IBD | APS Undergraduate Researcher

Histologic section of intestinal tissue isolated from healthy mice stained to visualize intestinal epithelial cells. Credit: Lance Peterson, Theresa Alenghat, and David Artis

The epithelium is a layer of cells that separates the inside of the human body from the external environment. In the gastrointestinal (GI) tract, these cells are known as enterocytes and must form a barrier against harmful pathogens present in the gut lumen, while at the same time aiding in the digestion and absorption of nutrients. It is important that all these functions of the epithelium are tightly controlled to maintain homeostasis. Dysregulation of these complex processes has been shown to lead to diseases such as inflammatory bowel diseases (IBDs) which affect over 1 million US residents (Kaplan, 2015). IBDs, which include Crohn’s disease and ulcerative colitis, are characterized by chronic inflammation of the GI tract leading to abdominal pain, weight-loss, fever and a loss of quality of life. While the exact cause of the disease remains incompletely understood, we know that the integrity of the barrier of our GI tract is crucial in IBD prevention (Martini, Krug, Siegmund, Neurath, & Becker, 2017).

At a young age, we are often told to eat our vegetables and that fiber is good for our digestive health, but what does that entail? Recently, we have shown that a dietary fiber known as rhamnogalacturonan (RGal) enhances gut barrier function. Furthermore, we have shown that RGal decreases disease severity in a mouse model of colitis. However, how RGal improves intestinal barrier function remains incompletely understood. My project over the summer aimed to characterize the mechanism through which RGal enhances epithelial barrier function. Specifically, my project aimed to evaluate the role of intracellular proteins known as protein kinase Cs (PKCs) in the modulation of barrier function in an intestinal epithelial cell line in response to RGal. Our lab used an apparatus called the Ussing Chamber to measure epithelial barrier permeability. In my project, I will treat my cells with various chemical inhibitors of PKCs in Ussing Chambers and then determine barrier permeability to small ions in response to RGal. If PKCs are involved in the modulation of barrier permeability in response to RGal, chemical inhibition of PKC will block the beneficial effect of RGal on barrier function. By understanding signalling pathways that enhance barrier function in inflammatory diseases in the GI tract, we have the potential to use dietary fibers such as RGal to leverage these pathways to treat active IBD.

There are two realities of research that I was able to experience this summer. First, I think one of the most rewarding things about my research is that we sometimes did not obtain the results that we expected to. Although this may seem counterintuitive, unexpected results in my project were always the most interesting because I was not only able to observe my supervisor’s stunned reaction, but those results were the ones that allowed us to come up with an alternative hypothesis and steer the project in a completely different direction than we initially planned. I think that the experiments that generate unexpected results are my favorite thing about science. Secondly, I think that the most important thing for people to realize about the day-to-day lives of scientists is that finding the cure for cancer or any other major disease does not happen every day. While understanding the bigger picture in the context of a particular disease and the rationale behind the experiments that we conduct is important and keeps us focused, the things that we study day-to-day often involve understanding the physiological role of a particular cellular protein or defining a cell signalling pathway. Although learning cell signalling pathways may sound a little less exciting than curing cancer, a single cell signalling experiment contributes to the overall body of knowledge which eventually leads to the development of a therapy.

This summer, I was incredibly fortunate to work with the people that I did. First, my supervisors Dr. MacNaughton and Dr. Baggio really allowed me to discover my passion for science. Every day, I am able to see their excitement about my work and their devotion to educating the next generation of scientists. Secondly, my lab mates were some of the most knowledgeable, supportive, and enthusiastic scientists that I know. Five years from now, I will not only remember the science from this summer, but I will still remember our debates about fruit with meat in salads, our arguments about whether or not the word ‘meth’ should be allowed in Scrabble (it shouldn’t), our common frustrations about failed western blots and our disagreements about how to pronounce words like ‘drama’ or ‘garage’.

Judie Shang attends the University of Calgary in Alberta, Canada where she is majoring in Biomedical Sciences. She is an Undergraduate Research Excellence Fellow (UGREF) and is working over the summer with Dr. Wallace MacNaughton at the University of Calgary where she is studying the effect of dietary fibre on the intestinal epithelium. After graduation, she plans to attend graduate school to study mucosal immunology.

Bile acid diarrhea is seen in approximately 10 million Irritable Bowel Syndrome (IBS) patients, and about 86% of Crohn’s patients. Furthermore, there are 90 million patients worldwide annually that have Bile Acid-Associated Diarrhea (BAD). Despite this high prevalence, the underlying mechanism associated with BAD is not completely understood. The primary goal of our research is to identify the mechanism behind bile acid-induced tight junction dysfunction that results in diarrhea. Bile is produced by the liver, released into the duodenum where it helps emulsify fat. Once its job is done, about 95% of bile acid is recycled back to liver, and less than 5% enters the colon. The primary bile acids enter the colon, where anaerobic bacteria convert the primary bile acids to secondary bile acids, and they aid with normal secretion of fluid. However, if the bile acid reabsorption is hindered, as in cases like Crohn’s disease and Celiac disease, there is an excess amount of bile acids that accumulate in the colon, resulting in fluid loss and diarrhea. It has been shown by us and others that bile acids with two hydroxyl groups, such as chenodeoxycholate (CDCA), can alter tight junctions when present in the luminal surface and stimulate chloride secretion when present in the basolateral surface to result in diarrhea. However, the bile acid with one hydroxyl group, lithocholate (LCA), does not alter secretion. In fact, it helps attenuate the barrier dysfunction caused by CDCA. So, how does CDCA travel from lumen to the basolateral surface? This summer, in collaboration with an organic chemist, Dr. Rubush, at my university, we tested the hypothesis that CDCA loosens tight junctions to travel paracellularly to reach basal surface. We synthesized and tested fluorescently tagged CDCA and LCA in order to visually track their movement in experiments. Our results thus far, show that there is a time dependent increase in CDCA content in the basolateral chamber of the transwell. I will continue to test if LCA behaves similarly. The reason for the yin and yang in bile acid action could be due to it structural variation, the lack of a hydroxyl group in the 7th position. Therefore, we are synthesizing 7, methyl CDCA to compare its effects on tight junctions with that of CDCA and LCA.

Being part of a research laboratory has made me realize that the process of doing science is filled with many unknowns and surprises. From having to deal with slow growing cells and lack of the expected band in a Western blot to problems getting the methyl group on the correct hydroxyl group and trying to increase the yield in chemistry lab, I was exposed to the challenges in day-to-day activities in a scientific lab. For example, a minor problem with the air conditioner in our school resulted in the decomposition of an intermediate reactant in the synthesis of methyl CDCA, which set us back by a week or so. Suddenly, our colon cell line started to grow slowly and this made us change the direction of our research temporarily and focus on performing ELISA assays on samples frozen previously.

My day-to-day life as a researcher consists of a combination of planned activities, such as making buffers, media and feeding cells every Monday, Wednesday, and Friday, and designing and running experiments on confluent cells at other times. At first, I thought research would be very repetitive with rigid timings and I would be alone doing experiments independently in a lab. To my surprise, my research timings were very flexible, and I collaborated with students and professors of different departments at my university. In fact, along with organic chemistry students in the summer research program at Benedictine University who synthesized the tagged bile acids, we gave a collaborative, interdisciplinary research presentation which was very well received by faculty and peers from the College of Science. As with life in general, there were good days when we got great data, and bad days when we had contaminated cells; but overall it is a very invigorating experience!

Ugne Dinsmonaite is a senior majoring in Biology at Benedictine University in Lisle, IL. She is a 2017 APS Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jayashree Sarathy’s lab at Benedictine University in Lisle, IL. Ugne’s fellowship is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Ugne plans to pursue a career in dentistry with research in craniofacial pathobiology.