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28 ROLE OF A-GLIADIN 31-49 PEPTIDE organ culture model. Small intestinal biopsies were placed on an organ culture grid and incubated for 16-18 hours in the presence or absence of a putatively toxic fraction. We measured enterocyte surface cell heights on sections of the biopsies. Using this technique, we reported that each of the gliadin fractions, that is a, b, gand was enterotoxic to coeliac small intestinal mucosa. Dr Kasarda subsequently went on to sequence gliadin. The now classic sequence of A-gliadin is known to be 266 amino 1 acids long . This is an unusual protein in that it is composed by 10-15% proline and 30- 35% glutamine residues; this is an efficient method of storing nitrogen for the growing plant. We subsequently went on to develop an in vivo method of assessing the toxicity of gliadin fractions. This involved passing a Quintron multiple biopsy capsule into the proximal small intestine to which was taped an infusion tube. This allowed test fractions to be infused and multiple small intestinal biopsies to be taken over eight hours. These could then be sectioned and assessed blindly for villous height, crypt depth ratio, epithelial surface cell height and intraepithelial lymphocyte count. Using this system, we tested on different days 10 mg, 100 mg followed by 500 mg, and 1g of gliadin. We showed that 10 mg produced no histological relapse, 100 mg minimal and 500 mg moderate histological relapse. With 1 g there was gross flattening of the mucosa which commenced between 1-2 hours, was maximal at 4 hours and started to recover by 6 hours. We observed flattening of the mucosa, with complete loss of the normal villous 2 architecture . We have also used electron microscopy to show increase in the number of lysosome-like bodies below the brush border, which occurs 1-2 hours after commencing gluten challenges, suggesting that these may be relevant to the pathogenesis of the condition. After a matter of hours, there is apoptosis and shedding of the surface enterocytes. We hypothesize that there is absorption of gluten protein between and through the enterocytes. Toxic gliadin peptides are presented by antigen presenting cells to gluten sensitive T cells which exacerbate the condition through the production of TH1 cytokines. We subsequently went on to perform in vivo experiments assessing the toxicity of peptide fractions corresponding to amino acids 31-49, 202- 220, 3-21 of A-gliadin. We used, as a positive control, unfractionated gliadin. We tested four subjects and showed that a fraction corresponding to amino acids 31-49 was 3 enterotoxic and caused flattening of the mucosa while the other two peptides did not . In a variety of different experiments we showed that there was an induction of TH1 cytokines and inducible nitric oxide, infiltration of the mucosa with T cells, a change in 4 the villous morphology and enterocyte surface cell height . Since gliadin is presented by antigen presenting cells to gluten sensitive T cells by HLA DQ2 or DQ8, we established a cellular binding assay to study the binding of gliadin peptides to DQ2. We demonstrated that the peptide, corresponding to amino acid residues 31-49 of A-gliadin which caused in vivo toxicity, bound to DQ2. The two other peptides which we tested in vivo, but which did not cause toxicity, did not bind to 5 DQ2 . To date, no group has demonstrated the presence of a small intestinal T cell clone that is sensitive to gliadin 31-49. However, a peripheral blood clone which responds to 6 this peptide has been demonstrated . Interestingly, the peptide has also been shown to 7 induce CD4 T cell infiltration into the buccal mucosa of coeliac disease patients . We therefore felt that there is good evidence that A-gliadin 31-49 is a coeliac toxic epitope, although it may be a minor one.
ROLE OF A-GLIADIN 31-49 PEPTIDE 29 References 1. Kasarda DD, Okita TW, Bernadin JE, Backer PA, Nimmo C, Lew E, et al. Nucleic acid (cDNA) and amino acid sequences of type gliadins from wheat (Triticum aestivum). Proc Nat Acad Sci 1984; 81: 4712-6. 2. Ciclitira PJ, Evans DJ, Fagg NLK, Lennox ES, Dowling RH. Clinical testing of gliadin fractions in coeliac patients. Clin Sci 1984; 66: 357-64. 3. Sturgess R, Day P, Ellis HJ, Lundin KE, Gjertsen HA, Konakou M, et al. Wheat peptide challenge in coeliac disease. Lancet 1994; 343: 758-61. 4. Kontakou M, Przemioslo RT, Sturgess RP, Limb GA, Ellis HJ, Day P, et al. Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge. Gut 1995; 37: 52-7. 5. Shidraw RG, Parnell ND, Ciclitira PJ, Travers P, Evan G, Rosen-Bronson S. Binding of gluten-derived peptides to the HLA-DQ2 (alpha 1*0501, beta 1*0201) molecule, assessed in a cellular assay. Clin Exp Immunol 1998; 111: 158-65. 6. Gjertsen HA, Lundin KEA, Sollid L, Eriksen JA, Thorsby E.. T cell recognition of gliadin peptides presented by the coeliac disease associated HLA-DQ (a1*0501, B1*0201) heterodimer. Human Immunology 1994; 39:243-52. 7. Lahteenoja H, Mäki M, Viander M, Raiha I, Vilja P, Rantala I, et al. Local challenge on oral mucosa with an alpha-gliadin related synthetic peptide in patients with coeliac disease. Am J Gastroenterol 2000; 95:2880-7.
Page 1: Perspectives on Coeliac Disease Vol
Page 4: The AIC Meeting was held in the Aul
Page 8 and 9: Italian Coeliac Society, Via Picott
Page 11 and 12: Preface Coeliac disease (CD) is a u
Page 13: Contents Current understanding of t
Page 16 and 17: 2 THE BASIS OF COELIAC DISEASE hapl
Page 18 and 19: 4 THE BASIS OF COELIAC DISEASE 1 pr
Page 20 and 21: 6 THE BASIS OF COELIAC DISEASE spre
Page 22 and 23: 8 THE BASIS OF COELIAC DISEASE Ackn
Page 24 and 25: 10 THE BASIS OF COELIAC DISEASE glu
Page 27 and 28: Catassi C, Fasano A, Corazza GR (ed
Page 29: T CELL RESPONSES TO GLUTEN PEPTIDES
Page 32 and 33: 18 A DOMINANT EPITOPE IN GLUTEN PEP
Page 47 and 48: COELIAC DISEASE IN THE SAHARAWI 33
Page 55: COELIAC DISEASE IN THE SAHARAWI 41
Page 58 and 59: 44 INFANT FEEDING PRACTICES AND COE
Page 66 and 67: Age (years) 52 INFANT FEEDING PRACT
Page 76 and 77: 62 MECHANISMS OF ORAL TOLERANCE the
Page 78 and 79: 64 MECHANISMS OF ORAL TOLERANCE pot
Page 80 and 81: 66 MECHANISMS OF ORAL TOLERANCE Epi
Page 82 and 83: 68 MECHANISMS OF ORAL TOLERANCE typ
Page 84 and 85: 70 MECHANISMS OF ORAL TOLERANCE 51
Page 86 and 87: 72 MECHANISMS OF ORAL TOLERANCE 23.
Page 91 and 92: GENETICALLY DETOXIFIED GRAINS 77 to
Page 93 and 94:
• • GENETICALLY DETOXIFIED GRAI
Page 95 and 96:
GENETICALLY DETOXIFIED GRAINS 81 3.
Page 97 and 98:
Catassi C, Fasano A, Corazza GR (ed
Page 99 and 100:
IMMUNOTHERAPY OF COELIAC DISEASE 85
Page 101 and 102:
IMMUNOTHERAPY OF COELIAC DISEASE 87
Page 103 and 104:
Catassi C, Fasano A, Corazza GR (ed
Page 105 and 106:
RECENT ADVANCES ON GLUTEN TOXICITY
Page 107 and 108:
Index A Antiendomysium antibodies (
Page 109:
INDEX 95 W Wheat, 7, 23, 27, 31, 53
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