Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients

et al. (2013) Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients. PLoS ONE 8(11): e80982. doi:10.1371/journal.pone.0080982 Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients Sara Vallejo-Diez 0 David Bernardo 0 Mara de Lourdes Moreno 0 Alba Muoz-Suano 0 Luis Fernndez- 0 Salazar 0 Carmen Calvo 0 Carolina Sousa 0 Jos A. Garrote 0 ngel Cebolla 0 Eduardo Arranz 0 Karol Sestak, Tulane University, United States of America 0 1 Mucosal Immunology Laboratory, IBGM, University of Valladolid-Consejo Superior de Investigaciones Cientificas , Valladolid , Spain , 2 Department of Microbiology and Parasitology, University of Seville , Seville, Spain, 3 Biomedal S.L, Seville , Spain , 4 Gastroenterology Service, Hospital Clinico Universitario , Valladolid , Spain , 5 Pediatric Service, Hospital Clinico Universitario , Valladolid , Spain , 6 Clinical Laboratory, Hospital Universitario Rio Hortega , Valladolid , Spain We studied whether celiac disease (CD) patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer) was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD), 17 CD patients on a gluten-free diet (GFD), 10 healthy controls (C) and 23 patients with other gastrointestinal pathology (GP)) and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients). Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA antiDGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD. a Current address; Antigen Presentation Research Group; Imperial College London; Northwick Park & St Mark's Campus; Harrow; United Kingdom - Funding: This work has been supported by grants (to EA) from the Instituto de Salud Carlos III FEDER (PI070244, PI10/01647), Junta de Castilla y Len (SAN126/VA31/09), AECID (A/019196/08), and Phadia Spain S.L (now ThermoFisher Scientific). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Some of the authors are inventors of the patent application Pptido inmunognico del gluten y sus aplicaciones (no P201131576). The patent application is owned by the University of Valladolid (Spain) and licensed to Biomedal S.L. This work has been partially funded by Phadia Spain S.L (now ThermoFisher Scientific). The authors declare that this does not alter their adherence to all the PLOS ONE policies on sharing data and materials. Celiac disease (CD) is a gluten-sensitive enteropathy that develops in genetically susceptible individuals following exposure to dietary wheat gluten and similar proteins from barley, rye and some varieties of oats [13] (Highlights S1). Prolamins constitute eighty percent of total gluten proteins. They are soluble in ethanol and rich in glutamine (Q) and proline (P) residues. Their names varies based on the source cereal (gliadin from wheat, secalin from rye, hordein from barley and avenin from oats) and they are classified in -, and -prolamins according to their electrophoretic mobility. The remaining 20% of the total gluten proteins are insoluble in ethanol and are divided in high molecular weight (HMW) and low molecular weight (LMW) glutenins. CD is characterized by villous atrophy, crypt hyperplasia and infiltration of inflammatory cells, both in the epithelium and in the mucosal lamina propria of the small intestine. The disease might affect approximately 1% of the Caucasian population. At present, the only treatment for CD is a life-long strict glutenfree diet (GFD), which in most cases leads to a complete remission of the disease. The inflammatory reaction appears to be driven by activation of Th1-like-CD4+ T cells that recognize gluten peptides modified by the enzyme tissue transglutaminase (tTG) in the context of human histocompatibility leucocyte antigen (HLA) region namely the HLA-DQ2/DQ8 molecules [4,5]. Deamidation is important for binding of gliadin-derived peptides to HLA DQ2/DQ8 molecules and subsequently for the stimulation of T cells [4]. Several gliadin-derived peptides have been identified as ligands for the disease-associated HLA-DQ molecules [6]. Whereas the T cell response in CD is relatively well understood, less is known about the B cell response [7]. Mucosal B cells are triggered to produce antibodies against food antigens, anti-gliadin (AGA), anti-deamidated gliadin peptides (DGP); and against self molecules as tTG. At the mucosal compartments humoral responses are mainly mediated by IgA antibodies so they are more specific than IgG antibodies as serological markers in gastrointestinal diseases like CD. The diagnosis of CD is based on 3 pillars: i) mucosal alterations as determined by histological evaluation of duodenal biopsy, ii) genetic susceptibility (HLA-DQ2/DQ8) and iii) a positive serology (antibodies against tTG and antiendomisium) [8]. Despite small bowel biopsy is still the gold standard for CD diagnosis, endoscopy is uncomfortable and expensive. Therefore, research has been focused on developing less-invasive markers for its correct diagnosis. Many approaches have led to the identification of several gluten peptides that can stimulate T cells from CD patients. Such peptides were found in -, - and -gliadins as well as in glutenins. While - and -gliadin-derived peptides are immunodominant in adults, responses to the LMW glutenins and -gliadins are frequently observed in children [9,10]. The study however of gliadin-derived peptides specifically generated in the duodenum of CD patients improves our current knowledge about gluten peptides since they may be used for development of specific serological markers with a clinical application and/or to target them in vivo to prevent their immunogenic properties in the celiac duodenum. We have previously described for the first time to our knowledge a specific pattern of gliadin-degrading metalloproteases in the duodenal mucosa of CD patients, not found in the duodenum from non-CD patients [11]. Therefore CD patients (both aCD and GFD (...truncated)