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.
