Mechanisms of Pain and Immune Processes

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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, autoimmune disorders such as Sjogrens Syndrome, or metabolic disorders such as 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 between 20 and 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 and in cancer patients treated with certain chemotherapeutic agents. 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 tracers expressed in mammalian cells. This assay is called luciferase immunoprecipitation systems (LIPS) and we have published papers showing that LIPS can readily, and without interference, measure antibodies in serum, plasma, cerebrospinal fluid, saliva and other bodily fluids. This translational research program addresses molecular and pathophysiological processes of nociceptive transmission and new ways to investigate chronic pain, autoimmune conditions and infectious diseases in human patients. (1) Our main goal is to understand the molecular and cell biological mechanisms underlying human chronic pain disorders. In addition, the assay methodology we established has great versatility due to its sensitivity, modularity, and use of recombinant DNA methodology to generate protein antigen tracers. Thus, numerous conditions have been examined as we evaluate the range of diseases and disorders that antibodies play a role in. These include various infectious diseases, with and without nervous system involvement, and various autoimmune disorders, all of which have nervous system symptomology in subsets of patients. , (2) Thus a second goal is to develop new, more sensitive and robust clinical tests for relevant diseases or conditions. Last year we extended the SjS study to include a comparison of salivary antibody levels to those in serum. Using only 5 microliters, the LIPS assay readily detected the major SjS autoantigens in saliva, yielding the same sensitivity and specificity as in serum. These results highlighted the feasibility of establishing non-invasive, saliva based assays for many types of human diseases and for monitoring of vaccine immune status for large populations of people. This year we published a report on a microfluidic platform for the LIPS assay that demonstrates proof of principle for a small point-of-care diagnostic device. The major Sjogren's antigens include the protein Ro52; this is an interferon-inducible member of the tripartite motif (TRIM) family of proteins. We have discovered a new Sjogrens autoantibody from the same TRIM family and have begun to examine this more broadly. We will begin to confirm these observations using serum from the SICCA Sjogren's registry maintained at the School of Dentistry, UCSF. We also identified antibodies to interferon gamma in some of the samples and also in some of the neuropathic pain samples. We generated a larger panel of anticytokine antibody probes. Antibodies to the various cytokines were not widely represented in either population. However, anti-interferon gamma antibodies do appear to play a role opportunistic infections and this collaborative research work defines a new mechanism of disease in certain patient populations. These results were recently published. 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 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 on several additional inter-institute and inter-institutional collaborations 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, especially pain, such as shingles and post-herpetic neuralgia. 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 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 a common denominator or general mechanism, 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. As time progresses, full multiple antigen profiling can be implemented to obtain a new level of understanding of many complex human disease states.