Team:UC Davis/Project

Project:
What is Celiac Disease?

        Celiac Disease is an autoimmune disorder that occurs inside the small intestine. When the body cannot properly digest gliadin (a component of gluten), this leads to an immune response on the surface of the small intestine (14). Different people have varying degrees of immune response such as bloating, diarrhea, and weight loss (14). An estimated one out of 133 Americans currently suffer from this disease (11). Considering the estimated 3.08 million people in America, this isn't a minor issue. In fact, it was one of our teammate's friends suffering from celiac who initially sparked our interest in designing this project.

What happens in the normal small intestine?
       
        The inside surface of the small intestine is covered with small microvilli, constituting the brush border membrane. In the normal small intestine, the brush border membrane is where polysaccharides, proteins, and fat droplets are digested into smaller parts that then get absorbed into the bloodstream.

What happens in the small intestine of people with celiac disease?

        The brush border characteristic to people with 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 attack. This immune response results in the inflammation of the surface of the intestine with loss of the normal cells required for absorption of sugars, protein, and fat from the diet.

        In addition to the array of symptoms illustrated above, the immune response 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).

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Current treatments:

        Currently, no cure has been found for this illness. The only way to avoid the symptoms of celiac disease is by adopting a gluten-free diet (14, 12, and 11). However, you are highly likely to find gluten in your everyday diet (in foods such as grains), 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 the stomach (before it reaches the 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--a newly-identified prolyl endoprotease--from Aspergillus niger, that was observed to "work optimally at 4-5pH and remains stable at 2pH"(8). Perhaps this enzyme will lead us to an alternative treatment for this disorder(8). Studies have shown that prolyl endoprotease from A.niger is able to "degrade gluten in vitro and under conditions similar to the ones present in the gastrointestinal tract." (8) ; However, 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(12). Using this method, 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).

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Our approach:

        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.

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

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

2. Sensing pH  and inducing cell death

• Finding an appropriate biological pH sensor
• Wiring the pH sensor into our system

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