Auto-Antibodies as Serum Biomarkers for Age-Related Macular Degeneration

描述（由申请人提供）：视网膜相关性黄斑变性（AMD）的特征在于从其最早期开始在布鲁赫膜（BM）和视网膜色素上皮（RPE）之间形成沉积物，称为玻璃疣。近年来，在生物学、实验和遗传学水平上已经确定了炎症在一般AMD和特别是玻璃疣的发病机制中的作用。玻璃疣具有高含量的炎性效应物和副产物，并且来自脉络膜（Ch）的树突状细胞（DC）（其是眼睛的常驻抗原呈递细胞之一）被募集并浸润BM和玻璃疣核心。在这些前提下，并基于来自文献的证据表明AMD患者具有比对照更高频率的针对Ret抗原，而且针对BM/Ch和RPE来源的抗原的循环抗体，我们假设自身抗体（Auto-Ab）是与眼部疾病状态相关的生物标志物，并且它们将在AMD患者的血清中可测量。为了检验这一假设的基本原则，本提案的目的是分析从患有AMD（n=188）和没有AMD（n=222）的参与者中收集的血清样本。其中，108名AMD受试者和222名未受影响的个体（平均年龄：79岁）是阿尔马研究的参与者，该研究是由P.I.在K23颁奖典礼上在此奖励期间将招募另外80名晚期AMD受试者。将对检查时采集的血清样本进行筛选，以确定是否存在针对从人类供体眼睛解剖的新鲜组织中生成的黄斑特异性Ret、RPE和BM/Ch匀浆的自身抗体。对血清自身抗体反应的匀浆蛋白进行免疫沉淀，并在2D凝胶上分离。将通过质谱法初步表征在AMD样品中唯一和/或更强烈观察到的最常见凝胶斑点的身份。基于我们的总体原理，我们预测来自AMD患者的血清将显示出比对照受试者更高频率的自身抗体，不仅针对Ret，而且尤其是针对RPE和/或BM/Ch抗原。如果我们的假设将被证明是正确的，在随后的R 01申请中，我们计划检验以下额外假设：（1）在AMD患者组中，抗Ret、抗RPE和/或抗BM/Ch自身抗体将在携带疾病易感遗传变异的受试者中更常见，以及（2）晚期AMD患者的自身抗体频率将高于早期AMD患者。我们进一步计划（3）详细表征所有鉴定的自身抗原，通过质谱法将自身抗体导向这些抗原，以及（4）通过对来自人供体眼睛的黄斑切片进行免疫组织化学实验，在组织水平上定位它们的表达。我们预测，最终，这一系列的研究将为我们提供有用的生物标志物，以进一步阐明在组织水平上参与AMD的途径，并确定潜在的预后和治疗目标。

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is characterized since its earliest stages by formation of deposits between the Bruch's membrane (BM) and the retinal pigment epithelium (RPE), termed drusen. In recent years, a role for inflammation in the pathogenesis of both AMD in general and drusen in particular has been established at the biological, experimental, and genetic level. Drusen have high contents of inflammatory effectors and by-products, and dendritic cells (DCs) from the choroid (Ch), which are among the resident antigen-presenting cells of the eye, are recruited to and infiltrate the BM and drusen cores. With these premises, and based on evidence from the literature indicating that AMD patients have a higher frequency of circulating antibodies directed against antigens not only of Ret, but also of BM/Ch and RPE origin than in controls, we hypothesize that auto-antibodies (Auto-Abs) are biomarkers relevant to ocular disease status and that they will be measurable in the serum of AMD patients. To test the basic tenet of this hypothesis, the Aim of this proposal is to analyze serum samples collected from participants with (n=188) and without (n=222) AMD. Of these, 108 of the AMD subjects and the 222 unaffected individuals (mean age: 79 years old) are participants in the ARMA Study, a study ancillary to Health ABC conducted by the P.I. during his K23 Award. Additional 80 subjects with advanced AMD will be recruited during this award. Serum samples collected at the time of examination will be screened for the presence of auto-Abs directed against macula-specific Ret, RPE, and BM/Ch homogenates generated from fresh tissues dissected from human donors eyes. Homogenate proteins reacting against serum auto-Abs will be immunoprecipitated and separated on 2D gels. The identity of the most common gel spots observed uniquely and/or more intensely in AMD samples will be preliminarily characterized by mass spectrometry. Based on our overall rationale, we predict that sera from AMD patients will show higher frequency of auto-Abs than control subjects not only against Ret, but also and especially against RPE and/or BM/Ch antigens. If our hypothesis will prove correct, in a subsequent R01 application we plan to test the additional hypotheses that (1) within the group of patients with AMD, anti-Ret, anti-RPE, and/or anti-BM/Ch auto-Abs will be seen more often in subjects harboring disease-predisposing genetic variants, and that (2) patients with advanced AMD will have a higher frequency of auto-Abs than patients with early-stage AMD. We further plan to (3) characterize in detail all the identified auto-antigens towards which the auto-Abs are directed by mass spectrometry and (4) localize their expression at the tissue level by conducting immunohistochemistry experiments on macular section from human donor eyes. We predict that, ultimately, this line of research will provide us with useful biomarkers to elucidate further at the tissue level the pathways involved in AMD, and to identify potential prognostic and therapeutic targets.