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复杂的相互作用。通过使用实验性脑疟疾（eCM）模型，我们报告了pantethine治疗通过抑制微颗粒（MP）的形成和脑血管泄漏来保护所有接受者免受eCM。atp结合盒a1缺乏阻止MP的形成，所有缺乏的动物都免于死亡。我们的初步数据表明，在eCM期间MP的形成明显增加，并与临床症状和疾病的组织学分析相关。通过我们的体外人血脑屏障（BBB）模型，我们观察到MPs是单核细胞的有效激活剂，并促进恶性疟原虫寄生的红细胞（bf - prbc）在体外通过粘附红细胞或HBEC粘附到人脑内皮细胞（HBEC）单层。我们随后用两种模型在患者中证实了我们的结果；我们报道了CM患者MPs的显著增加，并且血小板MPs的数量与昏迷深度和血小板减少有关。这一发现还表明，MPs在人CM凝血功能中起作用，这也与患者CM的发展有关，并强调了MPs和凝血功能在人CM发病中的重要性。将这些发现转化为人类CM的辅助治疗需要更好地理解MPs作为生物标志物的重要性，并确定其他mp特异性靶点进行干预。我们假设，在脑疟疾和MPs在CM中的功能过程中，微颗粒（MPs）数量的增加是通过以下方式引起的：(i)在脉管系统中引起促炎、促粘附和促凝状态，以及（ii）促进血脑屏障（BBB）的破坏。为了验证这一假设，我们使用了两种广为接受的模型：体内模型：小鼠或eCM感染柏氏假单胞菌；体外模型：黏附细胞群（即恶性疟原虫寄生的红细胞、血小板和白细胞）的HBEC单层。在目的1中，我们建议评估体内MPs的数量、它们的细胞来源、它们的表面和mp内蛋白，这些蛋白有助于在eCM过程中促进炎症和凝血特性。我们也在目的1中确定MP的体内动力学，命运，以及MPs是否直接参与eCM的发病机制。其中一些研究需要大量的患者，而在eCM中只有5只动物/组，因为寄生虫和宿主基因相同，环境受控；其他的研究在伦理上不能在人类身上进行。通过使用人类细胞，我们将在目的2中确定pf - prbc是否是MP生成的重要刺激物。在目的3中，我们将阐明MPs是否直接破坏血脑屏障，或者MPs是否增加细胞（Pf-pRBC、血小板和白细胞）粘附并间接破坏血脑屏障。提出的分析将导致在患者中进行明确的研究，以证实我们的假设，并可能最终导致抗mp辅助治疗的临床试验。这些研究意义重大，因为它们直接解决了导致数百万人死亡的一种重要但往往被忽视的疾病的致病机制，并确定了潜在辅助治疗的靶向机制，以拯救脑型疟疾患者。我们是唯一一个提出MP在CM发病机制中的创新作用的团队，提供了从动物模型到人体模型再到患者分析的结果来证实这一假设，确定了潜在的先导化合物，并开发了MP分析的新方法。因此，我们的方法是创新的。我们有广泛的初步数据来支持我们的研究，表明建议的研究是可行的。

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.