“ Sprue ” is a somewhat antiquated term referring generally to intestinal malabsorption - PowerPoint PPT Presentation

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

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

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


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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


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Celiac Disease

Celiac Sprue is a lifelong

disease, and if untreated it is associated

with increased mortality


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Celiac Disease

  • Triggered by ingestion of wheat gluten.


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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.


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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


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  • 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!


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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


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Fig 1. gliadin digested with gastric + pancreatic proteases (pepsin, trypsin, chymotrypsin, elastase, caboxypeptidase) followed by LC-ESIMS

“33-mer” = LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF


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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


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WQIPEQSR fragment used as a control


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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


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Fig. 2.

33-mer

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

Epitope within 33-mer

QLQPFPQPQLPY


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


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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.

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??

EC50 ~ 80 nM

(EC50 median effective concentration)


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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…”


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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)


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  • Fig 4.

  • 33-mer incubated in vitro with PEP

  • 33-mer digested in vivo in rat intestine with and without PEP

  • 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


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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.


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X-ray crystal structure of 33-mer bound to HLA-DQ2

Kim, et al. PNAS (2005)


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


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Questions?


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