Analysis of candidate retinal autoantigens in autoimmune uveitis

Morgan, James (2005) Analysis of candidate retinal autoantigens in autoimmune uveitis. PhD thesis, University of Nottingham.

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Autoimmune uveitis is a non-infective ocular inflammation of humans that potentially causes blindness. It is mediated primarily by autoreactive CD4+ Tlymphocytes that target antigens within the retina. Autoantibody responses play a secondary role. This thesis describes various investigations into humoral and cellular immune responses in autoimmune uveitis and assesses several methodologies for their suitability to applied human research.

Recombinant human retinal S antigen (RSAg), an important candidate autoantigen in uveitis, was cloned and expressed in bacterial and human cells. Purified RSAg was tested by ELISA against sera from uveitis patients and controls. The recombinant antigens performed well in ELISA. No significant differences in antibody titres were detected between the groups.

B-cell epitope preferences of anti-RSAg polyclonal antibodies were investigated by screening several random phage display libraries. One library produced results, but no defining epitope was identified for either uveitis or control sera. In uveitis research this technique might be better suited to delineating minimal epitope requirements of monoclonal antibodies.

As yet undiscovered uveitis autoantigens may exist. A human retinal complementary DNA library was constructed and screened with uveitis and control sera. Seven potentially autoantigenic peptides were identified and expressed as fusion proteins. At least one peptide displayed significantly higher ELISA readings for uveitis over control sera. The full potential of this technique is still to be realised.

Responses of peripheral CD4+ T-lymphocytes to antigen-specific stimulation were studied at the single-cell level using cytokine flow cytometry. A definite response to RSAg was detected in human uveitis and control lymphocytes using this method. This technique has great potential for identifying autoantigenic proteins/epitopes, and analysing resultant cytokine profiles in uveitogenic T-cells.

Several of the new strategies and techniques described here have already produced exciting findings. It is envisaged that they will make further significant contributions to applied human uveitis research in the near future.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Tighe, P.
Keywords: Uveitis; Cellular immune responses; Recombinant human retinal S antigen; Autoantigenic peptides; Cytokine profiles
Subjects: R Medicine > RE Ophthalmology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56713
Depositing User: Blore, Mrs Kathryn
Date Deposited: 13 May 2019 07:52
Last Modified: 07 May 2020 13:17

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