Abstract
Purpose: :
To analyze the percentage of anti-recoverin autoantibody positive horses in a cohort of horses with spontaneous equine recurrent uveitis (ERU) and negative controls. Recoverin, a retinal calcium-binding protein, belongs to the larger family of proteins which have an EF-hand calcium-binding domain. Recoverin participates in the recovery phase of visual excitation and in adaption to light.
Methods: :
Recoverin was identified as a potential autoantigen in equine recurrent uveitis (ERU) by a 2 DE Western blot screening, followed by tandem mass spectrometry for protein identification. In order to validate the immune reaction to recoverin and to determine the autoantibody frequency to recoverin in spontaneous and experimentally diseased horses, we first cloned, expressed and purified recoverin. Then, IgG and IgM antibody responses to recoverin were tested in Western blots, using sera of 102 ERU cases, 92 negative controls, 5 S-antigen immunized horses and 7 IRBP immunized horses (sera were kept from earlier experiments). Antibody binding was detected by enhanced chemiluminescence (ECL). Differences in autoantibody frequency between diseased and healthy horses were statistically analyzed using Fisher's Exact Test.
Results: :
Cloning and expression of recoverin was successful as verified by mass-spectrometry. From 102 tested ERU cases, 55.88 % (57 horses ) were recoverin-autoantibody positive. But from 92 tested controls, 52.17 % (48 horses) were also recoverin-autoantibody positive. The percentage of recoverin-autoantibody horses did not differ between spontaneous diseased and healthy cases, no matter how sera, secondary antibody and recoverin concentrations were changed. All 5 horses with experimental uveitis induced by immunization with S-antigen showed a positive autoantibody response to recoverin after immunization with S-antigen, as also seen in screening tests to whole retinal lysate. Sera taken before immunization were recoverin-autoantibody negative, indicating a change in immune response to recoverin after immunization with S-antigen. This could be caused by traces of recoverin in the preparation used for immunization or indicate epitope spreading to recoverin.
Conclusions: :
Recoverin-specific immune reactions do not seem to play a role in the pathogenesis of spontaneous equine recurrent uveitis. The immune response to recoverin in S-antigen immunized horses deserves further investigations.
Keywords: uveitis-clinical/animal model • autoimmune disease • protein purification and characterization