1. “Sprue” is a somewhat antiquated term referring generally to intestinal malabsorption

2. Celiac Disease One of the most common immune-mediated disorders Prevalence in U.S./Europe  1 % –Implies that approx. 3M afflicted in U.S. alone Causes destruction of villous structure in the intestine –Symptoms: diarrhea, fatigue, abdominal pain, malabsorption, neurological abnormalities

3. Celiac Disease Celiac Sprue is a lifelong disease, and if untreated it is associated with increased mortality

4. Celiac Disease Triggered by ingestion of wheat gluten.

5. Gluten “Gluten” is a general term for a composite of the storage proteins gliadin and glutenin. –These proteins (conjoined with starch) comprise ~80% of the total protein in wheat/rye/barley seed.

6. How does gluten cause negative effects? Certain Pro- and Gln-rich gliadin peptide fragments survive the digestion process & make it to the gut These peptides are deamidated by tissue transglutaminase (tTGase) APCs in HLA-DQ2 or –DQ8 positive individuals express these deamidated peptide fragments on class II MHC molecules The resulting CD4 + T-cell mediated immune response can eventually result in the development of celiac disease

8. Therefore, the only currently recognized treatment for celiac disease is complete abstinence from food grains containing gluten proteins –Bread, beer, cereals, many sauces… I tried a gluten-free diet for a month this year… Let’s just say that I wasn’t the nicest guy to be around during that month. It’s difficult!

10. Aims of this study Determine the physiologically stable regions of gliadin Investigate the biochemical basis of this stability Shed light on possible future treatments for celiac disease

11. “33-mer” = LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF Fig 1. gliadin digested with gastric + pancreatic proteases (pepsin, trypsin, chymotrypsin, elastase, caboxypeptidase) followed by LC- ESIMS

12. Significance of 33-mer Even after prolonged exposure to proteases, the 33-mer reamains intact Three previously identified epitopes (PFPQPQLPY, PQPQLPYPQ, and PYPQPQLPY) are found within this 33-mer “33-mer” = LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

13. WQIPEQSR fragment used as a control

14. Fig. 2. BBM digestions (A) 33-mer (B) Epitope within 33-mer BBM: brush border intestinal membrane enzymes  essential for breaking down any remaining peptides after gastric and pancreatic digestion

15. Fig. 2. 33-mer LQLQPFPQPQLPYPQPQLPYPQPQL PYPQPQPF Epitope within 33-mer QLQPFPQPQLPY

16. Implication  there’s something about the 33-mer which makes it highly resistant to physiological enzymatic degradation

17. Fig 3. Stimulation of 3 HLA DQ2-restricted T cell clones, derived from patients with celiac disease. Each of these T cell clones recognizes a class II MHC molecule presenting a distinct deamidated gluten epitope. EC 50 ~ 80 nM (EC 50  median effective concentration) Implication  the 33-mer is a very potent stimulator of these HLA DQ2 restricted T cell clones, even more so than the epitopes by themselves… Why??

18. Table 2. Respose of polyclonal T cell lines derived from patients with celiac disease to gluten epitopes (peptide X) and the 33-mer (bottom row) “… we interpret the combination of metabolic stability and multivalency of the 33-mer to endow it with exceptional toxic potency…”

19. Why is the 33-mer so resistant to gastrointestinal breakdown? This is likely due to the abundance and location of proline residues This gave the authors the bright idea to investigate the activity of a prolyl endopeptidase (PEP) on breakdown of the 33- mer… –(prolyl endopeptidases cleave peptides after proline residues)

20. Fig 4. (A)33-mer incubated in vitro with PEP (B)33-mer digested in vivo in rat intestine with and without PEP (C)Stimulation of HLA DQ2 restricted T cell clone by 33-mer after PEP and BBM treatment, followed by tTGase treatment PEP seems to effectively catalyze breakdown of the 33-mer

21. Overall implications of this study The 33-mer peptide, derived from gliadin, shows several characteristics suggesting that it is the primary initiator of the autoimmune response to gluten in patients with celiac disease. –Potential for much further study… vaccine?? Prolyl endopeptidase therapy may have the potential to detoxify gluten in celiac disease patients.

22. Kim, et al. PNAS (2005) X-ray crystal structure of 33-mer bound to HLA-DQ2

23. These are test results from a close family member, showing indications of celiac disease

24. Questions?