Pathophysiology of Acute Pain in Patients with Sickle Cell Disease

发作性疼痛是镰状细胞病（SCD）患者最常见的急性发病率和住院的主要原因。它是由镰状红细胞（RBC）诱导的微血管闭塞引起的，镰状红细胞是脱氧血红蛋白S（HbS）聚合的结果。导致急性镰状疼痛的因素包括游离DNA（cfDNA）和血红素的释放，其引发涉及炎症和血栓形成以及缺血-再灌注损伤的下游事件。cfDNA已显示存在于健康受试者的血浆中，但在特征在于通过坏死或凋亡增加的细胞死亡的疾病和病症中升高。 事实上，我们之前已经表明，SCD患者的cfDNA在急性疼痛发作期间急剧增加。在急性镰状疼痛期间，由于镰状红细胞和溶血的急性增加，还观察到血浆血红蛋白的显著升高。cfDNA和血红素（血红蛋白的分解产物）都充当损伤相关分子模式（DAMP）分子，引发内皮炎症、刺激中性粒细胞胞外陷阱（NET）形成、白细胞募集和微血管血栓形成。 尽管已经有几项稳态和急性SCD中的细胞因子和趋化因子的研究，但还没有关于炎症标志物如何与cfDNA的定量水平和谱相关的全面研究。在这项研究中，我们希望应用下一代测序（NGS）来分析稳态SCD患者血浆中的cfDNA，并在痛苦的危机期间获得对组织损伤起源的见解。与游离血浆DNA平行，我们建议测量溶血和炎症的标志物（细胞因子，趋化因子），并研究这些循环分子和血细胞（如中性粒细胞）之间的相互作用是否有可能调节疾病的进展和严重程度。此外，我们还想探索在稳态和急性疼痛期间SCD中是否存在独特的游离DNA和炎症特征。 总的来说，这项研究提供了一个机会，以评估新的生物标志物的镰状细胞疼痛危机，并预测疾病的严重程度和预后。这些措施可以使我们更好地了解血管闭塞，溶血和炎症相关事件和反应的作用，并作为临床终点，在未来的疾病发病机制和/或治疗干预镰状细胞病的研究。 该方案正在积极从NIH临床中心招募患者。首例患者于2017年3月17日入组。迄今为止，已入组69例患者，其中34例患者完成了研究的所有阶段，完成所有3个阶段的目标是40例患者。总入组目标为60例受试者。40名年龄和性别匹配的对照组已入组。对照组的总入组目标为40例受试者。

Episodic pain is the most common acute morbidity and the leading cause of hospitalization in patients with sickle cell disease (SCD). It is caused by microvaso-occlusion induced by sickled red blood cells (RBCs), an outcome of the polymerization of deoxygenated hemoglobin S (HbS). Factors that contribute to the acute sickle pain include the release of cell-free DNA (cfDNA) and heme that initiate a downstream of events involving inflammation and thrombosis, and ischemia-reperfusion injury. cfDNA has been shown to be present in plasma of healthy subjects, but elevated in diseases and conditions that are characterized by increased cell death through necrosis or apoptosis. Indeed, we have previously shown that cfDNA in patients with SCD increased dramatically during acute painful episodes. During acute sickle pain, marked elevation of plasma hemoglobin has also been observed due to the acute increase in sickled RBCs and hemolysis. Both cfDNA and heme (break down product of hemoglobin) act as damage-associated molecular pattern (DAMP) molecules, initiating endothelial inflammation, stimulation of neutrophil extracellular trap (NET) formation, leukocyte recruitment, and microvascular thrombosis. Although there have been several studies of cytokines and chemokines in steady state and acute SCD, there has been no comprehensive study of how the inflammatory markers correlate with quantitative levels and profile of cfDNA. In this study, we would like to apply next generation sequencing (NGS) to analyze cfDNA from the plasma of patients with SCD in steady-state, and during painful crises to derive insights on the origin of tissue damage. In parallel with the free plasma DNA, we propose to measure markers of hemolysis and inflammation (cytokines, chemokines), and to investigate if interactions between these circulating molecules and blood cells (e.g. neutrophils) have the potential to modulate the progress and severity of the disease. In addition, we would also like to explore if there is a distinctive cell-free DNA and inflammatory signature in SCD in steady-state and during acute pain. Overall, this study provides an opportunity to evaluate new biomarkers of sickle cell pain crisis and to predict disease severity and prognosis. These measures may allow us to better understand the role of vaso-occlusion, hemolysis, and inflammation-related events and responses and serve as clinical endpoints in future studies of disease pathogenesis and/or therapeutic intervention for sickle cell disease. The protocol is actively accruing patients from the NIH clinical center. The first patient was enrolled on March 17, 2017. To date, 69 patients have been enrolled with 34 patients completing all phases of the study, the target fro completion of all 3 phases is 40. Total enrollment target is 60 subjects. 40 age and sex matched controls have been enrolled. Total enrollment target for controls is 40 subjects.