Mechanisms of Pain and Immune Processes

概述：这项翻译研究计划涉及神经性疼痛的病理生理过程，以及慢性疼痛、自身免疫、感染性疾病的潜在融合，以及它们在人类患者中的交叉。慢性神经性疼痛可影响身体的任何部位，可由于各种侮辱、感染、自身免疫或代谢紊乱(如糖尿病周围神经病变)而发生。我们正在验证一种假设，即在一些患者中，慢性疼痛是由与周围神经或雪旺细胞或可能的中枢神经系统组件中的蛋白质产生的自身抗体相关的免疫病理过程启动和/或维持的。已知自身抗体是某些大纤维副肿瘤性周围神经病的罪魁祸首，大纤维也可导致神经病理性感觉障碍。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。为了验证痛性神经病变具有自身免疫成分的假设，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-荧光素酶融合作为示踪剂，在哺乳动物细胞中表达后获得。这项研究的目标是了解(A)人类慢性疼痛障碍潜在的分子和细胞生物学机制，以及(B)利用这一知识为疼痛和其他疾病设计新的治疗和诊断方法，使其能够适应。 我们建立的方法学，即荧光素酶免疫沉淀系统(LIPS)试验，可以强健而灵敏地检测血清、血浆、脑脊液或唾液中的抗体。我们的早期调查评估了抗体在其中发挥作用的一系列疾病和障碍，包括病毒和细菌感染，以及在部分患者中具有神经系统症状的自身免疫性疾病。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(Burbelo等人，2016)。我们的目的是确定嘴唇试验是否可以在适当的慢性疼痛患者中检测到这些蛋白的自身抗体。例如，我们完成了一项关于带状疱疹(带状疱疹)和患者的研究，在这些患者中，带状疱疹演变为一种称为带状疱疹后神经痛(PHN)的痛苦的神经疾病。我们在带状疱疹患者的亚群中检测到了一些中和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，而不仅仅是止痛药。在最近的一篇文章中，我们分别在死后的人三叉神经节和全血中证明了HSV1转录本和HSV1抗体。效价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HSV1读数与HSV1基因组以及所有转录本与HSV1潜伏期相关转录本(LAT)进行比对，这表明在死亡时没有一名受试者有病毒重新激活。我们可以在更大规模的头痛和偏头痛研究中使用这种方法。 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。最终，可以实施完整的多种抗原分析，以获得对许多复杂人类疾病状态的免疫成分的更深层次的了解。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表明，抗体针对的不是分泌的多肽或蛋白质，就是完整的质膜蛋白的胞外区域。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、受体和酶的胞外区域来寻找抗原靶标。这一概念极大地简化了寻找可能参与产生或维持慢性神经病理性疼痛状况的抗原的过程。

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.