Mechanisms of Pain and Immune Processes

概述：这项转译研究计划致力于研究伤害性信息传递的分子和病理生理学过程，以及研究慢性疼痛、自身免疫疾病、传染病及其在人类患者中的交叉点的新方法。慢性神经性疼痛可影响身体的任何部位，可由各种侮辱、感染和自身免疫或代谢紊乱(如糖尿病周围神经病变)引起。我们正在验证一种假设，即在一些患者中，慢性疼痛是通过与周围神经或雪旺细胞中产生的针对蛋白质的自身抗体相关的免疫病理过程来维持的。已知自身抗体是某些大纤维周围神经病的罪魁祸首，如副肿瘤性疾病。在疼痛是一个组成部分的情况下，我们假设存在对小直径痛觉(伤害性)C-或A-三角洲神经纤维产生的神经末梢中的蛋白质的自身抗体。为了验证疼痛神经病变与自身免疫成分有关的假设，我们建立了一种灵敏的、定量的液相发光检测方法，该方法使用重组蛋白抗原-荧光素酶融合蛋白作为示踪剂，这些融合蛋白在哺乳动物细胞中表达。这种分析方法被称为荧光素酶免疫沉淀系统(LIPS)，我们有多篇发表的论文表明，LIPS可以强健而灵敏地检测血清、血浆、脑脊液或唾液中的抗体。这项研究的目标是了解(A)人类慢性疼痛障碍的分子和细胞生物学机制，以及(B)利用这一知识设计新的疼痛障碍的治疗和诊断方法。 在这个项目中，探索了一种自体免疫假说。我们建立的检测方法具有很强的通用性，因为它的敏感性、模块化，以及使用重组DNA方法来产生蛋白质抗原示踪剂。因此，早期的调查评估了抗体在其中发挥作用的一系列疾病和障碍，包括有或没有神经系统受累的传染病、各种自身免疫性疾病，这些疾病可能在患者的亚群中出现神经系统症状，以及其他类型的筛查和方法变异。在一项研究中，我们从几名神经性疼痛患者的血清中鉴定了干扰素γ的抗体，然后产生了一组更大的抗细胞因子抗体探针，然而，这些其他细胞因子的抗体在神经病患者中并不广泛存在。将该小组的测试扩展到另一个患者群体显示，抗干扰素伽马抗体在机会性感染中发挥作用，这项合作研究工作在某些患者群体中定义了一种新的疾病机制。在许多神经自身免疫性疾病中，主要的自身抗原通常是质膜受体或离子通道。根据已发表的报告，我们已经为神经性疼痛障碍生成了几个这样的探针，特别是β-肾上腺素能受体和钾通道，目前正在进行测试，以确定嘴唇试验是否可以在适当的个人中检测到这种蛋白的抗体。我们目前正在研究一种PNS传染病疾病，带状疱疹或带状疱疹，这种疾病可能演变为一种痛苦的神经表现，称为带状疱疹后神经痛(PHN)。我们检测了一些中和抗细胞因子自身抗体的患者，有趣的是，所有这些患者都有PHN。这表明，一些PHN患者可能需要额外的干预来控制这种疾病，而不仅仅是止痛药。 该项目最引人注目的方面之一是数据集及其用途的渐进式分层和演变。随着我们增加测试抗原的数量，以及跨条件和跨疾病的检测，我们汇集了对免疫和自身免疫反应的全面评估。这是通过确定(A)对同源蛋白质和蛋白质片段的免疫反应的程度和特异性，(B)表明共同点或一般机制的抗原图谱的重叠，以及(C)参与细胞间或细胞内通讯的整个信号通路中的抗原性来实现的。一个例子是干燥综合征中TRIM蛋白家族的抗原性。另一个例子是基于自身抗原的狼疮红斑狼疮患者的新分类，我们通过多个自身抗原图谱进行了演示。公共卫生监测的新用途的一个例子是我们早先展示的唾液中自身抗体的测量，这突出了为许多类型的人类疾病建立基于唾液的非侵入性检测的可行性。基于唾液的监测可用于确定大量或脆弱人群或儿科人群的疫苗免疫状态。最终，可以实施完整的多种抗原分析，以获得对许多复杂的人类疾病状态的更深层次的了解。

Overview: This translational research program addresses molecular and pathophysiological processes of nociceptive transmission and new ways to investigate chronic pain, autoimmune conditions, infectious diseases and their intersection in human patients. Chronic neuropathic pain can affect any part of the body and can occur due to a variety of insults, infections, and autoimmune or metabolic disorders (e.g., diabetic peripheral 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 or Schwann cells. Autoantibodies are known culprits in certain large fiber peripheral neuropathies such as in paraneoplastic disorders. 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- or A-delta nerve fibers. To test the hypothesis that painful neuropathic conditions have an autoimmune component we established a sensitive, quantitative, liquid phase luminescence assay that uses recombinant protein antigen-luciferase fusion proteins as tracers, which are expressed in mammalian cells. This assay is called luciferase immunoprecipitation systems (LIPS) and we have multiple published papers showing that LIPS can robustly and sensitively measure antibodies in serum, plasma, cerebrospinal fluid or saliva. The goals of this research are to understand (a) the molecular and cell biological mechanisms underlying human chronic pain disorders, and (b) to use this knowledge to devise new treatments and diagnostics for pain disorders. In this project an autoimmune hypothesis is explored. The assay methodology we established has great versatility due to its sensitivity, modularity, and use of recombinant DNA methodology to generate protein antigen tracers. As a result earlier investigations evaluated a range of diseases and disorders that antibodies play a role in including infectious diseases, with and without nervous system involvement, various autoimmune disorders, which may have nervous system symptomology in subsets of patients and other types of screening and methodological variants. In one study, we identified antibodies to interferon gamma in serum from several neuropathic pain patients and then generated a larger panel of anti-cytokine antibody probes, however, antibodies to these other cytokines were not widely represented in the neuropathy patients. Extending testing of the panel to another patient population revealed that anti-interferon gamma antibodies play a role opportunistic infections and this collaborative research work defined a new mechanism of disease in certain patient populations. In many neural autoimmune disorders the major autoantigens are frequently plasma membrane receptors or ion channels. We have generated several such probes for neuropathic pain disorders, notably the beta-adrenergic receptor and a potassium channel based on published reports, and are currently testing to determine if the LIPS assay can detect antibodies to this protein in appropriate individuals. We are currently working a PNS infectious disease disorder, shingles or herpes zoster, that can evolve into a painful neurological manifestation called post-herpetic neuralgia (PHN). We detected some patients with neutralizing anti-cytokine autoantibodies and, interestingly, all of these had PHN. This suggests that some patients with PHN may require additional intervention to control the disorder other than just pain medications. One of the most compelling aspects of this project is the progressive layering and evolution of the datasets and their uses. As we increase the number of test antigens, and assay across conditions and diseases, we assemble comprehensive evaluations of immune and 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 common denominators or general mechanisms, and (c) antigenicity within an entire signaling pathway involved in inter- or intracellular communication. An example is the antigenicity of the TRIM family of proteins in Sjogren's Syndrome. Another example is a new autoantigen-based classification of patients with Lupus Erythematosis that we demonstrated by multiple autoantigen profiling. An example of a new use for public health monitoring is our earlier demonstration of autoantibody measurements in saliva, which highlights the feasibility of establishing non-invasive, saliva-based assays for many types of human diseases. Saliva-based monitoring could be applied to determinations of vaccine immune status for large or vulnerable populations of people or the pediatric population. Eventually, full multiple antigen profiling can be implemented to obtain a deeper level of understanding of many complex human disease states.