Autoimmune Autonomic Ganglionopathy: antibodies to the alpha3 ganglionic acetylcholine receptor and flow cytometric methods for their detection

Abstract

Autoimmune Autonomic Ganglionopathy (AAG) is a rare, immune-mediated disease of the autonomic nervous system. The incidence is unknown. All facets of the autonomic nervous system (parasympathetic, sympathetic and enteric) may be variably impacted, leading to heterogenous manifestations depending on which division is affected, and to what extent. Severe AAG manifests as complete paralysis of the autonomic nervous system – pandysautonomia. The uncommonness of this disorder, coupled with the diversity of clinical manifestations and lack of universally accepted diagnostic criteria has made AAG difficult to define and study. Over the last two decades, autoantibodies that bind to the ganglionic acetylcholine receptor (gnACHR) present on the surface of autonomic neurones have not only been found in approximately 50% of clinical diagnoses of AAG, they have been proven by extensive animal models and tumour cell line experiments to be directly pathological, exerting their effects by antibody-dependant cross-linking and internalization of receptors away from the neuronal surface. Antibodies to gnACHR are difficult to detect by immunoassays. Radioimmunoprecipitation (of whole, native receptor) was the first assay described and is currently the preferred method of detection. Luciferase immunoprecipitation (of isolated recombinant subunits) has also been described. Both assay formats have advantages, but neither are widely available, and both have high rates of false positivity when used in low pre-test populations. The work presented in this thesis describes alternative immunoassays for gnACHR autoantibody detection with emphasis on cell-based and microparticle-based assays, in addition to reviewing the clinical manifestations of patients with AAG in order to try and define better diagnostic definitions of the disorder. I have shown that the generation of a cell-based assay that detects the relevant pathophysiological effect exerted by these autoantibodies is not only feasible but can be implemented in a clinical diagnostic laboratory. The ease of performance of this assay and lack of radioactive material will provide a valuable asset to more clinical diagnostic laboratories and thus increase accessibility for this testing to the practising clinical neurologist who otherwise might not be able to routinely request these serological investigations.