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HLA-DQA1 and Celiac Disease

HLA-DQA1 and Celiac Disease. Presented by Cassie Bac. Image: http://www.scientificamerican.com/article.cfm?id=celiac-disease-insights. Background on Celiac Disease (CD). Autoimmune disease that creates a toxic immune response to gluten-- damaging the lining of the small intestine

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HLA-DQA1 and Celiac Disease

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  1. HLA-DQA1 and Celiac Disease • Presented by Cassie Bac Image: http://www.scientificamerican.com/article.cfm?id=celiac-disease-insights

  2. Background on Celiac Disease (CD) • Autoimmune disease that creates a toxic immune response to gluten-- damaging the lining of the small intestine • Affects 1 in 133 healthy individuals in the United States • Can appear at anytime throughout life! • No known cure No wheat, rye, or barley allowed!! Image: http://www.reallygreatfoods.com/

  3. What are the classic symptoms of CD? • Malnutrition • Abdominal cramping • Intestinal gas and stomach bloating • Chronic diarrhea • Weight loss or weight gain • Dermatitis Herpetiformis Images: http://healthhype.com; http://www.ehow.com/about_5141982_pediatric-celiac-disease-symptoms.html

  4. HLA-DQA1 protein structure • cell-cell recognition • cell surface receptors • immune system • cellular interaction

  5. How is HLA-DQA1 associated with CD? Peptide T-Cell T-cell Receptor Peptide DQA1 Cell Surface Antigen Presenting Cell Images: http://en.wikipedia.org/wiki/Gluten_immunochemistry

  6. What is still unknown? What mechanism causes the toxic immune system response in Celiac Disease? Image: http://jaymecarleton.com/secondnature/green-news/2011/04/toxic-toys-r-us/

  7. What do we know about gliadin? • Gliadin= a protein derived from gluten and the toxic factor involved in Celiac Disease! Question: How does gliadin act??

  8. Experimental Approach 1. Chemical Genetics How does gliadin affect HLA-DQA1 expression levels? How does gliadin affect the phenotype of the small intestine? • Phenotype How is gliadin acting at the molecular level? • Interactions

  9. Experimental Approach How does gliadin affect HLA-DQA1 expression levels? 1. Chemical Genetics How does gliadin affect the phenotype of the small intestine? • Phenotype How is gliadin acting at the molecular level? • Interactions

  10. Experimental Approach How does gliadin affect HLA-DQA1 expression levels? 1. Chemical Genetics How does gliadin affect the phenotype of the small intestine? • Phenotype How is gliadin acting at the molecular level? • Interactions

  11. (1) How does gliadin affect HLA-DQA1 expression levels? Method: Inject mouse intestinal cells with gliadin (+ other varieties) and measure HLA-DQA1 expression levels with microarray

  12. Why use a mouse model? Mouse protein domains H2-Aa Human protein domains HLA-DQA1 71.8% conserved protein structure Images: From SMART. HLA-DQA1 sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.pl; SMART. H2-Aa sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.pl

  13. Microarray data WT patient expression levels Celiac patient expression levels http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1

  14. (1) Hypothesis • The addition of gliadin to mouse intestinal cells should show decreased HLA-DQA1 expression.

  15. (1) Expected Data Celiac patient Celiac patient + HLA-DQA1 Celiac patient + Gliadin WT WT + gliadin Mouse Intestinal Cells - - HLA-DQA1 expression +/- - + = High HLA-DQA1 expression (+) = Low HLA-DQA1 expression (-)

  16. (2) How does gliadin affect small intestine phenotype? Method: Observe phenotype of mouse intestinal cells following gliadin treatment

  17. What do we know about intestinal cells? Microvilli line intestinal cell membrane; involved in absorption. Small intestine Images: http://en.wikipedia.org/wiki/Small_intestine; http://en.wikipedia.org/wiki/Microvillus

  18. CD and the small intestine... Image: Children’s Hospital Boston. 2009. http://www.youngmenshealthsite.org/gluten_free_diet.html

  19. (2) Hypothesis The addition of gliadin to mouse intestinal cells should cause physical damage to the microvilli. In addition, the cells that showed low HLA-DQA1 expression should show increased intestinal damage.

  20. (2) Expected Data Celiac patient Celiac patient + HLA-DQA1 Celiac patient + gliadin WT WT + gliadin HLA-DQA1 Expression Levels Mouse intestinal cells Images: http://health.uml.edu/thc/HealthIssues/Meghan_Donovan/Meghans_Homepage.html

  21. (3) How is gliadin acting at the molecular level? Method: TAP-tag Label gliadin and determine how it is affecting HLA-DQA1

  22. Gliadin may function in this way... Gliadin peptide? T-Cell Gliadin peptide? T-cell Receptor DQA1 Cell Surface Antigen Presenting Cell

  23. HLA-DQA1 protein interactions Question: Is gliadin regulating/binding HLA-DQA1? Image: From STRING 8.3. HLA-DQA1 evidence map. Retrieved from: http://stringdb.org/newstring_cgi/show_network_section.pltaskId=8jlOaZSDtoSa&interactive=yes&advanced_menu=_unassigned&network_flavor=evidenc

  24. What role do the predicted functional partners play? Question: Is gliadin regulating/binding any of these interacting proteins?

  25. (3) Hypothesis Using Tandem Affinity Purification, gliadin should be found to regulate HLA-DQA1 protein at the molecular level. Image: http://www.nature.com/nrm/journal/v4/n1/fig_tab/nrm1007_F1.html

  26. (3) Expected Data Mass spectrometry to analyze the protein-protein interactions Gliadin binds HLA-DQA1 Gliadin binds interacting proteins Image: http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/DrugChemicalResiduesMethodology/ucm113209.htm

  27. Future Directions • MUDPIT- Multidimensional Protein Identification Technology • Hypothesis: HLA-DQA1 protein observed in WT patient tissue samples, not observed in celiac samples. • Experiment: Take intestinal tissue samples from WT and celiac patients and observe specific protein identifications from the two samples.

  28. Future Directions • Microarray primary HLA-DQA1 interacting proteins- look at expression levels in WT vs. Celiac Disease patients. Are their levels expressed in similar patterns as HLA-DQA1?

  29. THE END Image: http://www.herbalgranny.com/2009/09/06/herbal-treatment-for-celiac-disease/

  30. References • University of Chicago Celiac Disease Center. Celiac Disease Facts and Figures. Print. • SMART- http://smart.embl-heidelberg.de/ • GeneBee- http://www.genebee.msu.su/genebee.html • http://knol.google.com/k/celiac‐disease# • Bizzaro, N., R. Tozzoli, D. Villalta, M. Fabris, and E. Tonutti. "Cutting‐Edge Issues in Celiac Disease and in Gluten Intolerance." Clinical Reviews in Allergy and Immunology 15.1 (1997): n. pag. Web. 7 Feb 2011http://www.springerlink.com.ezproxy.library.wisc.edu/content/j17wu631711u1027/fulltext.pdf. • http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001280 • PubChem- http://pubchem.ncbi.nlm.nih.gov/ • STRING- http://string.embl.de/ • Homologene- http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=AlignmentScores&list_uids=123820 • Microarrays- http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1

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