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

描述(申请人提供)：老年性黄斑变性(AMD)的特征是在Bruchs膜(BM)和视网膜色素上皮(RPE)之间形成沉积，称为玻璃体。近年来，炎症在AMD的发病机制中的作用已经在生物学、实验和遗传水平上得到证实。玻璃体含有大量的炎症效应物质和副产物，来自脉络膜的树突状细胞(DC)是眼部常驻的抗原提呈细胞之一，它们被募集到并渗透到骨髓和玻璃体核心。在这些前提下，根据文献中的证据表明AMD患者不仅针对Ret抗原，而且针对BM/CH和RPE来源的循环抗体的频率都高于对照组，我们假设自身抗体(Auto-Abbs)是与眼病状态相关的生物标记物，并且它们将在AMD患者的血清中被检测到。为了验证这一假说的基本原理，这项建议的目的是分析从患有AMD(n=188)和不患有AMD(n=222)的参与者那里收集的血清样本。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ARMA研究的参与者，这是一项附属于Health ABC的研究，由P.I.在他的K23奖期间进行。在此奖项期间，还将招募80名患有高级AMD的受试者。在检查时收集的血清样本将被筛选，以确定是否存在针对黄斑特异性Ret、RPE和BM/CH匀浆的自动抗体，这些匀浆来自于从人类供体眼睛解剖的新鲜组织。与血清AUTO-Abs反应的匀浆蛋白将被免疫沉淀并在2D凝胶上分离。在AMD样品中唯一和/或更强烈地观察到的最常见的凝胶点的身份将通过质谱学进行初步表征。根据我们的总体原理，我们预测AMD患者的血清不仅显示出更高的自身抗体频率，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R01应用中，我们计划测试额外的假设：(1)在AMD患者组中，抗-Ret、抗-RPE和/或抗BM/CH自动抗体将更常见于携带疾病易感基因变异的受试者，以及(2)晚期AMD患者比早期AMD患者具有更高的自动抗体频率。我们进一步计划(3)通过质谱学对所有已识别的自动抗体所针对的自身抗原进行详细的表征，以及(4)通过对人供体眼的黄斑切片进行免疫组织化学实验来定位其在组织水平的表达。我们预测，最终，这一系列研究将为我们提供有用的生物标志物，在组织水平上进一步阐明AMD涉及的途径，并确定潜在的预后和治疗靶点。

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.