Mechanisms of Pain and Immune Processes

总结 概述：这个转化研究计划解决了与神经性疼痛相关的病理生理过程，以及慢性疼痛，自身免疫，传染病及其在人类患者中的交叉点的潜在融合。慢性神经性疼痛可影响身体的任何部位，并且可由于各种损伤、感染、自身免疫或代谢紊乱（例如，糖尿病周围神经病变）。我们正在测试的假设，在一些患者中，慢性疼痛是启动和/或维持免疫病理过程相关的自身抗体产生的蛋白质在外周神经或雪旺细胞或可能的中枢神经系统的组成部分。已知自身抗体是某些大纤维副肿瘤性周围神经病变的罪魁祸首，大纤维也可导致神经性感觉迟钝。疼痛是一个组成部分，我们假设存在的自身抗体的蛋白质中发现的神经末梢产生的小直径疼痛感应（伤害性）C-或A-δ神经纤维。为了检验疼痛性神经病性病症具有自身免疫组分的假设，我们建立了一种灵敏的、定量的、液相发光测定法，该测定法使用重组蛋白抗原-荧光素酶融合物作为在哺乳动物细胞中表达后获得的示踪剂。本研究的目标是了解（a）潜在的人类慢性疼痛疾病的分子和细胞生物学机制，以及（B）使用这些知识来设计新的治疗和诊断疼痛和其他疾病，它可以适应。 我们建立的方法，荧光素酶免疫沉淀系统（LIPS）测定，稳健和灵敏地检测血清，血浆，脑脊液，或唾液中的抗体。我们的早期研究评估了抗体发挥作用的一系列疾病和病症，包括有和没有神经系统受累的病毒和细菌感染，以及在患者亚群中具有神经系统病理学的自身免疫性疾病。在许多神经自身免疫性病症中，主要的自身抗原通常是质膜受体或离子通道（Burbelo等人，2016）。我们的目的是确定LIPS检测是否可以检测到这些蛋白质的自身抗体在适当的慢性疼痛患者。例如，我们完成了一项关于带状疱疹（带状疱疹）和带状疱疹演变成一种称为疱疹后神经痛（PHN）的疼痛神经系统疾病的患者的研究。我们在带状疱疹患者的一个亚群中检测到一些中和性抗细胞因子自身抗体，有趣的是，所有有抗体的患者都有PHN。这一点很重要，因为它表明一些PHN患者可能需要额外的干预来控制这种疾病，而不仅仅是镇痛药物。在最近的出版物中，我们证明了HSV 1转录本和HSV 1抗体在死后的人三叉神经节和全血中，分别。滴度与转录本的符合率为100%。我们使用RNA-seq的事实使我们能够将HSV 1读段与HSV 1基因组进行比对，并且所有转录本与HSV 1潜伏相关转录本（LAT）进行比对，这表明没有受试者在死亡时有病毒再激活。我们可以将这种方法作为头痛和偏头痛的更大研究的一个组成部分。 该项目最引人注目的方面之一是其广泛的适用性以及数据集的逐步分层和演变。随着我们增加测试抗原的数量，并对各种病症和疾病进行检测，我们对免疫和自身免疫反应进行了全面的评估。这是通过确定（a）对正向同源蛋白和蛋白片段的免疫应答的程度和特异性，（B）指示共同的抗原或一般机制的抗原谱的重叠，和（c）参与胞间或胞内通讯的整个信号传导途径内的抗原性来实现的。最终，可以实施完整的多抗原分析，以更深入地了解许多复杂的人类疾病状态的免疫成分。我们最近的综述总结了我们目前对致病性自身抗体的看法。几乎所有自身抗体具有致病作用的自身免疫性疾病都显示抗体靶向分泌的肽或蛋白质或整合质膜蛋白的胞外结构域。后者通常是跨膜信号蛋白。我们现在可以专注于跨膜离子通道和受体的胞外结构域和酶在寻找抗原的目标。这一概念大大简化了对可能参与产生或维持慢性神经性疼痛病症的抗原的搜索。

Summary Overview: This translational research program addresses pathophysiological processes related to neuropathic pain and the potential confluence of chronic pain, autoimmunity, 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, autoimmune or metabolic disorders (e.g., diabetic peripheral neuropathy). We are testing the hypothesis that, in some patients, chronic pain is initiated and/or maintained by immunopathological processes related to autoantibodies generated against proteins in peripheral nerve or Schwann cells or possibly components of the central nervous system. Autoantibodies are known culprits in certain large fiber paraneoplastic peripheral neuropathies and large fibers can also contribute to neuropathic dysesthesias. 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 fusions as tracers obtained after expression in mammalian cells. The goals of this research are to understand (a) potential molecular and cell biological mechanisms underlying human chronic pain disorders, and (b) to use this knowledge to devise new treatments and diagnostics for pain and other disorders to which it can be adapted. The methodology we established, the luciferase immunoprecipitation systems (LIPS) assay, robustly and sensitively detects antibodies in serum, plasma, cerebrospinal fluid, or saliva. Our early investigations evaluated a range of diseases and disorders that antibodies play a role in, including viral and bacterial infections with and without nervous system involvement and autoimmune disorders that have nervous system symptomology in subsets of patients. In many neural autoimmune disorders, the major autoantigens are frequently plasma membrane receptors or ion channels (Burbelo et al., 2016). Our aim is to determine if the LIPS assay can detect auto-antibodies to these proteins in appropriate chronic pain patients. For example, we completed a study on shingles (herpes zoster) and patients in which shingles evolved into a painful neurological disorder called post-herpetic neuralgia (PHN). We detected some neutralizing anti-cytokine autoantibodies in a subpopulation of Zoster patients, and, interestingly, all of the patients with antibodies had PHN. This is important because it suggests that some patients with PHN may require additional intervention to control the disorder other than just analgesic medications. In a recent publication we demonstrated both HSV1 transcripts and HSV1 antibodies in post-mortem human trigeminal ganglion and whole blood, respectively. The correspondence between titer and transcript was 100%. The fact that we used RNA-seq allowed us to align the HSV1 reads to the HSV1 genome and all of the transcripts aligned with the HSV1 latency associated transcript (LAT), indicating that none of the subjects had viral re-activation at the time of death. We can use this approach as a component in a larger study of headache and migraine. One of the most compelling aspects of this project is its broad applicability and the progressive layering and evolution of the datasets. 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. Eventually, full multiple antigen profiling can be implemented to obtain a deeper level of understanding of the immune component of many complex human disease states. Our recent review summarizes our current thinking on pathogenic autoantibodies. Nearly all autoimmune disorders for which the autoantibody has a pathogenic action show that the antibodies target either a secreted peptide or protein or the extracellular domain of an integral plasma membrane protein. The latter are usually transmembrane signaling proteins. We can now focus on the extracellular domains of transmembrane ion channels and receptors and enzymes in the search for antigenic targets. This concept greatly simplifies the search for antigens that may participate in generating or sustaining a chronic neuropathic pain condition.