primary prevention of coeliac disease - Associazione Italiana ...

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.