氏疟原虫-ANKA感染和体外模型：具有贴壁细胞群的HBEC单层（即，恶性疟原虫寄生的RBC、血小板和白细胞）。我们在目标1中提出在体内评估MP的数量、它们的细胞来源、它们的表面和MP内蛋白，这些蛋白在eCM期间有助于它们的促炎和促凝血特性。我们还在目标1中确定MP的体内动力学、命运以及MP是否直接促成eCM发病机制。其中一些研究需要大量患者，而eCM中为5只动物/组，因为寄生虫和宿主遗传学相同且环境受控;其他研究在伦理上无法在人体中进行。通过使用人细胞，我们将在目标2中确定Pf-pRBC是否是MP产生的重要刺激物。在目标3中，我们将阐明MP是否直接破坏BBB或MP是否增加细胞（Pf-pRBC，血小板和白细胞）粘附并间接破坏BBB。拟议的分析将导致在患者中进行定义的研究，以证实我们的假设，并可能最终导致抗MP治疗的临床试验。这些研究意义重大，因为它们直接解决了一种重要但经常被忽视的疾病的致病机制，这种疾病导致数百万人死亡，并确定了一种潜在的预防性治疗机制，以拯救那些患有脑型疟疾的人。我们是唯一一个提出MP在CM发病机制中的创新作用的小组，提供了从动物模型到人类模型到患者分析的结果以证实假设，确定了潜在的先导化合物，并开发了MP分析的新方法。因此，我们的做法是创新的。我们有大量的初步数据支持我们的研究，表明拟议的研究是可行的。

DESCRIPTION (provided by applicant): Cerebral malaria (CM) pathogenesis is due to a complex interaction between the vascular, coagulation and immune systems, and parasite sequestration. By using the experimental cerebral malaria (eCM) model, we reported that pantethine therapy protects all recipients from eCM by inhibiting microparticle (MP) formation and brain vascular leak. ATP-binding cassette A1-deficiency prevents MP formation and all deficient animals are protected from death. Our preliminary data indicate that MP formation is markedly increased during eCM and correlates with clinical symptoms and histological analysis of disease. By using our in vitro model of the human blood brain barrier (BBB), we observed that MPs are potent activators of monocytes and facilitate the adhesion of Plasmodium falciparum-parasitized RBCs (Pf-pRBCs) to human brain endothelial cell (HBEC) monolayers in vitro by adhering to either the pRBCs or HBECs. We subsequently confirmed our results with the 2 models in patients; we reported a marked increase in MPs in patients with CM, and that the numbers of platelet MPs correlated with coma depth and thrombocytopenia. This finding also suggests that MPs function in human CM coagulopathy, which also correlates with the development of CM in patients, and underscores the importance of MPs and coagulopathy in CM pathogenesis in humans. Translation of these findings into adjunctive therapy for human CM requires an improved understanding of the significance of MPs as biomarkers and the identification of other MP-specific targets for intervention. We hypothesize that increased numbers of microparticles (MPs) are elicited during cerebral malaria and MPs function in CM by: (i) eliciting a pro-inflammatory, pro-adhesive, and pro-coagulatory state in the vasculature, and (ii) contributing to the breakdown of the blood brain barrier (BBB). To address this hypothesis, we use two well accepted models: an in vivo model: P. berghei-ANKA infection of mice or eCM and an in vitro model: HBEC monolayers with adherent cell populations (i.e., P. falciparum-parasitized RBCs, platelets, and leukocytes). We propose in aim 1 to assess in vivo the number of MPs, their cellular origin, their surface and intra-MP proteins that contribute to their pro-inflammatory and pro-coagulation properties during eCM. We also in aim 1 determine MP's in vivo kinetics, fate, and whether MPs directly contribute to eCM pathogenesis. Some of these studies would require large numbers of patients compared with 5 animals/ group in eCM because of the identical parasite and host genetics and controlled environment; other studies cannot be ethically performed in humans. By using human cells, we will determine in aim 2 whether Pf-pRBCs are important stimuli for MP production. In aim 3, we will elucidate whether MPs function directly to disrupt the BBB or whether MPs increase cell (Pf-pRBC, platelet, and leukocyte) adhesion and indirectly disrupt the BBB. The proposed analysis will lead to defined studies in patients to confirm our hypothesis and may ultimately lead to clinical testing of anti-MP adjunctive therapy. These studies are significant because they directly address pathogenic mechanisms of an important but often overlooked disease that kills millions of people and defines the mechanisms to target for a potential adjunctive therapy to rescue those presenting with cerebral malaria from their disease. We are the only group that has proposed this innovative role for MP in CM pathogenesis, provided results from animal models to human models to patient analysis to confirm the hypothesis, identified a potential lead compound, and developed new methods for MP analysis. Our approach is therefore innovative. We have extensive preliminary data to support our studies, indicating that the proposed studies are feasible.