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J. K. Zipplies, S. M. Hauck, H. Gerhards, M. Stangassinger, M. Ueffing, C. A. Deeg; Differentially Regulated Proteins in Sera of Horses With Spontaneous Recurrent Uveitis (ERU). Invest. Ophthalmol. Vis. Sci. 2007;48(13):5219.
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Equine recurrent uveitis (ERU) is a spontaneous disease with high prevalence. It is the only spontaneous animal model for human autoimmune uveitis. The composition of the serum proteome can reflect the physiological and pathophysiological state of an organism. Disease-induced changes to the serum proteome have the potential to act as biomarkers for diagnosis, to monitor disease progression, and to assess the clinical status of a patient. Identification of differentially regulated proteins in sera of horses with ERU could additionally help to detect the disease before the clinical signs occur and therefore identify individuals at risk of developing disease in a pre-clinical phase. In case of a clinical manifestation of the disease upcoming relapses could probably be detected by changes of serum proteome.
Serum samples of ERU cases and negative controls, as well as horses with induced uveitis (day 0 and after induced uveitis) were collected and immediately stabilized with protease inhibitors. To remove high abundant proteins as albumin and IgG, protein depletion was performed by resin binding on IgY-12 High Capacity Spin Columns (Beckman Coulter). 2DE was performed by first isoelectric focusing (IPG strips, pH 3-11; GE Healthcare) on a Multiphor for 14200 Vh at 20°C, and subsequent SDS-PAGE (9-15%). The gels were then silver-stained and spots were excised and prepared for mass spectrometry. We examined the patterns of protein expression profiles of complete and depleted sera of ERU diseased horses and compared them to the sera of eye healthy horses. Spots of interest were subsequently identified using MALDI-TOF-MS/MS.
Separating the serum proteome by 2DE gels led to a resolution of about 1037 spots per gel in non-depleted samples and in average 1021 spots in sera depleted of high abundant proteins. Differentially expressed spot could be detected in the group of horses with induced uveitis between day 0 and after the uveitic attack. Additionally, differentially regulated spots were detected between healthy negative control sera in comparison to spontaneous ERU cases. There is considerable overlap of regulated spots from spontaneous and induced uveitis patients. However, we also identified distinct proteins appearing exclusively in sera of patients from either induced or spontaneous uveitis.
Proteome analysis is useful to study the appearance of serum biomarkers involved in uveitis pathogenesis.
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