Peripheral Blood Exosome Lipids as Biomarkers of Disease Activity in Crohn’s Disease

摘要 炎症性肠病（IBD）患者的管理需要了解疾病状态， 治疗决定。常用的生物标志物，如临床疾病活动指数、C反应蛋白 (CRP)和粪便钙卫蛋白对肠道炎症的敏感性和特异性均未达到最佳水平1。数据 表明外周血外泌体（PBE）脂质组合物将活动性IBD与炎症性肠病区分开来。 正常患者（图1）。我们认为PBE脂质组合物将为临床医生提供高度敏感的 和特异性生物标志物来评估疾病活动而无需侵入性测试。识别细微疾病的必要性 源自“深度缓解”应作为治疗终点的共识结论。因此，在本发明中， 为了抑制亚临床炎症而对患者进行监测已经成为一种治疗方法， 目标3.外泌体是脂质包裹的亚细胞（60-80 nm）结构，在约20 - 30 μ g/ml时释放到外周血中。 来自肠上皮细胞（IEC）、活化的白细胞（例如中性粒细胞、巨噬细胞、树突细胞等） 和间充质细胞在炎症3.我们用了一台高分辨率轨道阱质谱仪， 分析来自患者（>25/组）的PBE的脂质组成。PBE脂质的主成分分析（PCA） 强度显示活动性IBD和正常对照之间数据点的广泛分离， 差异丰度分析显示，组内相关性高，组间相关性低 这表明可以分辨与疾病活动相关的不同脂质。在这个为期两年的项目中，我们 建议从1）中度-重度、2）轻度活动性和3）静止性克罗恩病（CD）中收集血浆 患者以及炎性（C.艰难梭菌感染）和4）正常对照。使用CD可以让我们测试 PBE组合物是否检测到轻度（在许多情况下，亚临床）疾病活动的水平，因为这鉴定了 没有深度缓解的患者，这是药物治疗的目标。在目标1中，基于PBE脂质的分类器将 开发用于区分轻度活性CD和重度活性CD以及对照。在目标2中，PBE脂质将 在治疗前和治疗后对个体患者进行纵向检查，以确定与临床症状相关的脂质。 反应这些信息也将被传递到目标1，以开发更强大的分类器。测试模式 反应性和难治性患者中的PBE脂质旨在提高分类器的稳健性。 目标1和目标2的研究通过纳入第二个临床研究中心（贝勒大学）而得到极大的加强， 提供样本以验证（或不验证）来自肯塔基州大学的数据。长期目标将是证明 鉴定“外泌体脂质特征”小组的研究，该小组提供了一组新的生物标志物， CD疾病活动水平，并为治疗决策提供信息。我们认为CD外泌体的产生 脂质生物标志物检测将使大多数患者不需要进行后续内窥镜检查。

Abstract Management of inflammatory bowel disease (IBD) patients requires knowledge of disease status to inform treatment decisions. Commonly used biomarkers such as clinical disease activity indices, C reactive protein (CRP) and fecal calprotectin achieve suboptimal sensitivity and specificity for intestinal inflammation1. Data provided indicate that peripheral blood exosome (PBE) lipid composition distinguishes active IBD from normal patients (Fig. 1). We contend that PBE lipid compositions will provide clinicians with a highly sensitive and specific biomarker to assess disease activity without invasive testing. The need to identify subtle disease derives from the consensus conclusion that “deep remission” should be the endpoint of therapy. Thus, monitoring of patients with intent to suppress subclinical inflammation has emerged as a treatment goal3. Exosomes are lipid-encased, subcellular (60-80 nm) structures released into the peripheral blood at from intestinal epithelial cells (IEC), activated leukocytes (e.g. neutrophils, macrophages, dendritic cells, etc.) and mesenchymal cells during inflammation3. We used an ultrahigh resolution Orbitrap mass spectrometer to analyze lipid compositions of PBEs from patients (>25/group). Principal component analysis (PCA) of PBE lipid intensities showed a wide separation of datapoints between active IBD and normal controls and a heatmap analysis of differential abundance revealed high within-group correlations and low between-group correlations suggesting that distinct lipids can be resolved that correlate with disease activity. In this two year project, we propose to collect plasma from 1) moderate-severe, 2) mildly-active and 3) quiescent Crohn’s disease (CD) patients as well as inflammatory (C. difficile-infected) and 4) normal controls. Using CD allows us to test whether PBE composition detects levels of mild (in many cases, subclinical) disease activity as this identifies patients not in deep remission, a goal of medical therapy. In Aim 1, PBE lipid-based classifiers will be developed to discriminate mildly-active from severely-active CD and controls. In Aim 2, PBE lipids will be examined longitudinally within individual patients before and after therapy to identify lipids correlate with clinical response. Such information will also be passed to Aim 1 to develop more robust classifiers. Testing patterns of PBE lipids in responsive and refractory patients is intended to improve the robustness of the classifiers. Studies in Aim 1 and 2 are greatly enhanced by the inclusion of a second clinical site (Baylor University) to provide samples to validate (or not) data from University of Kentucky. The long-term goal will be to justify studies to identify a “exosomal lipid signature” panel that provide a novel set of biomarkers for discriminating levels of CD disease activity and informing treatment decisions. We post that the creation of a CD exosome lipid biomarker test will obviate the need for follow-up endoscopy in the majority of patients.