Mechanisms of Pain and Immune Processes

概述：慢性神经性疼痛可影响身体的任何部位，包括口腔和面神经。神经性疼痛可由各种损伤、感染、自身免疫性疾病（如Sjogrens综合征）或代谢性疾病（如糖尿病神经病）引起。我们正在测试这样一种假设，即在一些患者中，慢性疼痛是由与周围神经中产生的针对蛋白质的自身抗体相关的免疫病理过程维持的。自身抗体在某些大纤维周围神经病变中是已知的罪魁祸首。如果疼痛是一个组成部分，我们假设存在针对神经末梢中发现的蛋白质的自身抗体，这些蛋白质来自小直径的痛觉（伤害性）c纤维或a - δ神经纤维。为了支持这一观点，有报道称，大约30%的SjS患者表现为小纤维神经病变，在上肢和下肢产生疼痛的感觉异常。类似的神经性疼痛在II型糖尿病和接受某些化疗药物治疗的癌症患者中显著发生。为了验证疼痛的神经性疾病有自身免疫成分的假设，我们建立了一种灵敏、定量的液相发光试验，使用在哺乳动物细胞中表达的重组抗原示踪剂，以测量血清、唾液或其他体液中抗体的存在。这个转化研究项目解决了伤害性传递的分子和病理生理过程，以及研究人类患者慢性疼痛状况的新方法。我们的目标是了解(1)人类慢性疼痛疾病的分子和细胞生物学机制，以及(2)利用这些知识设计新的疼痛疾病治疗和诊断方法。

Overview: Chronic neuropathic pain can affect any part of the body, including the oral cavity and facial nerves. Neuropathic pain can occur due to a variety of insults, infections, autoimmune disorders such as Sjogrens Syndrome, or metabolic disorders such as diabetes (diabetic neuropathy). We are testing the hypothesis that, in some patients, chronic pain is maintained by immunopathological processes related to autoantibodies generated against proteins in peripheral nerve. Autoantibodies are known culprits in certain large fiber peripheral neuropathies. Where pain is a component, we hypothesize the presence of autoantibodies to proteins found in nerve endings arising from small diameter, pain-sensing (nociceptive) C-fiber or A-delta nerve fibers. In support of this idea, it has been reported that approximately 30% of Sjogrens syndrome (SjS) patients exhibit a small fiber neuropathy that produces painful paresthesias in the upper and lower extremities. Similar neuropathic pain occurs prominently in Type II diabetes and in cancer patients treated with certain chemotherapeutic agents. To test the hypothesis that painful neuropathic conditions have an autoimmune component we established, a sensitive, quantitative, liquid phase luminescence assay, that uses recombinant antigen tracers expressed in mammalian cells, in order to measure the presence of antibodies in serum, saliva, or other body fluids. This translational research program addresses molecular and pathophysiological processes of nociceptive transmission and new ways to investigate chronic pain conditions in human patients. Our goals are to understand (1) the molecular and cell biological mechanisms underlying human chronic pain disorders, and (2) to use this knowledge to devise new treatments and diagnostics for pain disorders. In order to obtain sufficient throughput to examine large cohorts of normals and patients for multiple candidate antigens, we adapted the assay from a single tube format to a 96 well microtiter plates operating on our robotic pipeting platform. We also formed collaborations and assembled cohorts of different patient populations to establish baseline values in autoimmune disorders, infectious diseases and chronic pain and nervous system disorders. We have examined a known central nervous system autoimmune disorder called Stiff Person Syndrome. These patients have high titer autoantibodies to the enzyme glutamic acid decarboxylase (GAD65), which catalyzes the formation of the inhibitory neurotransmitter gamma-aminobutyric acid. We tested the major antigens (IA2, IA2b and GAD65) in Type 1 diabetes, which is the juvenile autoimmune form. These studies demonstrated that the non-radioactive luciferase immunoprecipitation assay is superior to the gold-standard radioactive assay in terms of sensitivity and specificity. We also performed an extensive analysis of autoantigens in Sjogrens Syndrome (SjS) patients (Ro52, Ro60 and La, and about 7 other antigens). We discovered two new antigens in sub-populations of SjS. One was against a nervous system protein, and another was against a gastric parietal cell protein. Over the past year we have extended the SjS study to include a comparison of salivary antibody levels to those in serum. Using only 5 microliters, the LIPS assay readily detected the major SjS autoantigens in saliva, yielding the same sensitivity and specificity as in serum. These results highlight the feasibility of establishing non-invasive, saliva based assays for many types of human diseases and for monitoring of vaccine immune status for large populations of people. In many neural autoimmune disorders the major autoantigens are frequently plasma membrane receptors or ion channels. To establish the basic parameters of receptor-based autoimmune disorders, we initiated a study on Myasthenia Gravis patients. This is a neurological autoimmune disorder against a membrane-bound, ligand-gated ion channel, the muscle nicotinic receptor (AChR). We established collaboration with the Myasthenia group at Johns Hopkins and demonstrated that LIPS detected autoantibodies that reacted with specific truncation mutants suggesting antigenic presentation of the extracellular domain in depends on intracellular folding of the second intracellular loop. These data provide a heuristic template for further studies of the AChR. We are currently working on several additional inter-institute and inter-institutional collaborations (such as the above Myasthenia study) to obtain well-characterized patients with Complex Regional Pain Syndrome (CRPS, a neuropathic pain disorder), other neuropathies, and other CNS and PNS disorders and infectious diseases that have neurological manifestations. We are working closely with groups at Rochester and Hopkins on SjS and salivary diagnostics and SjS patients with neurological symptoms, respectively. We have begun the analysis of neuropathic pain patients with CRPS using samples obtained from Rush University. We also are using this assay to explore the interrelationships between HIV, the virus causing Kaposis sarcoma and HIV-associated malignancies and painful peripheral neuropathies. The latter studies formed the basis of a Bench-to-Bedside award from the NIH Clinical Center. One of the most compelling aspects of this project is the progressive layering and evolution of the data set. As we increase the number of test antigens and assay across conditions and diseases, we assemble a comprehensive assessment of autoimmune responses. This is accomplished by determination of (a) the extent and specificity of immune response to orthologous proteins and protein fragments, (b) overlap in antigen profiles indicative of a common denominator or general mechanism, and (d) antigenicity within entire signaling pathways involved in inter- or intracellular communication. As time progresses, full multiple antigen profiling can be implemented to obtain a new level of understanding of many complex human disease states. In order to meet the increased demands of such broad spectrum autoimmunome profiling and take advantage of the layering, we are in the process of scaling up the throughput of the assay from 96 well plates to 384 well plates and then to 1536 well plates in collaboration with the NIH Chemical Genomics Center and the TRND group. This will conserve serum and allow us to examine individuals in much greater depth than what is presently possible.