Defining Antigenic Targets of Autoimmune Sensorineural Hearing Loss

描述(由申请人提供)：自身免疫性听力损失(AHL)是一种知之甚少的疾病，可导致严重和永久性的听力损失。AHL的内耳损害可以是抗体介导的，也可以是细胞介导的。我们关注的是抗体介导的听力损失，其中内耳靶点是一种多跨膜蛋白，称为胆碱转运蛋白2(CTL2)或溶质载体蛋白44A2(SLC44A2)(以下简称CTL2)。CTL2主要表达在Corti器官内的支持细胞上。在体内，内耳中结合这种蛋白的抗体会伴随着内耳毛细胞的丧失和实验动物的听力损失。因此，CTL2具有被抗体破坏的必要功能。临床AHL患者的抗体与内耳支持细胞结合的模式与CTL2抗体相同，对皮质类固醇治疗的反应率是对照组的三倍。在初步研究中，这些患者的抗体还与蛋白质印迹法上的重组人(Rhu)CTL2蛋白结合。本研究的总体目标是：1.探讨抗CTL2抗体与AHL的关系，建立一种灵敏、高通量的AHL检测方法，以确定该检测方法对自身免疫性CTL2抗体介导的听力损失的诊断和治疗是否有价值。2.利用已证实的多种SLC家族成员转运蛋白功能的体外实验方法，确定CTL2的功能，并推断该功能与内耳稳态、听力和平衡的关系。3.利用条件性CTL2基因敲除的转基因小鼠，研究CTL2基因在内耳发育和功能中的作用。为了实现这些目标，我们提出如下建议：在目标1中，我们将开发一种以不同构象呈现rHuCTL2的酶联免疫吸附试验，从而展示所有高抗原性表位。患者和rHuCTL2免疫小鼠的血清将进行测试，以确定是否存在单一的免疫优势表位，或者是否必须显示所有主要的高抗原域，以检测来自不同个体的抗CTL2抗体。利用这些信息，我们将开发一种高通量的ELISA法，可用于检测大量来自患者和对照组的血清。我们希望了解AHL患者中有多大比例的CTL2抗体，以及对照组中有多大比例的抗体。我们还将确定对照组和患者的抗体效价是否不同，以及是否存在与听力问题发展相关的特定表位抗体的偏好。CTL2是转运蛋白家族的成员，但其功能尚不清楚。在目标2中，我们将确定CTL2的转运蛋白功能。我们还将使用从转运蛋白功能实验中开发的数据来确定针对各种CTL2表位的抗体是否会在体外阻断CTL2的功能。在目标3中，我们将研究CTL2缺陷转基因小鼠的内耳发育、听力和平衡。

DESCRIPTION (provided by applicant): Autoimmune hearing loss (AHL) is a poorly understood disorder that can lead to profound and permanent hearing loss. Inner ear damage in AHL can be antibody mediated or cell mediated. We have focused on antibody mediated hearing loss in which the inner ear target is a multi-transmembrane protein called choline transporter-like protein 2 (CTL2) or solute carrier protein 44A2 (SLC44A2)(hereafter referred to as CTL2). CTL2 is prominently expressed on supporting cells within the organ of Corti. Binding of antibodies to this protein in the inner ear in vivo is accompanied by loss of inner ear hair cells and hearing loss in experimental animals. Thus, CTL2 has a necessary function that is disrupted by antibodies. Patients with clinical AHL who have antibodies that bind to inner ear supporting cells with the same pattern as CTL2 antibodies are three times more likely to respond to corticosteroid treatment. In preliminary studies antibodies from these patients also bind to recombinant human (rHu) CTL2 protein on western blots. The overall objectives of this study are: 1. To investigate the association of anti-CTL2 antibodies with AHL and to develop a sensitive and high through-put assay with a secondary objective to determine if the assay has value to diagnose and manage treatment of autoimmune CTL2 antibody mediated hearing loss. 2. To determine the function of CTL2 using proven in vitro approaches that have demonstrated the transporter function of numerous SLC family members and to deduce how this function is related to inner ear homeostasis, hearing and balance. 3. To investigate the in vivo role of CTL2 in inner ear development and function in a conditional CTL2 knock out in transgenic mice. To accomplish these objectives we propose the following: In Aim 1 we will develop an ELISA assay that will present rHuCTL2 in different conformations so that all of the highly antigenic epitopes will be displayed. Sera from patients and from rHuCTL2 immunized mice will be tested to determine if there is a single immunodominant epitope or if all of the major highly antigenic domains must be displayed to detect anti-CTL2 antibodies from different individuals. Using this information we will develop a high through-put ELISA that can be used to test large numbers of sera from patients and from control individuals. We expect to learn what proportion of AHL patients and what proportion of control individuals have antibodies to CTL2. We will also determine if controls and patients differ in the titer of antibody and if there is a predilection for antibodies to a particular epitope that is associated with development of hearing problems. CTL2 is a member of the transporter family, but its function is not known. In Aim 2 we will determine the transporter function of CTL2. We will also use the data developed from the transporter function experiments to determine if antibodies to various CTL2 epitopes will block CTL2 function in vitro. In Aim 3 we will study inner ear development, hearing, and balance in transgenic mice with defective CTL2.