Endothelial-reactive antibodies: a diagnostic test for Susac's syndrome

描述（由申请人提供）：苏萨克综合征（SS）是一种以脑病、视网膜分支动脉闭塞（BRAO）和听力损失为临床特征的神经-眼科疾病。它似乎是由影响大脑、视网膜和内耳的毛细血管前小动脉的微血管病变引起的。虽然磁共振成像（MRI）一致显示胼胝体中明显的白质紊乱，但MRI在大脑其他区域通常检测到的病变可能导致误诊为多发性硬化症（MS）。进一步使明确诊断复杂化的事实是，该疾病的三个组成部分很少同时发生。尽管自这种疾病被发现以来已有30多年的观察，但对其病因的了解却几乎没有进展。然而，用免疫抑制剂治疗SS的明显成功（但有差异）意味着这种疾病有免疫学基础。本提案中描述的实验，基于我们在10名SS患者血清中发现的3种内皮反应性抗体的一致模式，旨在测试SS患者携带循环内皮反应性自身抗体的假设，并且这些抗体的蛋白质靶点，一旦确定，可以用于开发明确的诊断测试。我们目前获得了40多名SS确诊患者的去鉴定血清样本，这些患者在采集血清时出现症状。我们将测试这些血清（以及MS患者和健康人的血清）是否存在内皮反应性抗体，方法是用培养的内皮细胞（EC）进行免疫荧光染色，将这些血清作为“一抗”。这些抗体的靶蛋白分子量将通过EC蛋白提取物的western blot测定，同样使用血清作为“一抗”。蛋白靶点将在2d凝胶上通过质谱鉴定EC蛋白提取物，并通过患者血清免疫印迹定位反应蛋白。作为一种原型诊断测试，这些蛋白（和选定的非反应性对照蛋白）将固定在微滴板的单个孔中，反应性抗体将通过比色ELISA在患者血清中定量。特异性将通过检测确诊SS患者和其他神经系统疾病患者（特别是多发性硬化症）以及健康个体的血清来评估。

DESCRIPTION (provided by applicant): Susac's syndrome (SS) is a neuro-ophthalmic disorder characterized by a clinical triad of encephalopathy, branch retinal artery occlusion (BRAO), and hearing loss. It appears to result from microangiopathy affecting the precapillary arterioles of the brain, retina, and inner ear. While magnetic resonance imaging (MRI) consistently shows a distinctive white matter disturbance in the corpus callosum, lesions commonly detected by MRI in other regions of the brain may lead to misdiagnosis as multiple sclerosis (MS). Further complicating definitive diagnosis is the fact that the three components of the disorder rarely occur simultaneously. Despite over 30 years of observation since the identification of this disorder, there has been little progress in the understanding of its etiology. However, the apparent (but variably) successful treatment of SS with immunosuppressive agents implies an immunologic basis for this disorder. The experiments described in this proposal, based upon our findings of a consistent pattern of 3 endothelial-reactive antibodies in sera of 10 SS patients, are designed to test the hypotheses that SS patients harbor circulating endothelial-reactive autoantibodies, and that the protein targets of these antibodies, once identified, can be exploited for the development of a definitive diagnostic test. We currently have access to de-identified serum samples from over 40 confirmed SS patients, symptomatic at the time of serum acquisition. We will test these sera (as well as sera of MS patients and of healthy individuals) for the presence of endothelial-reactive antibodies by immunofluorescent staining of cultured endothelial cells (EC) using the sera as the "primary antibody". Molecular weights of the protein targets of these antibodies will be determined by western blot of EC protein extracts, again using the sera as the "primary antibody". Protein targets will then be identified by mass spectrometry following resolution of EC protein extracts on 2-D gels and localization of reactive proteins by immunoblot with patient sera. As a prototype diagnostic test, these proteins (and selected non-reactive control proteins) will be immobilized in individual wells of a microtiter plate and reactive antibodies will be quantitated in patient sera by colorimetric ELISA. Specificity will evaluated by testing sera of confirmed SS patients and of patients with other neurological disorders (particularly multiple sclerosis), as well as of healthy individuals. PUBLIC HEALTH RELEVANCE: Susac's syndrome is a disorder characterized by visual defects, hearing loss, and a variety of brain dysfunctions (headaches, memory loss, personality changes, dementia). In the absence of a diagnostic test the disease is often misdiagnosed. The goal of this project is the development of such a test, which would facilitate prompt treatment and thus prevent or minimize residual defects associated with the disease.