RBC-derived microparticles function in cerebral malaria

描述(申请人提供)：脑型疟疾(CM)的发病机制是由于血管系统、免疫反应和寄生虫隔离之间的复杂相互作用。我们已经报道，给予恢复无生物利用度的化合物(一氧化氮[NO]供体、NO气体、精氨酸和瓜氨酸)可以保护实验性CM(ECM)的所有接受者；NO GAS和精氨酸目前正在进行临床试验，作为CM的辅助治疗。然而，没有生物标记物来衡量辅助治疗是否恢复了生物利用度。我们认为，红细胞衍生微粒(RMPs)在ECM过程中降低NO的生物利用度从而介导发病机制中起着关键作用。虽然游离血红蛋白(Hb)被迅速降解，但RMP中包含的Hb可以长时间循环，其清除能力是完整红细胞的1,000倍。确定RMPs在ECM发病机制中的作用是否代表了一个明确的R03先导性研究项目，该项目具有为RO1方案提供初步数据的巨大潜力。这一建议具有重要意义，因为它将提高对CM发病机制的理解和定义(需要开发其他辅助疗法，以保护CM患者免受宿主反应导致的死亡、认知障碍和精神疾病的影响，同时抗寄生虫治疗杀死恶性疟原虫)，确定正在进行临床试验的辅助疗法的作用机制，并确定追踪疟疾患者无生物利用度的生物标志物。我们小组在定义低NO生物利用度和微粒是ECM发病机制的关键方面发挥了核心作用。我们现在扩展我们的创新研究，将这两种必需的发病机制联系起来。我们假设，RMPs通过降低一氧化氮(NO)的生物利用度而在ECM的发展中发挥关键作用，这是由于血红蛋白对NO的清除。为了解决这一假设，我们使用伯氏疟原虫(PBA)感染小鼠作为CM的模型。我们的数据表明：(I)低NO生物利用度是ECM发病的关键，(Ii)MPS对ECM的发展至关重要，(Iii)人CM和ECM中RMP的数量显著增加。然而，RMPs在CM发病机制中的作用仍有待确定。因此，我们提出了一个有限的R03项目来检验这一假设。在目标1中，我们建议分析RMP和未包装的HB在(I)ECM敏感的，(Ii)ECM抵抗的，ECM保护的([iii]吸入NO和[iv]瓜氨酸)感染PBA的ECM敏感小鼠和(V)感染非ECM诱导P.berghei K173(Pbk)的ECM敏感小鼠中清除NO的能力。这些研究确定了RMPs与未包装Hb在ECM过程中低NO生物利用度的相对重要性。在目标2中，我们取消了红细胞中ABCA1的产生，ABCA1仅在这种细胞类型中防止MPS的形成；这些小鼠的存活与对照组的比较确定了RMPs在ECM发病机制中的重要性。我们还将比较等摩尔剂量的RMPs、未包装的OxHb、metHb和完整红细胞注射到目标1的(I)-(V)组的致病后果，以评估Hb状态在ECM发病机制中的重要性。通过将我们的研究结果与ECM保护性NO治疗和载体控制进行比较，我们不仅将确定吸入NO和瓜氨酸的新作用机制，而且还将阐明适合治疗的脑功能障碍的重要病理机制。这些研究意义重大，因为它们直接解决了一种重要但往往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治疗方法的作用机制，以拯救那些出现脑型疟疾的人。我们的团队由Gramaglia博士、Grau博士、Parks博士和Torbett博士组成，拥有执行拟议研究的专业知识和研究环境。

DESCRIPTION (provided by applicant): Cerebral malaria (CM) pathogenesis is due to a complex interaction between the vascular system, the immune response, and parasite sequestration. We have reported that administration of compounds that restore NO bioavailability (nitric oxide [NO] donor, NO gas, arginine and citrulline) protect all recipients fom experimental CM (ECM); NO gas and arginine are currently undergoing clinical trials as adjunctive therapy for CM. There are, however, no biomarkers to gauge whether the adjunctive therapy is restoring NO bioavailability. We propose that RBC-derived microparticles (RMPs) play a critical role in reducing NO bioavailability during eCM and hence mediating pathogenesis. While free hemoglobin (Hb) is rapidly degraded, Hb contained within RMPs circulates for extended periods of time and has a 1,000-fold greater NO scavenging than intact RBCs. Determining whether RMPs function in eCM pathogenesis represents a defined pilot RO3 research project with significant potential to provide preliminary data for a RO1 proposal. This proposal is significant because it will improve understanding and definition of CM pathogenesis (which is required to develop other adjunctive therapies aimed at protecting CM patients from the host response leading to death, cognitive impairment, and psychoses while the anti-parasite treatment kills Plasmodium falciparum), determine mechanisms of action of an adjunctive therapy undergoing clinical trials, and determine biomarkers for tracking NO bioavailability in malaria patients. Our group played a central role in defining that low NO bioavailability and microparticles are critical for eCM pathogenesis. We now extend our innovative research to link these 2 required mechanisms of pathogenesis. We hypothesize that RMPs play a critical role in the development of ECM by lowering nitric oxide (NO) bioavailability due to NO scavenging by hemoglobin. To address this hypothesis, we use the P. berghei- ANKA (PbA) infection of mice as a model for CM. Our data indicate that (i) low NO bioavailability is critical for eCM pathogenesis, (ii) MPs are critical for the development of eCM, and (iii) RMP numbers are markedly elevated in human CM and eCM. The contribution of RMPs to CM pathogenesis, however, remains to be determined. We therefore propose a limited RO3 project to test this hypothesis. In aim 1, we propose to analyze RMP's and unpackaged Hb's ability to scavenge NO in groups of (i) eCM-susceptible, (ii) eCM- resistant, eCM protected ([iii] inhaled NO and [iv] citrulline) mice infected with PbA and (v) eCM susceptible mice infected with non-eCM inducing P. berghei K173 (PbK). These studies determine the relative importance to low NO bioavailability during eCM of RMPs versus unpackaged Hb. In aim 2, we abrogate the production of ABCA1 in RBCs, which prevents the formation of MPs only in this cell type; comparison of survival of these mice to controls determines the importance of RMPs to eCM pathogenesis. We will also compare the pathogenic consequences of injecting equimolar doses of RMPs, unpackaged oxyHb, metHb, and intact RBCs into groups (i)-(v) of Aim 1 to assess the importance of the states of Hb to eCM pathogenesis. By comparing the results of our studies with eCM-protective NO therapy and vehicle control, we will not only define new mechanisms of action for inhaled NO and citrulline but also elucidate important pathological mechanisms in cerebral dysfunction that are amenable to treatment. These studies are significant because they directly address the pathogenic mechanisms of an important but often overlooked disease that kills millions of people and define the mechanisms of action of a potential therapy to rescue those presenting with cerebral malaria from their disease. Our team, comprising Dr. Gramaglia, Dr. Grau, Dr. Parks, and Dr. Torbett, has the expertise and research environment to perform the proposed studies.