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听力损失。AHL的内耳损伤可由抗体介导或细胞介导。我们专注于抗体介导的听力损失，其中内耳靶标是一种称为胆碱转运蛋白样蛋白2 （CTL2）或溶质载体蛋白44A2 (SLC44A2)（以下简称CTL2）的多跨膜蛋白。CTL2在Corti器官内的支持细胞上显著表达。体内内耳与该蛋白结合的抗体伴随着内耳毛细胞的丧失和实验动物的听力丧失。因此，CTL2具有被抗体破坏的必要功能。具有与CTL2抗体模式相同的内耳支持细胞结合的抗体的临床AHL患者对皮质类固醇治疗的反应可能性是其三倍。在初步研究中，这些患者的抗体在western blots上也能与重组人（rHu） CTL2蛋白结合。本研究的总体目标是：1。研究抗CTL2抗体与AHL的关系，并开发一种灵敏、高通量的测定方法，其次要目的是确定该方法是否具有诊断和管理自身免疫性CTL2抗体介导的听力损失治疗的价值。2. 利用已证实的体外方法确定CTL2的功能，这些方法已经证明了许多SLC家族成员的转运蛋白功能，并推断该功能与内耳稳态、听力和平衡的关系。3. 目的：探讨CTL2在转基因小鼠内耳发育中的作用及其在条件CTL2敲除中的功能。为了实现这些目标，我们提出以下建议：在目标1中，我们将开发一种ELISA检测，该检测将呈现不同构象的rHuCTL2，以便显示所有高抗原表位。将对患者和rHuCTL2免疫小鼠的血清进行检测，以确定是否存在单一的免疫显性表位，或者是否必须显示所有主要的高抗原结构域来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ELISA，可用于检测来自患者和对照个体的大量血清。我们希望了解AHL患者和对照个体中有CTL2抗体的比例。我们还将确定对照组和患者的抗体滴度是否不同，以及是否存在与听力问题发展相关的特定表位的抗体偏好。CTL2是转运蛋白家族的一员，但其功能尚不清楚。在目的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获得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功能。在Aim 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.