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C. A. Deeg, R. L. Kramer, S. M. Hauck, B. Amann, M. Ueffing; Changed Pbl Protein Expression Pattern in Spontaneous Recurrent Uveitis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1173.
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Equine recurrent uveitis (ERU), a spontaneous disease with high prevalence in horses, is mediated by autoreactive leukocytes crossing blood-retinal barrier. Reasons for emergence, migration and fate of these autoaggressive cells are essentially unknown to date. Specific objective of this study was the identification of differentially expressed proteins in peripheral blood-derived leukocytes (PBL) of ERU cases in comparison to controls by differential proteome analysis.
For this study, PBL of 37 healthy and 33 ERU diseased horses were examined. Proteins from cell lysates were labelled with fluorescent CyDyes (GE Healthcare) and subject to 2-D Difference Gel Electrophoresis (DIGE) comparing blood of ERU cases and healthy controls. After spot detection and quantification, we generated a spot map with identical spot boundaries for all gel images using DeCyder 6.5 (GE Healthcare). Spots were then excised and subsequently identified by mass spectrometry (MALDI-TOF/TOF or by LC-MSMS). Regulation of identified candidates was verified and quantified with Western blots. Expression pattern on equine PBL subsets was additionally investigated with flow cytometry, using equine specific antibodies to CD4, CD8, B cells, MPO, MHC II, Ki67 and alpha and beta integrins (all Serotec) for double staining.
We created a map of immune cell proteins and detected 50 differentially expressed proteins in diseased state in comparison to healthy samples. Twenty out of these 50 proteins could be identified by mass spectrometry. Ten proteins were downregulated in ERU and 10 proteins were upregulated. Higher abundant proteins on PBL of ERU cases were amongst others Scaffold attachment factor B, Lactotransferrin and NCK1 adaptor protein. Downregulated candidates comprised Protein Disulfid Isomerase, Transgelin 2, Serpin B10, Poly (rC) binding protein 2 isoform B, PWP1-interacting protein and Ezrin. Candidates were expressed on various lymphocyte subsets, but also on granulocytes. Function of these proteins point to changes in immune response, cell motility and transmigration of PBL in recurrent uveitis.
Understanding of initiation and maintenance of spontaneous recurrent uveitis requires knowledge about the properties of the participating cell subsets and their prerequisites to transmigrate into the eye. PBL proteome analysis involving fluorescent protein labelling and DIGE proofed efficient for identification of complex changes in immune cell protein expression patterns.
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