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Cornelia A Deeg, Nina B Burkhardt, Roxane L Degroote, Barbara Amann, Marius Ueffing, Stefanie M Hauck; Intravitreal leukocytes change plasma cell membrane expression pattern in spontaneous recurrent uveitis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2495.
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© ARVO (1962-2015); The Authors (2016-present)
Horses frequently (10%) develop spontaneous recurrent uveitis (ERU), an autoimmune mediated disease. Pathophysiology is driven by lymphocytes that enter the inner eye through outer blood-retinal barrier and destroy retinal proteins. In order to understand differences in cell surface phenotype of peripheral blood derived lymphocytes and intraocular lymphocytes, we chose a differential proteomics approach, determining plasma cell surface protein phenotypes in healthy and diseased cells.
Peripheral blood derived lymphocytes (PBL) were separated from horses with spontaneous equine recurrent uveitis, that were presented to Equine Clinic of LMU Munich for therapeutic vitrectomy. Intraocular lymphocytes (VL) were separated from vitreous obtained during pars plana surgery. Amount of PBL used in experiments was adjusted to total amount of cells obtained from intraocular surgery (around 1 x 104 cells) for comparison of protein expression. Cell surface proteins were covalently labeled with biotin, affinity purified and released by PNGase F. Detection, identification and quantification of respective proteins was carried out with label free mass spectrometry.
Our approach resulted in clear identification of 206 proteins in PBL and 212 proteins in VL preparation of plasma cell membrane proteins. Of these, 196 proteins were identified in both fractions. Overall, eight proteins were ≥ 5 fold enriched in immune cells separated from vitreous. In contrast, 14 proteins were enriched in PBL fraction (factor ≥ 5). Interestingly, upregulated proteins in VL comprise activated sushi domain containing 2, matrix metallopeptidase 25 and leukocyte cell adhesion molecule. Downregulated candidates were semaphorin 4D, atlastin GTPase 3, interferon (alpha, beta and omega) receptor 1 and Fas.
Cell surface analysis of ERU effector cells was suited to generate a cell surface proteome profile and to detect specific changes in intraocular lymphocytes of ERU cases. This will help to understand inflammation associated changes in a spontaneous autoimmune disease.
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