Defining Antigenic Targets of Autoimmune Sensorineural Hearing Loss

定义自身免疫性感音神经性听力损失的抗原靶标

• 批准号: 8513294
• 负责人: THOMAS E. CAREY
• 金额: $ 43.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513294
• 关键词: Adrenal Cortex Hormones; Affect; Animals; Antibodies; Autoantibodies; Autoimmune Process; Binding; Biological Assay; CCL21 gene; Carrier Proteins; Cells; Clinical; Data; Development; Diagnosis; Diagnostic tests; Disease; Ear; Enzyme-Linked Immunosorbent Assay; Epitopes; Equilibrium; Family; Family member; Hair Cells; Hearing; Hearing problem; Homeostasis; Human; Immunodominant Epitopes; In Vitro; Individual; Integral Membrane Protein; Knock-out; Knockout Mice; Knowledge; Labyrinth; Lead; Learning; Mammalian Cell; Mediating; Molecular Conformation; Mus; Organ of Corti; Patients; Pattern; Persons; Phage Display; Phenotype; Phospholipids; Process; Property; Proteins; Recombinants; Role; Sensorineural Hearing Loss; Serum; Supporting Cell; Testing; Time; Transgenic Mice; Western Blotting; Xenopus oocyte; base; cDNA Library; choline transporter; design; hearing impairment; in vivo; inner ear diseases; member; novel strategies; public health relevance; research study; solute

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.

描述（由申请人提供）：自身免疫性听力损失（阿勒）是一种知之甚少的疾病，可导致严重和永久性听力损失。阿勒的内耳损伤可以是抗体介导的或细胞介导的。我们关注的是抗体介导的听力损失，其中内耳靶点是称为胆碱转运蛋白样蛋白2（CTL 2）或溶质载体蛋白44 A2（SLC 44 A2）（下文称为CTL 2）的多跨膜蛋白。CTL 2在Corti器官内的支持细胞上显著表达。抗体在体内内耳中与这种蛋白质的结合伴随着实验动物内耳毛细胞的损失和听力损失。因此，CTL 2具有被抗体破坏的必要功能。具有与内耳支持细胞结合的抗体的临床阿勒患者与CTL 2抗体具有相同的模式，其对皮质类固醇治疗的反应可能性高三倍。在初步研究中，来自这些患者的抗体在蛋白质印迹上也与重组人（rHu）CTL 2蛋白结合。本研究的总体目标是：1.研究抗CTL 2抗体与阿勒的相关性，并开发一种灵敏、高通量的检测方法，次要目的是确定该检测方法是否具有诊断和管理自身免疫性CTL 2抗体介导的听力损失治疗的价值。2.使用已证实的体外方法（已证实许多SLC家族成员的转运蛋白功能）确定CTL 2的功能，并推断该功能如何与内耳稳态、听力和平衡相关。3.目的：在条件性CTL 2基因敲除的转基因小鼠中，研究CTL 2在内耳发育和功能中的体内作用。为了实现这些目标，我们提出了以下建议：在目标1中，我们将开发一种ELISA测定法，该测定法将以不同的构象呈现rHuCTL 2，以便展示所有的高抗原性表位。将测试来自患者和来自rHuCTL 2免疫小鼠的血清，以确定是否存在单个免疫显性表位或是否必须展示所有主要的高抗原性结构域以检测来自不同个体的抗-CTL 2抗体。利用这些信息，我们将开发一种高通量的ELISA，可用于测试大量的患者和对照个体的血清。我们希望了解阿勒患者和对照组中有多少比例的人具有CTL 2抗体。我们还将确定对照组和患者的抗体滴度是否不同，以及是否存在对与听力问题发展相关的特定表位的抗体偏好。CTL 2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CTL 2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各种CTL 2表位的抗体是否会在体外阻断CTL 2功能。在目标3中，我们将研究内耳发育，听力和平衡的转基因小鼠与缺陷的CTL 2。