Celiac Disease Research Project

[eroufs]

I am a senior in high school and I am working on a science fair project about celiac disease. I would ideally like to study the effects of gluten on the intestinal villi but this is obviously very difficult to do in vivo without biopies. I would like to compare conventional wheat to an heirloom species of wheat, such as einkorn, and see if the immune system reacts differently.
So my question essentially is: is there a way to study how the body would react to gluten without involving human subjects? I was brainstorming about culturing intestinal mucosa cells, but I am not sure exactly how I could do this...
Any ideas or comments are appreciated!
Thank you!

[SciB]

Hi,

That is a very interesting and worthwhile area of disease you have chosen to study but it is going to be a tough one to do a science fair project on. As you say, human research would be out of the question and also animal research. Experiments could be done on human intestinal epithelial cells but what you really are interested in is the immune response. Celiac disease or CD is an autoimmune disease that seems to occur in genetically predisposed persons. I don’t know how much you have studied immunology but one genetic alteration is in a protein that makes up the T lymphocyte receptor. These are white blood cells commonly called T cells that have the job of distinguishing between normal, nonharmful proteins such as those from wheat gluten and those from a pathogen such as influenza. 95% of the people with CD have a variant form of the T cell receptor (TCR) that causes their T cells to become activated when exposed to gluten protein. This triggers what is called an immune response and causes inflammation and tissue damage. That’s why people with CD suffer from problems in the upper part of the intestine.

Your question was whether an old variety of wheat such as einkorn might not cause a response in CD patients. That’s a great idea, now how can you test it? There are two ways that I can see where you can go with this depending on if you have access to a university lab and a grad student or post doc who is willing to spend time with you. Do you know any people doing research on CD? If so, try to get them interested in your project so you can work in their lab. There are human T cells that can be cultured in liquid in Petri dishes and you could expose them to various types of gluten protein and measure their response in several different ways. These experiments require specialized reagents and equipment not found in most high school labs.

The other way you can go, which does not require a lab, is to use the free online molecular biology software and protein databases to find the gluten peptide sequences that are being recognized by the TCR and compare them for different wheat varieties. You’ll have to do some serious googling and hunting on PubMed to find exactly which amino acid sequence is responsible for T cell activation in CD patients..

The potential problem with the database project is whether or not the einkorn genome has been sequenced or at least the gluten protein part of it. If you find that the gluten peptides from contemporary durum wheat are identical to those of old varieties of wheat then that tells you right away that it would be unsafe for a CD patient to eat the old wheat varieties.

I hope I have helped you to see the project possibilities without cluttering up the picture with too many technical details. I just wanted you to understand what you are dealing with better. I’m sure you will have lots more questions, so send more posts and we will try to steer you along the pathway to a winning project.

Good luck,

Sybee

[eroufs]

Thank you for your reply!
I am familiar with the details of how CD develops and affects the body, I have it myself. In fact, I believe the specific TCR genes you were referring to are HLA-DQ2 and HLA-DG8. What you said about studying the IMMUNE response to different types of wheat instead of the "intestinal response" makes complete sense to me. However when it comes to the actual science of immunology and genetics, my knowledge more limited. Is it possible to test human T cells, as you mentioned, that do have these genes so their reaction to the gluten protein mimics that of someone with CD? If so, what materials, procedures, and equipment is required to do this? My school lab is not equipped to do this, but there is a small possibility I could work at a local university lab. Before I contacted them, I wanted to have a strong research purpose and a general idea of what procedures I would need to do, what equipment to do it, and who would be the most helpful to my project (this makes it easier for everyone involved).
The possibility of doing online research is a very good backup in case doing the actual lab research falls through. I will begin to look into this and if I do get the opportunity to work in a lab, it will still be a very interesting addition to any experiments I do!
Thanks again!

[caraskl]

Hi,

You might want to research HLA-DQA1 and HLA-DQB1 genes, because variants of these genes are associated with an increased risk of developing celiac disease. HLA stands for Human Leukocyte-Antigen, and members of the HLA gene family produce proteins that distinguish the body's normal cells and proteins from foreign viruses and bacteria. Proteins encoded by HLA-DQA1 and HLA-DQB1 are involved in forming an antigen-binding heterodimer that attaches to immune cells in order to facilitate the recognition and attack of foreign cells by immune cells. Although certain haploytpes are associated with an increased risk for immune dysfunction, other factors besides genetic ones may contribute to celiac disease. Further information can be found at http://ghr.nlm.nih.gov/gene/HLA-DQA1. If you want to compare gene sequence information, you may want to visit NCBI BLAST.

In addition, you might also want to compare the gene sequences of different varieties of wheat using NCBI BLAST. The six most common types of wheat used to make bread in the U.S. are hard winter read wheat, hard spring red wheat, soft winter read wheat, hard winter white wheat, and soft spring white wheat. Compared to these modern varieties of wheat, einkorn wheat is lower in gluten and higher in micronutrients http://nourishedkitchen.com/good-questi ... nt-grains/. Genetically, Einkorn wheat differs from most varieties of bread wheat, in that Einkorn wheat is a diploid with two complements of seven chromosomes, whereas bread wheats are hexaploids with three sets of chromosomes.

In sum, comparing the genetics of different varieties of wheat and HLA genes may provide some insight as to why an some people develop celiac disease.

[eroufs]

This is very helpful information to me! As I start to do more background research for my project, I need to develop a better understanding of all of these factors. These links give me a fabulous place to start! In regard to the specific genetic characteristics that predispose someone to CD, what is the actual gene sequence responsible? Is it HLA-DQA1 and HLA-DQB1 or HLA-DQ2 and HLA-DQ8? There was some explanation in the first link you gave me, but I am still a bit confused. If I understood it correctly, it says that these two haplotypes are found in 30% of the population but only 3% develop CD. Why is this?
Thank you!

[deleted-140482]

Hi eroufs,

You have a very interesting topic developing. With regards to your question about the actual gene sequence predisposing to CD, I think I can help clarify somewhat. The name of the genes of interest are HLA-DQA1 and HLA-DQB1. Everyone has these genes, but the exact sequence of the gene will vary from person to person. These are called alleles. So there is a specific allele of HLA-DQA1 that might predispose one to CD (according to the article linked to in the last post, HLA-DQA1 05:05 (or 05:01). Carrying one of these alleles alone is probably not sufficient to cause any significant increase in your chances of getting CD, but when someone has both this 05:05 allele of HLA-DQA1 AND the 02:02 allele of HLA-DQB1, this is called having a DQ2 haplotype, and predisposes one to having CD. The same is true for DQ8, which is the combination of different allele variants that also predispose to CD. Did that help clarify the difference between the GENE and the allele variants, and the DQA1 gene and the DQ2 haplotype?

As for why these two haplotypes are found in 30% of the population, but only 3% develop CD, that is a very important and interesting question that scientists are actively trying to understand. In a general sense, you are talking about the penetrance of CD, which is the proportion of individuals carrying a particular variant of a gene (i.e., an allele) that then express the associated trait. With some very genetic disorders, if you carry the mutation, you WILL develop the disease. For example, anyone who carries certain alleles in the cystic fibrosis gene, will get cystic fibrosis. Many other disease are much more complicated, though. Cardiovascular disease, for example, has a number of causes, and while certain alleles will predispose you to high cholesterol, other factors, like diet and exercise also affect your chances of having a heart attack, so even if you carry these alleles, you might never get a heart attack.

Celiac Disease is more like cardiovascular disease than cystic fibrosis. While we know that certain alleles in the HLA genes predispose to CD, there are also other factors, probably largely environmental, that seem to affect whether a person actually ends up with CD. What these factors are is not entirely known yet, but we do know that there is a role for the bacteria that colonize your gut in CD, and there may be any number of other factors, like exposure to certain inflammatory stimuli at certain ages, additional genes, etc. This is definitely an active area of research, so if you do some reading on it, you may find something new that helps to answer the question.

I hope this helps and good luck finding a project on CD that is interesting to you!
JMP

[deleted-210226]

Very interesting discussion on an interesting question!

If you wanted to go in more of a social science direction, and were willing to obtain preapproval from the institutional review board (https://www.sciencebuddies.org/science- ... ects.shtml), another topic is how celiac disease is affecting the lives of those who have it. E.g., you could develop a structured survey and/or interview questions and then identify and survey some people who have CD regarding their experiences.

Just a thought - both the genetic database and laboratory research ideas are great!

[eroufs]

Hello!
Thank you to everyone who has posted replies to my questions!
I have attempted to get into several labs in my area without any success. I am willing to travel (if reasonable) to get access to a lab if anyone is willing or knows someone who is willing to help me with my project!
Also, could someone give me an idea of exactly what equipment, cultures, procedures, etc. I would need to study the effects of gluten on immune cells from a subject with Celiac? If I have a better idea of what my lab testing would potentially look like, it would help me figure out what route my project is going to to take at this time...
Any other background information or ideas are GREATLY appreciated!
Thanks!

[SciB]

Hi,

You are proposing a project that will use blood from humans and that requires special permission from an institutional review board in the university where the lab is located. Here’s what that involves: http://en.wikipedia.org/wiki/Institutional_review_board

Many labs already have IRB permission to use human subjects so you should look for labs in universities that have an associated college of medicine where biomedical research is done.

Once you obtain the blood from volunteers you will need to separate the lymphocytes from the other cells and there are methods to do that: http://www.youtube.com/watch?v=Cl4sTwKFe9k

The cells you obtain are known as mononuclear cells and contain the immune cells you want to test with gluten protein: http://en.wikipedia.org/wiki/Peripheral ... clear_cell

Lymphocytes can be separated from macrophages if desired because the macrophages adhere to plastic culture dishes. You can grow these cells for several weeks in special culture media in disposable plastic flasks or Petri dishes. Cell culture is a specialized technique but one that is very common in biomedical research labs. It takes practice but if you pay attention and are careful it is not difficult. There are lots of youtube videos showing how to do it.

The hardest part of your project in my opinion will be deciding what to measure for the lymphocyte response to gluten protein. This is something you will need to discuss with a prospective mentor as there are several highly specialized techniques.

This is definitely going to be a relatively long and difficult project. I would allow a month or more just to learn the methods then a couple more months to get the cells and begin the testing. When you talk to a prospective mentor, one of the first things they will ask you is how much time you can devote to the project. Cells in culture have to be fed every couple of days and replated when they grow too dense. The actual experiments can take many hours and often several days to complete.

Good luck and we’ll be here to help you along.

Sybee