Mechanisms of Pain and Immune Processes

疼痛和免疫过程的机制

• 批准号: 7967114
• 负责人: Michael J. Iadarola
• 金额: $ 66.7万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967114
• 关键词: AIDS neuropathy; Acute; Acute Pain; Address; Adolescent; Affect; Animal Model; Animals; Antibodies; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Diseases of the Nervous System; Autoimmune Process; Autoimmune Responses; Autonomic nervous system; Award; B-Lymphocytes; Binding; Biological; Biological Assay; Biological Markers; Biological Models; Biopsy; Body part; Butyric Acids; C Fiber; Caliber; Carboxy-Lyases; Catalytic Domain; Cells; Cholinergic Receptors; Clinical; Clinical Protocols; Clinical Research; Collaborations; Communicable Diseases; Communication; Complex; Complex Regional Pain Syndromes; Data; Data Set; Dense Core Vesicle; Dental Schools; Diabetes Mellitus; Diabetic Neuropathies; Diagnostic; Disease; Elements; Enzymes; Epitopes; Evolution; Exhibits; Face; Facial nerve structure; Fiber; Florida; Functional disorder; Future; Gastric Emptying; Gastric Parietal Cells; Gated Ion Channel; Glutamate Decarboxylase; Goals; Gold; HIV; Human; Human T-lymphotropic virus 1; Immune; Immune response; Immune system; Immunoprecipitation; Infection; Injury; Institutes; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Ion Channel; Knowledge; Life; Ligand Binding; Liquid substance; Lower Extremity; Luciferases; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Membrane; Metabolic Diseases; Methodology; Methods; Modeling; Molecular; Multiple Sclerosis; Muscle; Myasthenia; Myasthenia Gravis; National Institute of Dental and Craniofacial Research; National Institute of Neurological Disorders and Stroke; Nerve; Nerve Endings; Nerve Fibers; Nervous system structure; Neuraxis; Neurobiology; Neurologic; Neurologic Manifestations; Neuroma; Neuropathy; Neurotransmitters; Nicotinic Receptors; Nociception; Non-Insulin-Dependent Diabetes Mellitus; Numbness; Operative Surgical Procedures; Oral cavity; Orthologous Gene; Pain; Pain Disorder; Painful Paresthesias; Pancreas; Parietal; Patients; Pattern; Peripheral; Peripheral Nerves; Peripheral Nervous System Diseases; Phase; Population; Postherpetic neuralgia; Predictive Value; Process; Protein Fragment; Proteins; Proteome; Radioactive; Recombinants; Reporting; Research; Research Ethics Committees; Research Project Grants; Risk; Robotics; Role; Saliva; Sampling; Sensory; Serum; Signal Pathway; Sjogren' s Syndrome; Specificity; Spinal Cord; Stiff-Person Syndrome; Stomach; Structure of trigeminal ganglion; Symptoms; Syndrome; System; Testing; Time; Tissues; To autoantigen; Tracer; Translational Research; Trauma; Trigeminal Neuralgia; Tube; United States National Institutes of Health; Universities; Work; base; bench to bedside; chronic neuropathic pain; chronic pain; cohort; cysteine sulfinic acid; drug development; gastrointestinal; human disease; immunogenicity; insight; interest; luminescence; nerve injury; nervous system disorder; neurophysiology; optic nerve disorder; painful neuropathy; patient population; programs; receptor; relating to nervous system; sarcoma; therapeutic development; transmission process

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, or autoimmune disorders such as Sjogrens Syndrome or 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. 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 saliva or serum. This translational research program addresses molecular and pathophysiological processes of nociceptive transmission and new ways to effectively 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. Over this past year we 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-amino butyric acid. We used recombinant methods to identify the major epitope of GAD65, which mapped to the conserved catalytic domain. Immunogenicity extended to this domain in GAD67 (a closely related ortholog and partially antigenic) but not to the next most homologous decarboxylase, cysteine sulfinic acid decarboxylase, despite a high level of sequence similarity. We also 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 sentitivity and specificity. Interestingly IA2 is found in large dense core vesicles which store insulin in the pancreatic B-cells. These same large dense core vesicles are found in pain sensing peptidergic C-fibers, and we shall test all chronic pain cohorts for autoantigens to IA2 and IA2b. 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. Examination of the clinical patient information from these seven neural antigen positive patients showed that 2 SjS patients had peripheral neuropathy, one had trigeminal neuralgia and 2 others had significant CNS involvement. Since it been reported that approximately 20% of SjS patients exhibit peripheral neuropathy, optic neuropathy and autonomic nervous system dysfunction, future studies will address whether there is a relationship between positive anti-neural antibodies and neurological disease in SjS patients. We then examined antibodies to this protein in a small cohort of neuropathic pain patients where we observed that 14% had antibodies to this neural protein. By comprison, none of the controls for either SjS or CRPS exhibited a titer to either the parietal or neural antigens. These are very encouraging data that support the hypothesis of immune system involvement in peripheral neuropathic pain disorders. The identification of a parietal cell antigen is also compelling. Eighteen percent of the SjS patients exhibited high titers. The SjS patients frequently complain of gastric problems and characterization of gastric emptying time and other gastrointestinal parameters is being conducted by Dr. Nikolov as part of our current SjS clinical protocol on autonomic function. It will be very interesting to determine if the gastric symptoms associate with the subpopulation that has gastric autoimmune presentation. In both Stiff Person Syndrome and SjS, it is hypothesized that one of the major autoantigens is a membrane receptor or ion channel. 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. We established collaboration with the Myasthenia group at Johns Hopkins. The Hopkins IRB recently approved our clinical protocol and we already have samples that we are measuring. 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 NIDCR intramural groups on Sjgrens syndrome and diabetes and we have extended the SjS-neurological symptoms study with additional patients from the Dental School at University of Florida. We have formed a working relationship with the NINDS groups of David Goldstein (CRPS and other autonomic nervous system autoimmune problems) and Henry McFarland and Steve Jacobson (multiple sclerosis and HTLV1 infection). 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, which is currently being pursued. 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.

概述：慢性神经性疼痛会影响身体的任何部位，包括口腔和面部神经。神经性疼痛可能是由于多种损伤，感染或自身免疫性疾病（例如Sjogrens综合征或糖尿病（糖尿病神经病））引起的。我们正在检验以下假设：在某些患者中，慢性疼痛通过与外周神经中蛋白质产生的自身抗体有关的免疫病理学过程维持。在某些大型纤维周围神经病中，自身抗体是已知的罪魁祸首。在疼痛是成分的地方，我们假设存在于小直径，疼痛感（伤害感受）C纤维或A-二尔特神经纤维引起的神经末端发现的蛋白质自身抗体。为了支持这一想法，据报道，大约30％的Sjogrens综合征（SJS）患者表现出很小的纤维神经病，在上肢和下肢会产生疼痛的异常。类似的神经性疼痛在II型糖尿病中显着发生。为了测试疼痛的神经性疾病具有我们确定的自身免疫性成分的假设，即一种灵敏的，定量的，液相发光测定法，该测定使用在哺乳动物细胞中表达的重组抗原示踪剂，以测量唾液或血清中抗体的存在。该转化研究计划介绍了伤害感受传播的分子和病​​理生理过程，以及有效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）人类慢性疼痛障碍的分子和细胞生物学机制，以及（2）使用这些知识来设计新的治疗方法和诊断疼痛障碍。 为了获得足够的吞吐量来检查多种候选抗原的大量正常人和患者，我们将测定法从单管格式调整为在机器人移液平台上运行的96个井的微量滴定板。 我们还成立了不同患者人群的合作和组装人群，以建立自身免疫性疾病，传染病，慢性疼痛和神经系统疾病的基线值。 在过去的一年中，我们检查了一种已知的中枢神经系统自身免疫性疾病，称为僵硬的人综合症。 这些患者具有高滴度自身抗体，可促谷氨酸脱羧酶（GAD65），该酶催化抑制性神经递质γ-氨基酸的形成。我们使用重组方法来识别映射到保守催化域的GAD65的主要表位。 免疫原性扩展到GAD67（紧密相关的直系同源物和部分抗原），但没有与下一个最同源的脱羧酶，半胱氨酸硫酸脱羧酶脱羧酶，尽管序列相似，但均不足。我们还测试了1型糖尿病的主要抗原（IA2，IA2B和GAD65），即少年自身免疫性形式。 这些研究表明，就疾病和特异性而言，非放射性荧光素酶免疫沉淀测定法优于金标准的放射性测定。有趣的是，IA2在大型密集的核囊泡中发现，这些核囊泡将胰岛素存储在胰腺B细胞中。 这些相同的大型核心囊泡是在疼痛感测肽纤维C纤维中发现的，我们将测试所有慢性疼痛队列的自身抗原对IA2和IA2B。 我们还对Sjogrens综合征（SJS）患者（RO52，RO60和LA以及大约7种抗原）进行了自身抗原的广泛分析。 我们在SJS子群中发现了两种新抗原。 一种是针对神经系统蛋白的，另一种是反对胃顶细胞蛋白。 检查了这七个神经抗原阳性患者的临床患者信息，表明2例SJS患者患有周围神经病，一个患有三叉神经痛，另有2例患有CNS显着。 由于据报道，大约20％的SJS患者表现出周围神经病，视神经病变和自主神经系统功能障碍，因此未来的研究将解决SJS患者中阳性抗神经抗体与神经系统疾病之间的关系。然后，我们检查了一小群神经性疼痛患者中对该蛋白的抗体，我们观察到14％的神经蛋白具有抗体。由成员组成，SJS或CRP的任何控件都没有表现出对顶叶或神经抗原的滴度。 这些非常令人鼓舞的数据，支持免疫系统参与周围神经性疼痛障碍的假设。顶叶抗原的鉴定也令人信服。 18％的SJS患者表现出很高的滴度。 SJS患者经常抱怨胃排空时间和其他胃肠道参数的表征，Nikolov博士是我们当前关于自主功能的SJS临床方案的一部分。 确定胃症状是否与具有胃自身免疫性表现的亚群相关，这将非常有趣。 在僵硬的人综合症和SJ中，都假设主要的自身抗原之一是膜受体或离子通道。为了建立基于受体的自身免疫性疾病的基本参数，我们开始了对肌无力重症患者的研究。这是一种针对膜结合的配体门控离子通道的神经自身免疫性疾病，肌肉烟碱受体。我们与约翰·霍普金斯（Johns Hopkins）的Myasthenia Group建立了合作。 霍普金斯IRB最近批准了我们的临床方案，我们已经有了我们正在测量的样本。 目前，我们正在研究其他几个互助和机构间合作（例如上述肌无动物研究），以获取具有复杂区域疼痛综合征（CRP，神经性疼痛障碍），其他神经病以及其他CNS和PNS疾病和感染性疾病的良好特征性的患者。 我们正在与NIDCR室内组紧密合作，以了解SJGRENS综合征和糖尿病，并通过佛罗里达大学牙科学校的其他患者扩展了SJS神经学症状研究。 我们已经与David Goldstein的Ninds组建立了工作关系（CRP和其他自主神经系统自身免疫性问题）以及Henry McFarland和Steve Jacobson（多发性硬化症和HTLV1感染）。我们还使用该测定法探索HIV，病毒引起Kaposis肉瘤和与HIV相关的恶性肿瘤和疼痛周围神经病之间的相互关系。 后者的研究构成了NIH临床中心的基础，目前正在追求NIH临床中心。 该项目最引人注目的方面之一是数据集的渐进分层和演变。随着我们增加了跨疾病和疾病的测试抗原和测定的数量，我们组装了对自身免疫反应的全面评估。 这是通过确定（a）对直系同源蛋白质和蛋白质片段的免疫反应的程度和特异性来完成的，（b）在抗原特征中重叠，指示了共同的分母或一般机制，以及（d）与细胞间或胞内通信有关的整个信号途径中的抗原性。随着时间的流逝，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新水平。