J S Fraser

Coeliac disease: in vivo toxicity of the putative immunodominant epitope

Background: Peptides from α-gliadins have been used to characterise the immunodominant coeliac toxic epitope. A peptide corresponding to amino acid residues 57–73 of A-gliadin causes peripheral blood mononuclear cells from coeliac patients to secrete interferon γ (IFN-γ); gluten specific small intestinal T cell clones proliferate in response to peptides corresponding to residues 57–68 and 62–75 of α-gliadins. We wished to investigate whether a peptide corresponding to residues 56–75 of α-gliadins exacerbates coeliac disease in vivo. Methods: Four adults with coeliac disease, all of whom were on a gluten free diet, underwent three challenges. Peptic-tryptic gliadin (PTG 1 g) served as a positive control. The test peptide and a negative control peptide were studied on separate occasions. The peptides were instilled into the duodenum and biopsies were taken before the infusion, and two, four, and six hours after commencing the infusions, using a Quinton hydraulic multiple biopsy capsule. Biopsy specimens were assessed blindly for villus height to crypt depth ratio (VH:CD), enterocyte cell height (ECH), and intraepithelial lymphocyte (IEL) count. We used the Mann-Whitney U test, with 95% confidence intervals, for statistical analysis. Results: VH:CD and ECH fell, and IEL increased significantly 4–6 hours after commencing infusions with both PTG and the test peptide in all subjects. The negative control peptide caused no significant changes to villus morphology, enterocyte height, or IEL count in any patient. Conclusion: We have confirmed that the putative immunodominant epitope, a peptide corresponding to residues 56–75 of α-gliadins, exacerbates coeliac disease in vivo.

Investigation of the putative immunodominant T cell epitopes in coeliac disease

Background: Coeliac disease (CD) is an enteropathy mediated by gluten specific T cells which secrete interferon γ (IFN-γ) when stimulated by gluten peptides presented by HLA-DQ2 or DQ8 molecules. Residues 62–75 of α2 gliadin have been proposed as the immunodominant epitope in the majority of CD patients. Deamidation by tissue transglutaminase (tTG) of the glutamine (Q) at position 65 to glutamic acid (E) is essential for T cell stimulation. Aims: To investigate the antigenicity of this peptide and to establish whether its T cell activating properties can be downregulated by the formation of altered peptide ligands. Patients: Individuals with known CD. Methods: Peptide G4 corresponding to α2 gliadin residues 62–75, Q-E65 and analogues, substituting each amino acid, except E65, in turn for alanine residues, were synthesised. Small intestinal biopsies were obtained from patients. Biopsies were cultured overnight with a peptic/tryptic digest of gliadin (PTG). Lymphocytes were cultured and restimulated with tTG treated PTG. A T cell line was cloned and clones tested for stimulation and IFN-γ production in response to G4 and its analogues. Results: Some high activity clones were isolated with, for example, a stimulation index (SI) of 15 to G4 and secreting 327 pg/ml of IFN-γ. Substitution of amino acids at several positions abolished or downregulated stimulation and IFN-γ production. Conclusions: Peptide G4 is highly immunogenic. Certain amino acid substitutions in peptide G4 abolish T cell reactivity while others are partial agonists which may have potential in immunomodulation in this condition.

CTLA-4/CD28 gene region is associated with genetic susceptibility to coeliac disease in UK families

Coeliac disease (CD) is a malabsorption disorder characterised by a small intestinal enteropathy that reverts to normal on removal of dietary gluten. Susceptibility to disease has a strong genetic component. Ninety percent of patients in northern Europe have the HLA class II alleles DQA1*0501 and DQB1*0201, which encode the cell surface molecule HLA-DQ2.1 However, haplotype sharing probabilities across the HLA region in affected sib pairs suggest that genes within the MHC complex contribute no more than 40% of the sib familial risk of CD, making the non-HLA linked gene (or genes) the stronger determinant.2 Attempts have been made to identify these loci using genome wide linkage studies. Zhong et al 3 performed an autosomal screen in 45 affected sib pairs from the west coast of Ireland, using 328 microsatellite markers. They found evidence of linkage with lod scores of greater than 2.0 in five areas: 6p23 (separate from HLA), 7q31.3, 11p11, 15q26, and 22cen. A larger genome wide search involving 110 affected Italian sib pairs using 281 markers found no evidence of linkage in these five areas.4 It did, however, propose a novel susceptibility locus at 5qter, important in both symptomatic and silent CD, and another at 11qter, which appeared to differentiate the two forms. In UK families an initial genome wide search,5 followed by a study of 17 candidate regions6 identified five areas with lod scores of greater than 2.0: 6p12, 11p11, 17q12, 18q23, and 22q13. Of these, 11p11 replicates one of the loci identified by Zhong et al 3 and it is likely that this area contains an important non-HLA susceptibility locus. However, in general the results of these studies are disappointingly inconsistent. A number of candidate genes have been investigated in linkage and association studies. Of these, the only region with repeatedly …

Characterizing one of the DQ2 candidate epitopes in coeliac disease: A-gliadin 51-70 toxicity assessed using an organ culture system.

Objective To investigate, using an organ culture system, in-vitro toxicity of region 51–70 of A-gliadin (SQQPYLQLQPFPQPQLPYSQ), a peptide overlapping some of the sequences recently characterized as DQ2-restricted T-cell epitopes in coeliac disease. Methods Jejunal biopsies obtained from each of ten coeliac patients (eight treated, two untreated) and two non-coeliac patients were cultured in vitro for 18 h in the presence of A-gliadin amino acids 51–70 (200 μg/ml), organ culture medium only, peptic–tryptic digest of gliadin (1 mg/ml) or ovalbumin (1 mg/ml), the last two acting as positive and negative controls, respectively. Morphometric analysis involved measuring the cell height of 30 enterocytes, selected at random from the middle third of different villi for each section. Mean enterocyte cell heights (ECH) were compared with values for specimens cultured in medium alone. Levels of tissue transglutaminase antibody in biopsy supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). Results In eight of ten coeliac patients, A-gliadin 51–70 was significantly toxic, causing a 30% decrease in ECH when compared with medium alone. In two of ten subjects, the peptide did not show any toxic effect. In all ten cases, we found that both positive and negative controls worked as expected. The peptide was non-toxic in the non-coeliac individuals. Tissue transglutaminase antibody titre in the supernatant was not found to be related to mucosal damage. Conclusions We showed that the peptide corresponding to amino acids 51–70 of A-gliadin is characterized by in-vitro toxicity to the jejunal coeliac mucosa, correlating with recent findings of an immunological role of similar peptides. The lack of response in two of ten subjects suggests that this epitope may not be relevant in all cases of coeliac disease.