primary prevention of coeliac disease - Associazione Italiana ...

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20 A DOMINANT EPITOPE IN GLUTEN PEPTIDES ? truncated or destroyed. Hence, a variety of data support the hypothesis that coeliac disease could be initiated by a Th1-like HLA-DQ2 restricted CD4 T cell response focused on particular (probably deamidated) gluten peptide/s. However, current methods relying upon T cell clones, or challenge of coeliac tissue in vivo or ex vivo are incapable of defining whether peptides are dominant or subdominant in the immunopathogenesis of coeliac disease. In vivo gluten challenge and peripheral blood epitope mapping to define T cell 20 epitope hierarchies Antigen challenge of sensitized subjects results in activation of cognate memory T cells. Memory T cells tend to reside at anatomical sites where their cognate antigen was previously encountered. Recently, memory T cells have been divided into “effector” - + 21 (cytokine-secreting, CCR7 ) and lymph node-homing subtypes (CCR7 ) . We + reasoned that gluten challenge in healthy HLA-DQ2 coeliac subjects following a strict gluten free diet would reactivate gluten-specific memory T cells. The initial intestinal inflammation documented by others may be driven by effector memory T cells, but subsequent appearance of T cells in peripheral blood, perhaps homing back to the gut, may reflect proliferation of gluten-specific T cells in lymphoid tissue during the days after antigen exposure. Hence, the kinetics and frequency of epitope-specific peripheral blood T cells might reveal dominant versus subdominant gluten epitopes. Synthetic 15mer peptides overlapping by 10 aminoacids corresponding to the composite aminoacid sequence (rather than cDNA-derived sequence) of A-gliadin (Fig. 1) with or without in vitro deamidation by tTG were studied in overnight ex vivo A 1. VRVPVPQLQP QNPSQQQPQE QVPLVQQQQF PGQQQ QFPPQ QPYPQPQPFP SQQPYLQLQP B P A 61. PQ PQLPYPQ PQ SFPPQQPY PQPQPQYSQP QQPISQQQ AQ QQQQQQQQQQ B [..] P R Q Q[..] A111. QQQILQQILQ QQLIPC MDVV LQQHNIAHAR SQVLQQSTYQ LLQELCCQHL WQIPEQSQCQ B R GS Q Q A171. AIHNVVHAII LHQQQ KQQQQ PSSQVSFQQP L QQYPLGQGS FRPSQQNPQA B [..] [..] Q S A221. QGSVQPQQLP QFEEIRNLAL QTLPAMCNVY I APYC TIAPF GIFGTN B P [..] 14 Fig. 1. Aminoacid sequence of A-gliadin used to derive overlapping 15mer peptides in 20 gluten challenge studies (A), and residues that deviate from the consensus sequence derived from 61 Genbank Triticum aestivum a - and a/b-gliadin cDNAs using ClustalW (B) ([..] indicates polymorphic insertion).
A DOMINANT EPITOPE IN GLUTEN PEPTIDES ? 21 interferon gamma (IFNg) ELISpot assays using peripheral blood mononuclear cells (PBMC). IFNgELISpot assays define the frequency of cells secreting IFNg, and are capable of detecting peptide-specific T cell frequencies as low as 5-10 per million + PBMC. Blood was collected from HLA-DQ2 healthy non-coeliac and coeliac subjects after gluten free diet for at least 4 weeks, and then in the 12 days after commencing gluten challenge (200 g white bread daily for ½ day [n=1], 3 days [n=10], or 10 days [n=1]). Short-term gluten challenge was generally well tolerated by coeliac subjects, 11/12 subjects were able to consume 200g gluten bread for 3 or more days, and 8/12 had only mild symptoms. One subject had abdominal cramps and vomited within 3 hours of the first 2 slices of bread. All symptoms resolved within 1 to 3 days after ceasing gluten challenge. Prior to gluten challenge, no A-gliadin peptide elicited responses in the IFNg ELISpot. However, in 11/12 coeliac subjects and 0/4 healthy control subjects there was induction of IFNgELISpot responses on day 4-8 for one pool of overlapping peptides only when treated with tTG. In all cases, IFNginduction was attributed to 2 peptides overlapping by 10 aminoacids (A-gliadin 56-75). Aminoacid sequencing demonstrated that only one glutamine residue in A-gliadin 56-75 was susceptible to tTG-mediated deamidation (Q65). Truncations of A-gliadin 56-75 treated with tTG indicated that residues 64-68 (PQLPY) were critical for bioactivity, and that the 17mer QLQPFPQPQLPYPQPQS (57-73) was the optimal peptide. Bioactivity of QLQPFPQPELPYPQPQS was identical to tTG-treated QLQPFPQPQLPYPQPQS, demonstrating that bioactivity of this peptide was dependent upon a single deamidated glutamine residue (QE65). Immunomagnetic bead depletion of PBMC prior to addition of peptide showed that this peptide specific immune response was due to CD4 T cells E expressing the b, 7 but not the aintegrin protein, indicating that A-gliadin 57-73 QE65 specific T cells express the abintegrin 4 7 associated with homing to the intestinal lamina 22 propria . Pre-incubation of PBMC from HLA-DQ2 homozygous subjects with antibody specific for HLA-DQ (but not HLA-DR or HLA-DP) blocks A-gliadin 57-73 QE65 responses, indicating HLA-DQ2 restriction. In one subject who consumed only 2 slices of bread, IFNg-secreting T cells specific for A-gliadin 57-73 QE65 were induced on day 6 and persisted until day 12. In another subject who consumed 4 slices of bread daily for 10 days, A-gliadin 57-73 QE65- specific T cells were also present on day 6-8. IFNgsecretion was only induced by A- gliadin 57-73 QE65 in subjects who consumed bread for 3 days. However, PBMC collected on day 11 from the subject who consumed bread for 10 days secreted IFNgin response to 6 out of 10 pools of tTG-treated A-gliadin peptides (one pool included A- gliadin 57-73). Hence, T cell epitope spreading occurs as early as 10 days after commencing antigen challenge. These studies indicated: 1. In A-gliadin, there is a hierarchy of T cell epitopes with only one dominant T cell epitope. 2. T cells specific for the dominant epitope are present in peripheral blood only
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 42 and 43: 28 ROLE OF A-GLIADIN 31-49 PEPTIDE
Page 45 and 46: Catassi C, Fasano A, Corazza GR (ed
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 89 and 90:
Catassi C, Fasano A, Corazza GR (ed
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:
Page 99 and 100:
IMMUNOTHERAPY OF COELIAC DISEASE 85
Page 101 and 102:
IMMUNOTHERAPY OF COELIAC DISEASE 87
Page 103 and 104:
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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