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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010T-Cell Epitopes in Autoimmune Bullous Skin DisordersKatalin Uray 1 , Márta Marschalkó 2 , Hajnalka Szabados 1 ,Antal Blazsek 2 , Ferenc Hudecz 1,3 , Sarolta Kárpáti 2 , and Szilvia Bősze 11 Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös LorándUniversity, Budapest, H-1117, Hungary; 2 Department of Dermato-Venereology and SkinOncology, Semmelweis University, Budapest, H-1085, Hungary; 3 Department ofOrganic Chemistry, Eötvös Loránd University, Budapest, H-1117, HungaryIntroductionAutoimmune bullous skin diseases, such as bullosus pemphigoid (BP) and pemphigusvulgaris (PV), are rare, severe, sometimes life-threatening skin disorders. Serologicallythey are characterized by autoreactive serum antibodies directed against different adhesionmolecules of the epidermis and the dermoepidermal basement zone [1]. The antibodybinding affects the adhesive function of these molecules resulting in detachment of the cellsand subsequent blister formation [2,3].In the pathogenesis of both pemphigus and pemphigoid, autoreactive T-cell responseplays a crucial role, because initiation and perpetuation of B-cell response needs therecognition of T-cell epitopes. In both types of diseases the T-cells recognize epitopes fromthe extracellular domain of desmoglein 3 (Dsg3) [4-6] or NC16a domain of collagen XVII(BP180) proteins [3,6], producing different cytokines, e.g. interferon- (IFN- ).We have selected potential T-cell epitope regions within the proteins BP180 (502-515)and Dsg3 (96-112). The synthetic peptides were used to stimulate the peripheral bloodmonomorphonuclear cells (PBMC) of patients and healthy controls, and the amount ofproduced IFN- was determined by ELISA.Results and DiscussionPeptides were synthesized by solid phase peptide synthesis method using standardFmoc/tBu chemistry on Rink-amide MBHA resin. The peptides were RP-HPLC purified,then characterized by ESI-MS and amino acid analysis, showing the expected composition(Table 1).PBMC from the peripheral blood of healthy donors (D1, ♂30y, D2, ♀ 34y) andpatients diagnosed with pemphigus vulgaris (D3, ♂74y, D4, ♀30y) were isolated asdescribed [7]. PBMC were cultured at 1.5-1.8 x 10 5 /well in 96-well U-bottom plates. Dsg3and BP180 peptides were added at concentrations 0.05 and 0.025 mM [8]. As nonspecificpositive control, phytohaemagglutinin (PHA-P) and Enterotoxin B, Staphylococcal (SEB)were used at c = 5 µg/mL. After 20 or 90 h of incubation, supernatants were harvested.IFN- content was measured by sandwich ELISA. As capture antibody human anti-IFN-(BD 551221) was used. The INF- level of PBMC supernatant was determined usingbiotinylated mouse anti-IFN- (BD 554550) as detection antibody.IFNγ concentration (pg/ml)16001400120010008006004002000D1 healthy control ♂30yD3 PV patient ♂74yD4 PV patient ♀30ymedium control peptide #1 peptide #2 peptide #3 peptide #4 peptide #5Fig. 1. IFN- level of supernatants obtained from PBMC of donors after 20 hrs incubationwith 0.025mM peptide, determined by ELISA.476