Inflammatory bowel disease (IBD) is a disease condition in which a person's intestines have persistently inflammation. It affects both children and adults worldwide. IBD occurs because the body's defense system, known as immunity, reacts incorrectly to the bacteria and viruses in our gut. We do not fully understand why the body's defenses overreact in this way. Research has suggested that differences in each person’s DNA (i.e., genetics) might play a role in the development of IBD. Therefore, we aim to determine how these genetic differences lead to IBD. Our previous research discovered that a specific type of genetic variants can together send a "false alarm" in our body’s defense system. This makes the body think it is fighting a virus when it is attacking its own parts. We have observed that these genetic variants are commonly possessed by IBD patients. Thus, we hypothesize that these "false alarms" may be a cause in at least a subset of IBD patients. Our proposed research will test this hypothesis. We plan to develop a method to predict whether these "false alarms" will occur, using information from a person's DNA. If we are successful, we will be able to identify IBD patients who experience these "false alarms" by examining their DNA. Specifically designed medicines to quiet the "false alarms" will be used to treat IBD in these patients.

Ulcerative Colitis (UC) is a public health challenge worldwide, particularly in children, which presents atypical phenotypes, limited approved therapies, higher metabolic demands, and decreased metabolic reserve capacity that exacerbate tissue damage. Preliminary data: Recent studies have shown that intestinal organoids derived from patients with UC can mimic the histological and functional features of primary colitic tissues. Rosen's lab has confirmed this finding in epithelial colonoids isolated from pediatric patients with active UC, identifying atrophic morphology and poor response to bioenergetic demands during the differentiation that reflects mitochondria damage. In collaboration with Rosen’s lab, during my first year as a postdoctoral researcher in Thakor Lab, I studied the effects of administering MSC-EVs to epithelial colonoids from pediatric patients with active UC and found a reduction in hypermetabolic activity and a re-establishment of bioenergetic requirements to levels observed in control colonoids. Aims: In this proposal, I will use a pulsed-focused ultrasound (pFUS) technology developed by the Thakor lab to augment the regenerative phenotype of MSC-EVs and study the capacity of these primed MSC-EVs to regulate the mitochondrial bioenergetic functions in epithelial colonoids isolated from children with active UC. In Aim 1, I will define the effects of MSC-EVs on mitochondrial bioenergetics by directly measuring oxygen consumption rate, basal and maximal respiration, spare respiratory capacity, and ATP-liked respiration in colonoids derived from patients with active UC (treated versus non-treated) and colonoids from patients without UC. In Aim 2, I will expand upon these results by identifying how mitochondrial gene expression changes in response to therapy and correlating this with the bioenergetic results from Aim 1.

Crohn’s disease (CD) is a predominant form of inflammatory bowel disease characterized by chronic inflammation in the gut that affects millions of people worldwide. In CD, cells from the immune system attack the intestines, leading to persistent inflammation resulting in gut damage and perturbing normal function, making it difficult to absorb nutrients and thrive. In healthy individuals, specific immune cells called regulatory-T cells (Treg) maintain immune regulation in different tissues including in the gut, but these cells are less functional in CD patients. The CD is more prevalent in the pediatric population as compared to ulcerative colitis and is less likely to be responsive to conventional therapies. Further, gut damage induced by uncontrolled inflammation often requires surgery and hospitalization and is a source of poor quality of life and suffering for many children as well as their families. Thus, pediatric CD patients are in urgent need of treatment option that could alleviate the disease symptoms with minimum side effects and supports development and growth. In this study, I propose to develop gut-specific-engineered Treg-like cells (GI-CD4LVFOXP3) which will be a patient-specific “living drug” that can control excess inflammation in the gut and support gut repair. Comparable engineered Treg-like cells (CD4LVFOXP3) developed in our laboratory are in Phase I clinical trials for a different disease, also involving the gut, supporting the applicability of these cells in the clinic. I will generate GI-CD4LVFOXP3 by inducing the expression of gut-targeting proteins (receptors) on the surface, which will direct the GI-CD4LVFOXP3 to the gut. I will test the function of these cells using gut samples from CD patients. These gut samples are manipulated in vitro to mirror the gut environment preserving CD disease perturbations that we aim to resolve. Successful completion of this study will provide key pre-clinical data toward a new therapeutic for pediatric CD.

Crohn’s disease (CD) is a predominant form of inflammatory bowel disease characterized by chronic inflammation in the gut that affects millions of people worldwide. In CD, cells from the immune system attack the intestines, leading to persistent inflammation resulting in gut damage and perturbing normal function, making it difficult to absorb nutrients and thrive. In healthy individuals, specific immune cells called regulatory-T cells (Treg) maintain immune regulation in different tissues including in the gut, but these cells are less functional in CD patients. The CD is more prevalent in the pediatric population as compared to ulcerative colitis and is less likely to be responsive to conventional therapies. Further, gut damage induced by uncontrolled inflammation often requires surgery and hospitalization and is a source of poor quality of life and suffering for many children as well as their families. Thus, pediatric CD patients are in urgent need of treatment option that could alleviate the disease symptoms with minimum side effects and supports development and growth. In this study, I propose to develop gut-specific-engineered Treg-like cells (GI-CD4LVFOXP3) which will be a patient-specific “living drug” that can control excess inflammation in the gut and support gut repair. Comparable engineered Treg-like cells (CD4LVFOXP3) developed in our laboratory are in Phase I clinical trials for a different disease, also involving the gut, supporting the applicability of these cells in the clinic. I will generate GI-CD4LVFOXP3 by inducing the expression of gut-targeting proteins (receptors) on the surface, which will direct the GI-CD4LVFOXP3 to the gut. I will test the function of these cells using gut samples from CD patients. These gut samples are manipulated in vitro to mirror the gut environment preserving CD disease perturbations that we aim to resolve. Successful completion of this study will provide key pre-clinical data toward a new therapeutic for pediatric CD.

This project aims to identify new drug candidates for pediatric inflammatory bowel disease (IBD) treatment by understanding mitochondrial dynamic, which includes mitochondrial fusion and fission. This project focuses on mitochondrial fission protein (Fis1), a critical molecule for pathological mitochondrial fission, as a potential target to develop new drug candidates. This project will investigate therapeutic effects of novel Fis1 inhibitors and their underlying mechanisms using cells and preclinical IBD model.

"The human gastrointestinal tract is home to an incredibly dense population of microbes (the microbiome) which play an important role in inflammatory bowel disease (IBD). One of the most concrete ways we know that the microbiome influences IBD is through the production of drug-like small molecules. These molecules interact with human cells within the gut, influencing intestinal health, but also are absorbed into the body and can influence the set point of the immune system. Probiotics are a class of living biological products (mainly bacteria) that are commonly used to resolve intestinal symptoms. Although very safe,  robiotics have not shown a clear benefit in patients with IBD. I propose that the major reason why probiotics are not successful as therapies is because they do not have the capability to produce molecules that improve intestinal health. My goal is to develop a new class of engineered probiotics for the delivery of beneficial microbial metabolites in the gut. The greatest barrier to generation of these engineered probiotics is that the genes and pathways required for production of beneficial metabolites are not known. Here, I will leverage a new high-throughput mass spectrometry platform that I have developed to define the microbial genes responsible for production of a beneficial microbial metabolite, 3-indoelpropionic acid (IPA). Next, I will engineer these IPA producing genes into a probiotic strain known to colonize the human gut. I will then test whether IPA production by this strain can improve mucosal health in preclinical animal models of IBD. Results from this proposal will help develop a first-in-class engineered probiotic to treat pediatric IBD.

Patients with chronic inflammatory bowel diseases (IBD) often need life-long medications to keep their disease and symptoms under control. Medications are often initially tested in adults. As a result, medication dosing for children is commonly based on findings from adult studies. The most commonly used and most effective medication class in patients with IBD is called anti-tumor necrosis factor-a (anti-TNF) medication. While these medications are effective, there is a wide variability in patient response. Certain factors can make a medication work less well or can increase the likelihood that a patient may stop responding to the medication itself. Our group previously designed mathematical computer programs (models) that help us to predict the right amount of medication that a child will need. These models capture a child’s unique medication needs to optimize the chance that the child’s body will respond with minimal side effects. While helpful, these models are not perfect.

Our objective is to improve these models to help make decisions about medication choice and dosing for each individual child. To improve these models, we hypothesize that we will need to examine the individual bacteria and chemicals (metabolites) in the bowel and stool. To answers these questions, we will use blood, stool and tissue from the intestine (biopsies) of children who started anti-TNF therapy and were followed until a repeat colonoscopy after 1 year of therapy.

This research will have 2 steps. In step-1 we will aim to identify individual bacteria and chemical patterns and study the association between high vs normal drug loss. In Aim 2, we will use artificial intelligence models using different biological data (blood markers, bacteria and chemicals (in the gut and tissue)) and evaluate which model best predicts bowel healing.

In this project, we will develop a novel MRI technique that can lead to a faster and safer diagnosis of pediatric inflammatory bowel disease. The technique will halve the acquisition time and/or double the spatial resolution by incorporating an optimized RF pulse, a novel data sampling approach, and a deep learning reconstruction framework into the conventional MRI technique.

A smart toilet could seamlessly integrate urine and stool analyses as part of routine toileting events for the "digitalization" of healthcare, which takes a process from human-driven events to machine-driven. The integrated Bowel-symptom Diagnostics (iBD) toilet is a standard toilet outfitted with gadgets on top of the bowl. This system will be equipped with artificial intelligence and Internet of Things technologies to continuously monitor human excreta from pediatric inflammatory bowel disease (IBD) patients. Stool colors, mucoid stool, bloody stool, and any anal diseases can be assessed during excretory events longitudinally with computer vision. Along with digital biomarker measurements, fecal calprotectin levels will be longitudinally, passively measured as stool samples undergo molecular analysis in the platform in real-time, which will be particularly valuable to detect IBD flares.

Respiration is vital to all cellular processes, including the ability to produce new cells for the maintenance of optimal bodily function. This is especially true for mammalian intestines that are composed of rapidly multiplying epithelial cells that need to be shed and replenished every 3-5 days to maintain the intestinal barrier against foreign invaders. The mitochondria direct the respiratory capabilities of all cells, and this ability is compromised in the colon of pediatric patients with ulcerative colitis (UC). Since mitochondrial function is directly linked to the replenishment of the intestinal barrier, it is vital to decipher genetic and environmental drivers of disease that may modulate mitochondrial function to perpetuate disease in UC. Mammals are inevitably exposed to sulfites from natural foods and food additives. Sulfites are by-products of certain amino acid breakdown and can be an indirect product of intestinal bacteria. It is estimated that many children may be exposed to sulfites at a concentration that is over 300% of the adequate daily intake. Sulfites easily diffuse into the mitochondria where they distort the normal energy-producing capability of the mitochondria, hence, they are regarded as toxic and subsequently converted into sulfates which could be excreted or used in other physiological processes. Recent studies, however, suggest that a key gene (Mocs1) vital in the removal of sulfites is less abundant in the colon of pediatric UC patients compared to healthy controls.

This study seeks to understand how sulfite exposure impacts mammalian mitochondrial function and intestinal replenishment in colon samples derived from healthy and pediatric UC patients. We also propose to evaluate how the lack of the Mocs1 gene changes mitochondrial dynamics and intestinal renewal in the pediatric colon. Overall, the proposed study will initiate discussions and solutions surrounding sulfite exposure for the personalized management of UC, especially in children.

This proposal aims to leverage the cutting-edge technologies of yeast display and TRACeR (see details in the proposal) to efficiently engineer protein binders with high labeling stability, target specificity and affinity, tissue penetration to celiac disease related gliadin peptides associated to the human leukocyte antigen (HLA)-DQ2.5 (DQA1*05/DQB1*02) complexes. Successful development of gliadin peptide/DQ2.5 binders will enable the direct targeting of gliadin antigen presenting cells at the small intestine (both lesion sites and adjacent lymph nodes) in situ, deepening the understanding of celiac disease pathogenesis, and bridging the gap of gliadin antigen presentation to T cell-mediated enteropathy. The development of the proposed binder will also open avenues leading to improved diagnostic methods (for histological analysis) and therapeutic (for pathogenic T cell blockage/suppression) options for celiac disease. 

In the current project I will apply our novel air-liquid interface (ALI) organoid culture system into pediatric IBD research by generating patient specific pediatric IBD ALI organoid. The ALI organoids are derived from patient endoscopic biopsies, incorporating diverse endogenous innate and adaptive immune cells, epithelial cells and stromal cells. The proposed 3 aims will help us to better understand the IBD related features in culture and dissect the contributions of T cells and epithelial DUOX2 and reactive oxygen species in pediatric IBD pathophysiology.

In the current project I will apply our novel air-liquid interface (ALI) organoid culture system into pediatric IBD research by generating patient specific pediatric IBD ALI organoid. The ALI organoids are derived from patient endoscopic biopsies, incorporating diverse endogenous innate and adaptive immune cells, epithelial cells and stromal cells. The proposed 3 aims will help us to better understand the IBD related features in culture and dissect the contributions of T cells and epithelial DUOX2 and reactive oxygen species in pediatric IBD pathophysiology.

The microbiota is a key factor in the etiology and development of Crohn's disease, but virtually nothing is known about how symbiotic protists affect disease development. However, all characterized symbiotic protists of the trichomonad lineage stimulate type 2 immunity in the murine small intestine through excretion of the metabolite succinate, but I have discovered a new species which instead stimulates a type 1 immune response. I hypothesize that these divergent immunological impacts drive opposing effects on Crohn's-like ileitis, and thus that human-associated trichomonads may act as either probiotics or pathobionts for pediatric Crohn's disease in children.