Team:TU-Eindhoven/Description

Description

“If you are feeling really sick, you do everything to feel better” - Interviewed patient

Here you will find the total description of our project, beginning with our team formation and motivation, continuing all the way to our final product and future research. Throughout the page, you will find links to the rest of our wiki for more detailed descriptions and information.

The Team

In late January, early February we started off as a small team of six MSc. (Bio)medical Engineering students looking to solve a problem in the field of healthcare. Soon after, we found two more well motivated and nimble-witted students willing to join us in this adventure, completing our team for this year.

After weeks of deliberation, reading papers and consulting established individuals in the field of healthcare, such as our supervisors, we came across several interesting articles. One particular article provided by the supervisors described a novel approach for abdominal imaging that utilises gene expression and ultrasound equipment, which we could relate to Inflammatory Bowel Disease (IBD). We quickly learned of the severity and prevalence of this disease, as well as its inadequate golden standard for monitoring. Many of us were personally motivated about this topic as we either have distant relatives or know people that suffer to some degree from IBD. This has resulted in a project on which we worked with great passion and vigor.

The Team

Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is an umbrella term used to describe disorders that involve chronic inflammation of your digestive tract, and includes types, such as Crohn's disease and Ulcerative Colitis. Crohn's disease is characterized by inflammation of the lining of your digestive tract, and often involves the deeper layers of the digestive tract. Ulcerative Colitis on the other hand, is a condition that is characterized by continuous inflammation and sores (ulcers) along the superficial lining of your large intestine and rectum. Both diseases typically result in symptoms, such as diarrhea, rectal bleeding, abdominal pain, fatigue and weight loss. IBD can be debilitating and some-

times leads to life-threatening complications. Both diseases are characterized by flare-ups followed by periods of remission [1].

The exact cause of IBD is unknown, but IBD is the result of a defective immune system. A properly functioning immune system attacks foreign organisms, such as viruses and bacteria, to protect the body. In the case of IBD, the immune system responds incorrectly to environmental triggers, which causes inflammation of the gastrointestinal tract. External factors that may trigger IBD include smoking, lifestyle, diet, medication, vitamin D deficiency, and transition to a western-style diet [2,3].

Incidence & Prevalence

Worldwide, over 6.8 million people suffer from IBD. The direct health-care cost is estimated to be €4.6–5.6 billion annually in Europe [4,5], and is further touched upon in the Integrated Human Practices segment The Need. The incidence of IBD rose steadily in the twentieth century in the Western world, but IBD was relatively rare in developing nations. However, over the past few decades, newly industrialized countries in Asia, South America and the Middle East have documented the emergence of IBD. The incidence of IBD in these newly industrialized countries is still considerably lower than that in the Western world; yet, the rate of the rise in the incidence of IBD is considerably higher. Furthermore, the newly industrialized countries of

India and China each have a population exceeding one billion people. These large populations in conjunction with expanding urbanization and westernization might mean that the number of cases of IBD in newly industrialized countries could, at some point, overtake the number of cases in the Western world [4,6].

State of the Art

The current golden standard for monitoring disease activity in IBD patients consists of two components: a digital survey system and a home fecal calprotectin test. Patients are requested to complete a monthly survey, which contains questions on all facets of this complex disease. This includes disease activity, extraintestinal manifestations, medication use, stratification and side effects. In addition, the survey contains questions about more general aspects to get a better overall picture of the condition of the patient, such as work productivity, physical exercises, smoking, nutrition and psychosocial factors. When the disease is in remission, patients fill out the survey once per three months. In case of a flare up, patients are requested to fill out the survey weekly [7–9].

In addition to the survey, the patient has to perform a home fecal calprotectin test, such as IBDoc. This is a lateral flow, point-of-care device—similar to the COVID-19 self test—that measures the calprotectin levels in patients' stools. Calprotectin is present in several bodily fluids at levels proportional to the degree of inflammation [10]. In case of a positive test result, the patient must go to hospital for a follow-up, which often results in an endoscopy and biopsy being performed [3,11]. This means the patient’s colon will be inspected using a long tubular camera, and a tissue sample will be taken. In the days prior to this procedure, the patient must take strong laxatives and abstain from eating to prepare the intestines for the procedure.

Shortcomings

As briefly explained, the current monitoring procedure—i.e. the fecal calprotectin test—lacks specificity, resulting in unnecessary follow-up examinations such as endoscopies that are invasive, expensive, complex, and time consuming [10]. For instance, several trained staff members, typically two nurses and a gastroenterologist, are required to perform an endoscopy. It also requires patients to interact with and measure out fecal samples, often multiple times. Patients are required to use strong laxatives and fasten prior to follow-up examination, on top of having to alter their diets days prior to examination, as described in our Integrated Human Practices segment The Need [12]. This calls for an additional intermediate procedure that can time-efficiently rule out these false-positive test results, in a non-invasive, cost-effective, easy-to-use manner.

Our Solution - IBDetection

This paper, written by Bourdeau et al. and published in 2018 [13], describes the development of acoustic reporter genes (ARG), and was provided by our supervisors as a novel approach for the use in ultrasound imaging, which is further described in the Integrated Human Practices segment The Science. The ARG encode for structural & scaffolding gas vesicle proteins, which when expressed oligomerize into gas vesicles in the bacterium's cytoplasm. The gas vesicles could be collapsed acoustically and showed clear ultrasound contrast after image-analysis. More information on ARG can be read on our Proof of Concept page. The paper described induction of ARG protein expression using IPTG, and was able to characterize a clear dose-response from the ultrasound measurements.

We theorized that if we can couple this signal output to a molecularly detectable signal input that is suitable for our user case, i.e. the intestines of an IBD patient, we would have a living cell-based sensor design on our hands with the potential to be a non-invasive alternative for detecting intestinal inflammation.

This paper, written by Daeffler et al. and published in 2017 [14], describes the use of a two-component protein sensing system for the use of detecting metabolites that are characteristic for intestinal inflammation with high specificity, more specifically: thiosulfate and tetrathionate. More details on the sensing part can be read on our Proof of Concept page and in the Integrated Human Practices segment The Science. The paper used fluorescence in the form of sfGFP expression as output, characterizing the system and it’s dose-response. Detection of tetrathionate was highly specific compared to a range of similar molecules.

Continuing our theorizing, and identifying tetrathionate as the suitable biomarker for our user case, we postulated that the integration of this highly specific sensing part with the novel ARG would indeed result in a sensor design that is not only non-invasive, but also highly specific for IBD detection.

Concept - IBDetection

Integrating the two aforementioned papers, we envisioned a bacterium—for our proof-of-concept using E. coli BL21 (DE3) as the engineered organism—with the two-component sensing part to detect the intestinal inflammation marker tetrathionate. Then, this sensing part would activate the expression of ARG, resulting in the production of gas vesicles, which can be detected non-invasively using ultrasound imaging. Bacteria with this recombinant system would then be incorporated into a pill, which can be ingested orally by the patient, that delivers the live bacteria to the patient’s intestine. There, they would migrate through the colon and act as living sensors for a monitoring period. How the pill is envisioned to work is described in detail in the Integrated Human Practices segment The Pill and on our Implementation page. This should culminate in a solution for the current lack of specificity in the home calprotectin test and offer a non-invasive, fast-imaging additional intermediate procedure to rule out false positives test results, while being cost-effective and easy-to-use.

Goals

Non-Invasive

Due to the nature of an ultrasound scan, the patient will not be subjected to insertion of any measuring equipment into the body during the procedure, unlike during an endoscopy. The patient will simply orally ingest a pill, just like ibuprofen, and will not be required to abstain from eating or take laxatives prior to examination. More on this can be found on our Implementation page.

Specific

The two-component sensing system described in the paper above was characterized and tested versus a wide range of intestinal metabolites, receptor-ligands and tetrathionate analogues at well over physiological concentrations to test for specificity of our transmembrane receptor protein TtrS. Of these molecules, only tetrathionate yielded a response [14]. More information about the specificity of our system can be read in our Integrated Human Practices segment The Science and on our Proof of Concept page.

Easy-To-Use

During discussions with experts, it was brought up that endoscopies can only be performed by gastroenterologists, a nurse cannot carry out an endoscopy. An ultrasound scan on the other hand, is easier to use. Some practice and training is required, but image-analysis can be considerably automated. In addition, it is no longer needed to follow fasting instructions or take strong laxatives, and instead, simply ingesting a pill is a considerable improvement in the ease-of-use for the patients.

Cost-Effective

Taking an endoscopy is expensive, and average costs usually fall between 700 and 800 dollars. Ultrasound procedures on the other hand, only tend to cost less than 120 dollars, with an average procedure of the intestine costing around 90 dollars. Given the low specificity of the calprotectin test, ruling out false positives can be very cost effective, especially when considering the gain in quality of life. A cost analysis was performed and included on our Implementation page.

Fast Imaging

Conducting an endoscopy takes around 30 minutes, and requires a gastroenterologist as well as two nurses to perform and prepare [15]. An ultrasound measurement can be done by a single person and takes under 30 minutes to perform, typically around 20 minutes for the abdominal area [16,17]. This effectively competes with the current method and is generally faster while requiring less staff to perform. More on this is further touched upon on our Implementation page.

Project - IBDetection

To integrate the sensing part from the two-component system with the ARG to use as output as described above, two design strategies—design A & B—were devised to achieve the functioning protein network as shown below. More on the design strategies, and why they were necessary can be read on our Proof of Concept page. The complete cascade starts with the transmembrane receptor protein TtrS that binds to the biomarker tetrathionate with high specificity. Our regulator protein TtrR is subsequently phosphorylated and forms a homodimer, which acts as a transcription factor for our ARG. This leads to transcription and translation of our gas vesicle proteins, and eventual oligomerization into gas vesicles, which can then be detected using ultrasound technology. All mechanisms are described in much greater detail on the Proof of Concept page, and supplementary part information is included on the Parts page. In addition, we also wrote an extensive model to analyse and estimate and study the behaviour of this mechanism, which can be found on our Model page.

This mechanism is envisioned to be incorporated into gut-safe bacteria, which in turn will be incorporated into a pill design that releases them alive to the intestine, where they will migrate down through the colon and act as living sensors. We discussed with many stakeholders, including doctors, patients and field-specific companies on the application of our product in industry, the delicate choice of bacteria and intricate pill design. In addition, we delegated a lot of our attention to the safety and ethics of our product, as well as GMO legislation, to gain insight on and take into account the many details on devising and developing a product that would utilise live GMOs for the purpose of healthcare & monitoring.

Over the course of the project, we have validated our individual components, and worked hard on developing a proof-of-concept to show that the mechanism functions in a range of bacteria, including our E.coli BL21 (DE3). This illustrates the huge potential of being able to implement our entire system in any desired bacterium. More on these results can be read on our Results page. This culminated in the development of a new composite part BBa_K3972004, which describes a tetrathionate sensor with transferred TtrR and pTtrB activated ARG on the same plasmid, as described on our Parts page. Parallel to our proof-of-concept, we also set out to add to the iGEM library by improving the existing part BBa_K801100. We improved this Red Fluorescent Protein (RFP) coding part by exchanging the old ribosome binding site (RBS) for a five prime untranslated region (5’UTR), which can be read about on our Part Improvement page.

Potential

In practice, we expect the potential of IBDetection to be significant in both industry to save on costs and labour, and for the patient due to the non-invasive nature, fast methodology and ease-of-use. This is because in addition to its inherent benefits, no laxatives have to be taken, and no fasting is required beforehand. This potential is further elaborated upon on our Implementation page. Results from patient consultations and questionnaires were positive, with very realistic requirements that would have to be met, such as gut microbiome safety. Results from the lab and our model also pointed to a promising future prospect, with components working as anticipated, and predictions being optimistic for effective ultrasound imaging.

Future Prospects

The future prospect will primarily consist of further establishing and characterizing our proof-of-concept, especially in different—more suitable—cell types, such as L. Plantarum WCFS1. Additional mechanisms could be introduced to ensure full control over safety, depending on future GMO-usage requirements. Naturally, patenting and particularly pill development are on the future agenda, and are described in detail on our Implementation page. In the end, we aim to provide the industry with a cost-effective and easy-to-use solution for the current lack of specificity in the home calprotectin test and offer a non-invasive fast-imaging additional intermediate procedure to rule out false positives with IBDetection.

References

1. Inflammatory bowel disease (IBD). [cited 26 Sep 2021]. Available: https://www.mayoclinic.org/diseases-conditions/inflammatory-bowel-disease/symptoms-causes/syc-20353315

2. Rizzello F, Spisni E, Giovanardi E, Imbesi V, Salice M, Alvisi P, et al. Implications of the Westernized Diet in the Onset and Progression of IBD. Nutrients. 2019;11. doi:10.3390/nu11051033

3. Gajendran M, Loganathan P, Catinella AP, Hashash JG. A comprehensive review and update on Crohn’s disease. Dis Mon. 2018;64: 20–57.

4. Kaplan GG. The global burden of IBD: from 2015 to 2025. Nat Rev Gastroenterol Hepatol. 2015;12: 720–727.

5. GBD 2017 Inflammatory Bowel Disease Collaborators. The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol Hepatol. 2020;5: 17–30.

6. Kaplan GG, Ng SC. Understanding and Preventing the Global Increase of Inflammatory Bowel Disease. Gastroenterology. 2017;152: 313–321.e2.

7. eHealth. 17 Nov 2020 [cited 26 Sep 2021]. Available: https://www.crohn-colitis.nl/over-crohn-colitis/behandeling-van-crohn-en-colitis/ehealth-en-ibd/

8. de Jong M, van der Meulen-de Jong A, Romberg-Camps M, Degens J, Becx M, Markus T, et al. Development and Feasibility Study of a Telemedicine Tool for All Patients with IBD: MyIBDcoach. Inflamm Bowel Dis. 2017;23: 485–493.

9. de Jong MJ, van der Meulen-de Jong AE, Romberg-Camps MJ, Becx MC, Maljaars JP, Cilissen M, et al. Telemedicine for management of inflammatory bowel disease (myIBDcoach): a pragmatic, multicentre, randomised controlled trial. Lancet. 2017;390: 959–968.

10. Ricciuto A, Griffiths AM. Clinical value of fecal calprotectin. Crit Rev Clin Lab Sci. 2019;56: 307–320.

11. Spiceland CM, Lodhia N. Endoscopy in inflammatory bowel disease: Role in diagnosis, management, and treatment. World J Gastroenterol. 2018;24: 4014–4020.

12. How to prepare for a colonoscopy. [cited 6 Sep 2021]. Available: https://www.webmd.com/colorectal-cancer/prepare-for-colonoscopy

13. Bourdeau RW, Lee-Gosselin A, Lakshmanan A, Farhadi A, Kumar SR, Nety SP, et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature. 2018;553: 86–90.

14. Daeffler KN-M, Galley JD, Sheth RU, Ortiz-Velez LC, Bibb CO, Shroyer NF, et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol Syst Biol. 2017;13: 923.

15. Moore LE. The advantages and disadvantages of endoscopy. Clin Tech Small Anim Pract. 2003;18: 250–253.

16. Abdominal ultrasound. [cited 17 Oct 2021]. Available: https://www.mayoclinic.org/tests-procedures/abdominal-ultrasound/about/pac-20392738

17. Radiological Society of North America (RSNA), American College of Radiology (ACR). Ultrasound - Abdomen. [cited 17 Oct 2021]. Available: https://www.radiologyinfo.org/en/info/abdominus