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Title: Use of proteomic techniques to investigate the aetiology of ankylosing spondylitis
Author: Wright, Cynthia Anne
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2009
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Mechanisms of tolerance by which the human body distinguishes self from non-self serve to protect us from pathogens, and are central to the understanding of immunology. Breach of self- tolerance can result in inappropriate immune responses targeting one's own proteins, cells 0 tissues and lead to autoimmune disease. Ankylosing Spondylitis (AS) is a systemic, rheumatic autoimmune disease, which affects up to 0. 1% of the population. Despite a strong association with the MHC class I allele, HLA-B27, the aetiology of AS is unknown. The work in this thesis describes the use of existing and the development of new proteomic methods to characterize and quantify differences in protein expression in both cellular an humoral components of the immune system in patients with AS. Monocytes from AS patients have been characterized by 2D-gel electrophoresis and label-free quantitative nano-UPLC-MSE mass spectrometry analysis, which has revealed differential expression in proteins from the Ubiquitin Proteasome Pathway (UPP) as well as an up-regulation of 13 proteins that are induced by Tumour Necrosis Factor. An in vitro proteasomal digest assay demonstrated that one protein upregulated in the UPP, the proteasomal activator PA2S, increase the generation ofHLA•B27•restricted epitopes. In parallel, novel methods for the MALDI-TOF analysis of endogenous and tryptic plasma peptides were developed with the aim of identifying diagnostic biomarkers in AS plasma. The depletion of abundant plasma proteins followed by plasma fractionalion based on isoelectric focusing or nano-UPLC•MSE mass spectrometry analysis was also employed to this end Preliminary plasma cytokine profiling showed a specific upregulation of interleukin-27 in AS patients. Levels of AS plasma matrix-metallo proteins (MMPs) analysed demonstrated a~ upregulation of two collagenases, however this find ing was not supported by plasma MM enzymatic activity assays. Finally, in collaboration with the Harvard Institute of Proteomics, the novel finding 0 autoantibodies in AS patients was characterized and quantified by the use of Nucleic Acid Programmable Protein Arrays. 44% of AS patients studied demonstrated a broad, non-specific autoantibody response and multiple AS patients showed responses to common putative autoantigens. The AS cohort autoantibody response was more specific compared to that from the RA cohort and autoantibodies from AS patients showed a bias towards antigens involved i6 skeletal and connective tissue disorders. Taken together, evidence for differential expression of proteins in the Ubiquitin Proteasome Pathway and a broad, non-specific autoantibody response in AS patients support the hypothesis ~f a role in antigen presentation for HLA-B27 in Ankylosing Spondylitis. The work in this thesis demonstrates that clinical proteomics is a viable method for the investigation of au toimmuI1f diseases, provides entry points to target cellular and molecular mechanisms for further investigation of disease pathogenesis, disease diagnosis and therapeutic benefit.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available