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

描述（由申请人提供）：Susac综合征（SS）是一种神经眼科疾病，其特征为脑病、视网膜分支动脉阻塞（BRAO）和听力丧失的临床三联征。它似乎是由影响脑、视网膜和内耳毛细血管前小动脉的微血管病引起的。虽然磁共振成像（MRI）始终显示胼胝体中有一个独特的白色物质紊乱，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）。进一步复杂的明确诊断是，这三个组成部分的疾病很少同时发生。尽管自发现这种疾病以来已有30多年的观察，但对其病因的理解进展甚微。然而，用免疫抑制剂治疗SS明显（但不成功），这意味着这种疾病的免疫学基础。本提案中描述的实验，基于我们在10例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，以及这些抗体的蛋白质靶点，一旦确定，可用于开发确定性诊断试验。我们目前可以获得来自40多名确诊SS患者的去识别血清样本，这些患者在血清采集时有症状。我们将测试这些血清（以及MS患者和健康个体的血清）的内皮反应性抗体的存在下，通过免疫荧光染色培养的内皮细胞（EC）使用的血清作为“一抗”。这些抗体的蛋白质靶标的分子量将通过EC蛋白质提取物的蛋白质印迹来确定，再次使用血清作为“一抗”。然后，在2-D凝胶上分离EC蛋白提取物并通过患者血清免疫印迹定位反应性蛋白后，通过质谱法鉴定蛋白质靶点。作为原型诊断试验，将这些蛋白质（和选定的非反应性对照蛋白质）固定在微量滴定板的单个威尔斯孔中，并通过比色ELISA定量患者血清中的反应性抗体。特异性将通过检测确诊SS患者和其他神经系统疾病（特别是多发性硬化症）患者以及健康个体的血清进行评价。 公共卫生相关性：苏萨克综合征是一种以视觉缺陷、听力损失和各种脑功能障碍（头痛、记忆力丧失、人格改变、痴呆）为特征的疾病。在缺乏诊断测试的情况下，这种疾病经常被误诊。该项目的目标是开发这样一种测试，这将有助于及时治疗，从而防止或最大限度地减少与疾病相关的残留缺陷。

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.