Team:UC Davis/Project

        Celiac Disease is autoimmune disorder that occurs inside the small intestine. When the body cannot digest gliadin (component of gluten) properly, this leads to an immune response in the surface of the small intestine (14). Furthermore, different people have different reactions to this immune response such as bloating, diarrhea, and weight loss (14). It has been estimated that about one out of 133 Americans are currently suffering from this disease (11); including one of our teammate's friends, and that was what sparked our interest in designing this project.

What happens in the normal small intestine?
       
        In the normal small intestine, the brush border membrane on the surface digests starches and other sugars, proteins, and fat droplets and lets their breakdown products pass into the bloodstream.

What happens in Celiac disease small intestine?

        The brush border membrane in the small intestine of people suffering from Celiac Disease allows large molecules such as gliadin to go straight into the blood stream without directing them through the transcellular route. Once gliadin passes through, Antigen Presenting Cells (APCs) recognize gliadin as a foreign object and will attack. This immune response results in inflammation of the surface of the intestine with loss of the normal cells required for absorption of sugars, protein, and fat from the diet.

The immune response not only causes abdominal pain, bloating, diarrhea and weight loss it may also damage intestinal villi, which are important for absorbing nutrients. The destruction of villi eventually leads to a lack of absorption of different nutrients, which can lead to malnutrition (14). Some people affected with this disorder can also suffer from autoimmune thyroid disease, autoimmune liver disease, and rheumatoid arthritis (diseases in which body immune system attacks healthy cells/tissues) (14).

Current treatments:

        Currently, no cure has been found for this illness. The only way to avoid some of celiac disease’s symptoms is by adopting a gluten free diet (14, 12, and 11). However, you are more likely to find gluten in your everyday diet, and people with celiac disease must purchase gluten-free substances from specialized grocery stores. "Oral supplementation with prolyl oligopeptidases that can digest and detoxify gluten has therefore been proposed as a potential therapeutic approach."(8). However, enzymes studied earlier were not able to degrade gluten inside stomach (before it reaches small intestine) because they were "irreversibly inactivated by pepsin and acidic pH, both present in the stomach."(8)

        Nevertheless, over the past years, researchers have discovered an enzyme from Aspergillus niger, a newly identified prolyl endoprotease, that was observed to "work optimally at 4-5pH and remains stable at 2pH"(8) and maybe this enzyme will lead us to an alternative treatment for this disorder (8). Studies have shown that prolyl-endoprotease from Aspergillus niger is able to "degrade gluten in vitro and under conditions similar to the ones present in the gastrointestinal tract." (8) ; but due to licensing restrictions we have opted not to work with this protein.

        Recently (year 2007), a study has suggested an alternative approach by combining a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata); with gastric activity and complementary substrate specificity there is a possibility of increasing the safe threshold of ingested gluten (12). One of the advantages of this “combination product is that both enzymes are active and stable in stomach and can therefore be administered as lyophilized powders or simple capsules or tablets” (12).

Note: Moreover, this study was first “evaluated via in vitro digestion of whole-wheat bread and then confirmed by in vivo studies in rats (12), unlike other earlier studies which were performed on synthetic gluten oligopeptides, recombinant gliadin proteins, or uncooked gluten” (12).

        Gliadin induces an immune system response when it is absorbed into the blood stream through the small intestine. We plan to take advantage of this by breaking down gliadin in the stomach before it reaches the small intestine. We have developed a secretion system to release an enzyme that can break down gliadin. Also, in order to prevent our delivery system from taking residence anywhere outside of the stomach, we plan to incorporate the difference in pH between the stomach and small intestine with a pH-inducible apoptosis system. By adopting these two systems, we will create a pH-moderated secretion system.
        Unlike other suggested treatments requiring the consumption of capsules or tablets, our method of using E. coli as our delivery system is more cost- and time-efficient.

Our project is split into two parts:
1.Induced Secretion

• Gene sequence for secretion system
• Adding secretion
• Assay for measuring protein localization and activity

• Finding an appropriate biological pH sensor
• Wiring the pH sensor into our system
• Assay for measuring the behavior of the pH sensor