Crohn’s disease and ulcerative colitis are forms of inflammatory bowel disease (IBD) that affect more than 6.8 million adult and pediatric patients worldwide. Because no cure is available to date, treatment is limited to reducing IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms that mediate symptoms are unclear, there is mounting evidence that enzymes produced by bacteria in the gut are important regulators of disease onset and severity. The composition of bacteria in children’s guts is more dynamic compared to adults making them more susceptible to new bacterial activity. 

Therefore, we hypothesize that enzymes produced by the commensal microbiota and pathogenic bacteria cleave protease-activated receptors (PARs) to mediate various forms of pediatric IBD. These proteases are thus therapeutic targets for pediatric IBD. To test our hypothesis, we have developed strategies to screen for, and identify modulators of PAR2 in the human gut lining. In this project, we will use human organoids, cells and animal models to confirm the importance of specific bacterial enzymes in mediating human disease symptoms. We will also use our expertise in developing enzyme inhibitors to identify new tools and therapeutic lead molecules to further assess the importance of enzyme-triggered signaling pathways. In parallel, strains that decrease susceptibility to IBD by disarming PAR2 will be explored as potential probiotics. Ultimately, this work will help to define the mechanism by which specific bacterial enzymes in the gut impact the pathogenesis of IBD while also creating new avenues for treatment of this complex disease.

Inflammatory bowel disease (IBD) is a gastrointestinal disorder that presents in children as well as adults and is associated with abnormal composition and overall abundance of gut microbes [1,2,3]. Anti-inflammatory drugs such as anti-TNF-alpha are the only FDA-approved therapy for children with IBD, yet response rates vary. In pediatric patients who respond to the drug, the composition of the gut microbiome is more similar to healthy individuals, whereas in patients who do not respond to the drug, the composition remains abnormal [1,2]. Based on this observation, we think that gut bacteria probably utilize some survival strategies to adapt to the inflamed intestinal environment of patients with pediatric IBD and that if the inflammation is decreased by the anti-TNF-alpha treatment, the gut microbes are closer to what we see in healthy normal people. However, we don’t know what bacterial factors contribute to these adaptive traits. In this research, we will examine how bacterial genetic elements help microbes adapt to changes in the intestinal environment following anti-TNFalpha treatment, aiming to uncover links between these elements and treatment outcomes.

The study has two key objectives. First, we will identify microbial genetic elements involved in environmental adaptation in the gut microbiome of patients with pediatric IBD. To do so, we will analyze metagenomic data from 59 patients at multiple time points during anti-TNF-alpha treatment. Second, we aim to identify specific genetic elements related to therapy success. To this end, we will compare the abundance of genetic elements before and after treatment within the same individual, considering the results of treatment. This will allow us to better understand how these elements contribute to bacterial adaptation and influence treatment outcomes, drug dosing, and disease progression.

Pediatric Crohn's disease (CD) is on the rise, affecting an increasing number of children and having a profound impact on their overall well-being and family life. The primary goal in treating pediatric CD is to alleviate symptoms, reduce the risk of future complications, and ultimately enhance the quality of life for these young patients. However, the available tests for disease evaluation are either invasive or have poor compliance, highlighting the ongoing and critical need for new laboratory tests to facilitate early diagnosis and monitor treatment effectiveness. Blood tests represent the safest and cost-effective strategies to monitor many diseases. These tests detect certain chemicals that circulate in the body which tell us how active the immune system is, how healthy the gut barrier is, and whether the gut microbiome is healthy or altered. However, no large-scale, comprehensive clinical studies of chemicals in the blood of pediatric CD patients have been performed. Our lab at Stanford recently performed the largest study to date of chemicals in the blood of pediatric patients with CD. In the current proposal, we have the following two objectives: 1) To identify chemicals in blood that distinguish CD patients from non-CD controls and that predict the development of complications; 2) To identify chemicals in blood that provide insight into disease severity and have utility as new tests to monitor treatment effectiveness. Here, we will use advanced approaches in computational biology to identify new and clinically actionable chemical markers that can be used as non-invasive tests to diagnose pediatric CD and monitor treatment response. Ultimately, this work could guide physician’s use of therapies to improve the outcomes in pediatric CD patients and propose new therapeutic targets for personalized medicine.

Ulcerative colitis (UC) is a chronic disease that causes inflammation and damage to the colon. It affects about 1.25 million people in the United States, including 42,000 children. The inner lining of the intestines, called the intestinal epithelium, acts as a barrier protecting the body from harmful microbes in the gut. When this barrier is disrupted, it can lead to chronic inflammation, which is a major factor in UC. For children, UC can disrupt growth and development, significantly impacting their quality of life. Current treatments often provide inconsistent results, and no cure exists, making the search for new therapies especially important for pediatric patients.

Patients with UC show changes in their lipid metabolism, and recent evidence suggests that this disruption could impair the mitochondria in the intestinal epithelium. This mitochondrial dysfunction may worsen inflammation in the colon. This proposed research focuses on understanding how lipid metabolism may contribute to UC. A key player in fat metabolism is the protein PPARα. Our preliminary research shows that PPARα is overactive in UC, potentially leading to excessive lipid oxidation and fueling inflammation. To investigate this, we will use in cell models, specifically patient-derived colonoids, to study how PPARα affects lipid metabolism and inflammation at the cellular level. These colonoids closely mimic the disease environment in UC patients, providing insights that are highly relevant to human disease. In addition, we will use pre-clinical animal models, specifically mouse models with intestine-specific knockout of PPARα, to study how this protein affects the entire intestinal system in UC. By depleting PPARα in these mice, we aim to see whether it reduces the inflammation and damage typically seen in UC.

Together, these two complementary models will help us better understand PPARα’s role in UC and explore whether targeting it could lead to more effective treatments for children suffering from this debilitating disease.

Autoimmune diseases, including Celiac Disease (CD), are on the rise and are expected to become even more prevalent in the future. CD is a lifelong condition caused by an immune reaction to gluten, a protein found in wheat, barley, and rye. When people with Celiac Disease consume gluten, the resulting immune response damages their small intestine, causing symptoms like abdominal pain, bloating, and diarrhea. Despite the increasing number of people affected by CD, our understanding of the condition is hindered by the difficulty in directly studying the small intestine, where most damage occurs. Current research largely relies on stool samples, which do not adequately represent the small intestinal environment. To address this issue, our study will use a novel, safe, non-invasive device called CapScan. This capsule, which can be swallowed, is designed to collect luminal samples from different parts of the small intestine as it moves through the digestive system. The capsule is then recovered from the stool for analysis. This innovative approach enables us to directly study the hard-to-reach regions of the gut, providing unparalleled insights into the gut environment in CD. Our research will include newly diagnosed or uncontrolled CD patients and a matched group of healthy individuals as controls. We will analyze markers of inflammation from both the gut and blood samples in both groups, as well as microbiome structure and metabolite profile, investigating how these features change over time and vary across different locations in the intestine.

The primary goal of this study is to develop a more thorough understanding of gut-related inflammation in autoimmune diseases, with a specific focus on CD. By gaining a better understanding of the gut's role and the microbiome in particular in the development and progression of these conditions, we hope to lay the foundation for the development of new, targeted treatments that can improve the quality of life for people living with CD and other autoimmune disorders.

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.

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.

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.

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.

"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.

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 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. 

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.

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.

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 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.

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.

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.