Lee A. Denson - US grants

DESCRIPTION (adapted from the application) Dr. Denson will devote the majority of his time to clinically-oriented basic research. This award will provide for protected time for research, additional didactic training, and the purchase of reagents. It will support pursuit of the immediate research aims, as well as the establishment of an independent laboratory. The Department of Pediatrics has assured that 80% of Dr. Denson's time over the next five years will be dedicated to research activities. The hepatocyte's response to cytokines includes a reduction in the synthesis of key metabolic proteins; this constitutes the negative acute phase response (APR). Adverse clinical consequences include impairments of linear growth, hepatocyte transport, and glucose and drug metabolism, due to down-regulation of regulatory genes. The primary objective of this proposal will be to determine the cytoplasmic signaling mechanisms by which cytokines suppress expression of clinically important hepatic genes, including the growth hormone receptor and albumin. Our prior studies have demonstrated suppression of hepatocyte transporter and albumin promoter-driven luciferase activity by tumor necrosis factor alpha (TNF-a) or interleukin-1 Beta (IL-1B). Retinoid response elements have been shown to mediate coordinate reduction of hepatocyte transporter expression by IL-1B and ceramide. The goal of AIM I will be to identify cytokine response elements and associated transcription factors which regulate growth hormone receptor and albumin expression in HepG2 cells and primary rat hepatocytes. The goal of AIM 2 will be to characterize the cytoplasmic signal transduction mechanisms mediating cytokine suppression of these transactivators and associated target genes. Initial studies will define the role of ceramide signaling in acute phase down-regulation of these genes. The goal of AIM 3 will be to confirm the significance of cytokine signaling pathways identified in Aims I and 2 to in vivo acute phase gene regulation. Wild type and IL-1B and TNF-a receptor deficient mice will be treated with endotoxin and effects upon cytoplasmic signaling proteins and target regulatory transactivators and genes will be determined. Clarification of these molecular mechanisms will contribute to a broader understanding, of the regulatory links between several important hepatic metabolic pathways in both health and disease, and may ultimately lead to more specific treatments for cytokine-induced complications of inflammatory diseases such as linear growth failure.

DESCRIPTION (provided by applicant): Children with cholestatic liver diseases including extrahepatic biliary atresia (EHBA) exhibit an acquired growth hormone (GH) resistance, in which GH secretion is normal, but hepatic expression of the GH receptor (GHR) and synthesis of anabolic target genes including IGF-I is reduced. Consequences include poor linear growth, muscle wasting, and increased post-transplant morbidity and mortality. The molecular basis for impaired GH signaling in obstructive cholestasis is not known. We hypothesize that down regulation of the GHR by TNFalpha and up regulation of Suppressors of Cytokine Signaling 3 (SOCS-3) by IL-6 may combine to reduce GH signaling in this setting. We will test this hypothesis in the following aims: Aim 1: Characterize cytokine regulation of liver Ghr and Socs-3 gene expression. Cytokine response elements in the mouse Socs-3 promoter will be identified by examining regulation of gene promoter reporter constructs in cultured hepatocytes. We will then determine whether TNFalpha down regulates Ghr expression via Sp1/Sp3 de-phosphorylation leading to reduced DNA binding affinity, and whether IL-6 up regulates Socs-3 expression by increasing STAT3 phosphorylation and nuclear abundance. These data will identify mechanisms by which cytokines regulate target genes involved in GH signaling via alterations in transcription factor phosphorylation. Aim 2: Identify mechanisms of GH resistance in obstructive cholestasis. Our preliminary studies have demonstrated that bile duct ligated (BDL) mice exhibit GH resistance which reproduces that observed in children with EHBA. This is associated with hepatic up regulation of TNFalpha and IL-6, down regulation of the Ghr, and up regulation of Socs-3. Abundance of these proteins relative to GH activation of STAT5 will be examined in sham, pair fed, and BDL wild type (WT), TNF receptor 1 (TNFR1) null, and IL-6 null mice. These data will determine whether disruption of TNFalpha or IL-6 signaling will restore GH action in obstructive cholestasis. These findings will have implications for therapeutic strategies to ameliorate acquired GH resistance in diseases complicated by liver inflammation, including chronic cholestatic liver diseases

Children with Inflammatory Bowel Disease (IBD) frequently develop an acquired growth hormone (GH) resistance. The inflammatory cytokine, Tumor Necrosis Factor alpha (TNF) has been implicated, although the underlying molecular mechanisms are not known. Consequences can include reductions in linear growth, muscle mass, bone density, and intestinal healing. We hypothesize that down regulation of the GH receptor (GHR) by TNF inhibits GH signaling in colitis. We will test this hypothesis in the following aims: In Aim 1, we will determine whether GHR expression is reduced in children with IBD and in murine experimental colitis. The functional significance of TNF induced alterations in GHR expression will then be assessed in liver and colon cell lines. We will identify TNF response elements in the mouse GHR gene promoter, and characterize signal transduction mechanisms by which TNF regulates nuclear abundance and DNA binding of the cognate transcription factors. In Aim 2, we will identify TNF dependent mechanisms of GH resistance in children with IBD and in murine experimental colitis. Abundance of the GHR relative to GH signaling and related parameters of growth, body composition, and colon histopathology will be determined folllowing administration of an anti-TNF antibody. In Aim 3, we will determine whether beneficial effects of TNF neutralization in colitis require STATSb activation, by examining the effect of anti-TNF antibody administration to STATSb deficient mice with colitis. The overarching hypothesis of this work is that a global defect in GH dependent STATSb activation leads to growth failure and impaired mucosal healing in IBD. These studies will clarify the direct importance of reduced STATSb activation in altering GH signaling in colitis. If the proposed studies support the utility of TNF blockade in reversing GH resistance in colitis, these findings should translate into improved clinical therapies for children with IBD.

0-11 years old; 1, 2-Dehydrocortisone; 1,7-Dihydro-6H-purine-6-thione; 21+ years old; 3H-Purine-6-thiol; 5-Aminosalicylic Acid; 6 MP; 6-Mercaptopurine; 6-Purinethiol; 6-Thiohypoxanthine; 6-Thiopurine; 6-Thioxopurine; 6H-Purine-6-thione, 1,7-dihydro- (9CI); 7-Mercapto-1,3,4,6-tetrazaindene; Adrenal Cortex Hormones; Adult; Arthritis, Juvenile Chronic; Arthritis, Juvenile Rheumatoid; Asacol; CRISP; Child; Child Youth; Childhood; Children (0-21); Chronic; Chronic Childhood Arthritis; Chronic Kidney Failure; Chronic Renal Disease; Clinical; Clinical Trials; Clinical Trials, Unspecified; Cola; Colon; Computer Retrieval of Information on Scientific Projects Database; Controlled Clinical Trials, Randomized; Corticoids; Corticosteroids; Crohn's disease; Crohn's disorder; Dehydrocortisone; Delta(1)-Cortisone; Deltacortisone; Deltadehydrocortisone; Disease; Disease remission; Disorder; Endocrine Therapy; Enteritis, Granulomatous; FLR; Failure (biologic function); Frequencies (time pattern); Frequency; Funding; GHN; Generalized Growth; Genus Cola; Goals; Grant; Growth; Growth Hormone; Growth Hormone 1; Growth and Development; Growth and Development function; Healed; Height; Hormonal Therapy; Human, Adult; Human, Child; Institution; Intestinal; Intestines; Investigators; JRA; Kidney Failure, Chronic; Maintenance; Maintenances; Mercaptopurine; Mercapurin; Mern; Mesalamine; Mesalazine; Metacortandracin; Monosomy X; Morgagni-Turner syndrome; Morgagni-Turner-Albright syndrome; NIH; National Institutes of Health; National Institutes of Health (U.S.); Numbers; Patients; Pentasa; Pituitary Growth Hormone; Population; Prednisone; Prednisonum; Pregna-1,4-diene-3,11,20-trione, 17,21-dihydroxy-; Purine, 6-mercapto-; Purine-6-thiol (8CI); RMSN; Randomized; Randomized Controlled Clinical Trials; Remission; Remission Induction; Renal Failure, Chronic; Research; Research Personnel; Research Resources; Researchers; Resources; Rowasa; STH; Shereshevskii-Turner syndrome; Somatotropin; Source; Therapeutic Corticosteroid; Tissue Growth; Turner syndrome (TS); Turner's Syndrome; Turner-Albright syndrome; Ullrich-Turner syndrome; Ullrich-Turner syndrome (UTS); United States National Institutes of Health; Week; XO syndrome; adult human (21+); bowel; children; chromosome XO syndrome; chronic kidney disease; clinical investigation; clinical significance; clinically significant; delta-Cortisone; disease/disorder; eleocolitis; failure; genital dwarfism; granulomatous enterocolitis; hGHN; healing; hormone therapy; improved; juvenile arthritis; juvenile idiopathic arthritis; m-Aminosalicylic Acid; meta-Aminosalicylic Acid; monosomy X syndrome; new therapeutics; next generation therapeutics; novel therapeutics; ontogeny; ovarian dwarfism; ovarian short stature syndrome; pediatric; primary ovarian insufficiency; pseudonuchal infantilism; randomisation; randomization; randomized trial; randomly assigned; regional enteritis; restoration; somatotropic hormone; youngster

Project Summary: It is likely that there are several immunogenetic forms of IBD, with CD and UC representing the broadest clinical classifications. While therapeutic options have increased over the past decade, our ability to target newer biologic therapies to specific subgroups of patients has lagged behind. Our recent studies have shown that CD patients with elevated GM-CSF auto-antibodies (GM-CSF Ab) are at high risk for progressive disease requiring surgery, and that neutrophil antimicrobial functions are reduced in this subset of patients. We hypothesize that: 1) elevated GM-CSF Ab will predict increased risk for complicated disease and surgery; 2) GM-CSF Ab level is a familial trait; and 3) GM-CSF Ab suppress neutrophil function by inhibiting GM-CSF bioactivity. We will test these hypotheses in the following Aims: Aim 1. Validate GM- CSF Ab antibody prediction of CD behavior. The serum concentration of neutralizing GM-CSF Ab will be determined in a prospective cohort of CD patients and related to disease behavior. These data will determine whether neutralizing GM-CSF Ab increase risk for complicated CD behavior requiring surgery, relative to current serologic markers and therapies. Aim 2. Determine the heritability of serum GM-CSF Ab levels. The serum concentration of GM-CSF Ab will be determined in affected and unaffected first degree relatives of index CD patients and the intra-class correlation coefficient and estimate of familial aggregation for GM-CSF Ab will be determined. These data will determine whether neutralizing GM-CSF Ab are inherited as a quantitative trait influenced by CARD15 and IRGM genetic polymorphisms. Aim 3. Examine GM-CSF Ab regulation of neutrophil function. The serum concentration of GM-CSF Ab will be determined and related to basal and GM-CSF dependent neutrophil STAT3/5 activation, cell survival, and antimicrobial functions including CD11B activation, adhesion, phagocytosis, microbial killing, and oxidative burst. These data will determine whether GM-CSF Ab suppress neutrophil STAT5 activation and antimicrobial function, while promoting STAT3 activation and cell survival.

DESCRIPTION (provided by applicant): This Training Grant in Pediatric Gastroenterology is designed to provide an intensive basic or patient based research experience that is essential to prepare clinician-scientists for productive and independent careers in academic and investigative medicine. The Program has a demonstrated track record of recruiting well-trained clinicians and providing them with research opportunities, an educational curriculum and mentoring to enable them to achieve successful academic careers. The program will support 7 postdoctoral fellows at Cincinnati Children's Hospital Medical Center. These fellows will spend two years with a Faculty Advisor in the laboratory or a structured clinical research program. The Faculty Advisors consist of 44 investigators with research interests relevant to pediatric gastroenterology. The thematic focus areas central to the Program: are 1) Chronic liver disease, 2) Digestive organ (liver and intestinal) failure and transplantation, 3) Inflammatory and diarrheal diseases, and 4) Obesity. The presence of a fully aligned Digestive Disease Research Core Center (PSO) further enhances the academic environment and focus of the training program. The Faculty has a demonstrated record of productive collaboration and extramural funding. A group of well- qualified prospective (associate) Faculty Advisors has also been identified to ensure continued growth of the program. The Administrative Core of the Program includes a Program Director and Associate Program Director (co-PI) who work both together both strategically and operationally to ensure a strong program. Didactic course work is required for trainees in both basic and clinical research and this is supplemented with appropriate seminars, journal clubs, a course in the responsible conduct of research, and other enrichment offerings. Those learning how to perform patient based research are expected to complete a Master of Science Degree. The record of our Trainees in staying in academic medicine (75%), and performing research (61%) is a measure of our success. We have also fulfilled our commitment to training women (44%) and minorities (22%). There is a pool of highly qualified candidates applying to and/or already committed to entering our clinical training program. Thus, when considered with our institutional resources and past success, the Program is poised to extend the training opportunities to increase the number new investigators in Pediatric Gastroenterology. PUBLIC HEALTH RELEVANCE: There is a critical shortage of investigators in pediatric gastroenterology who can advance new knowledge to improve the outcomes for children with digestive diseases. Our training program focuses on 1) Chronic liver disease, 2) Liver and intestinal failure and transplantation, 3) Inflammatory bowel disease, eosinophilic gastrointestinal and diarrheal disorders, and 4) Obesity. We will provide mentored training to pediatricians who will become the next generation of investigators in pediatric gastroenterology.

DESCRIPTION (provided by applicant): Ulcerative colitis (UC) is one of the chronic intestinal disorders known as inflammatory bowel disease (IBD). It affects hundreds of thousands of Americans with about 25-30% being children. UC causes considerable suffering with abdominal pain, diarrhea, bleeding, and weight loss and for unknown reasons is often more severe in children than adults. Longstanding disease in addition carries a high risk of colon cancer. While current medical therapies often relieve symptoms, there is currently no cure and many patients require corticosteroids (steroids) or potent immunsuppressive medications (IM) with major risks, or, ultimately surgery to remove the colon. There is a paucity of controlled data on the treatment of UC in children and virtually no studies that provide guidance to clinicians as to which children are going to do well and who is going to do poorly and need increasing medication exposure and possible surgery. We hypothesize that a combination of clinical, genetic, and immunologic tests performed at diagnosis may allow construction of a model for individualized treatment and thereby improvement of current outcomes. Specifically, we will conduct a clinical trial of standardized medical therapy for 410 children newly diagnosed with UC at 17 pediatric IBD centers in North America (Predicting Response to Standardized Pediatric Colitis Therapy: The PROTECT Study). Using a clinical protocol that is designed to provide optimal care and be responsive to disease severity, as well as state of the art technology to monitor medication adherence, we will follow this cohort for 2 years with a primary clinical outcome of clinical remission at one year without the concurrent use of steroid medications or the need for more potent immunsuppressive medications or surgery. Biospecimens including blood, stool, and colonic tissue will be obtained at diagnosis and during follow-up, and pre-specified clinical, genetic, environmental, and immune factors will be determined and tested for their impact upon mucosal inflammation, and the primary clinical outcome of steroid-free remission. The collective results of the PROTECT study will have a significant and sustained impact upon the field by: 1) providing data regarding response to standardized therapy and a more optimal adaptive study design for a clinical trial of early IM use in pediatric UC patients unlikely to achieve steroid-free remission with mesalamine alone, 2) discovering fundamental new knowledge regarding the role of common genetic and environmental factors in the differentiation and function of effector and regulatory lymphocytes, and 3) establishing a repository of clinical, genetic, and immune data, and biospecimens, which can be used by for future ancillary studies. Ultimately, results of PROTECT will be integrated with those of an ongoing prospective study of 1000 pediatric Crohn's Disease (CD) patients sponsored by the Crohn's and Colitis Foundation of America (CCFA), the RISK study, to provide a powerful new platform for discovering mechanisms which drive pathogenesis and clinical outcomes in pediatric IBD. PUBLIC HEALTH RELEVANCE: The goal of the PROTECT study is improve the long-term outcome of children with ulcerative colitis by discoveries to guide clinical decision making. We plan to develop a model of clinical, genetic, and immunologic information that will allow clinicians to formulate individualized treatment plans to achieve clinical remission and minimize exposure when possible to toxic medications or the need of surgery.

DESCRIPTION (provided by applicant): Ulcerative colitis (UC) is one of the chronic intestinal disorders known as inflammatory bowel disease (IBD). It affects hundreds of thousands of Americans with about 25-30% being children. UC causes considerable suffering with abdominal pain, diarrhea, bleeding, and weight loss and for unknown reasons is often more severe in children than adults. Longstanding disease carries a high risk of colon cancer. Ideally, UC is adequately controlled with mesalamine, a 5-aminosalicylate with a favorable benefit: risk profile. Unfortunately, mesalamine alone is often not sufficient requiring step-up therapy with corticosteroids (steroids), potent immunosuppressive medications (IM) with major risks, or surgery to remove the colon. There are a lack of controlled data on the treatment of UC in children and virtually no studies that provide guidance to clinicians as to which children are going to do well with mesalamine alone and who is going to do poorly and need increasing medication exposure or surgery. We hypothesize that a combination of clinical, genetic, and immunologic tests performed at diagnosis may allow construction of a model for individualized treatment and thereby improvement of current outcomes. Specifically, we will conduct a clinical trial of standardized medical therapy for 430 children newly diagnosed with UC at 24 pediatric IBD centers in North America (Predicting Response to Standardized Pediatric Colitis Therapy: The PROTECT Study). Using a clinical protocol that is designed to provide optimal care and be responsive to disease severity, as well as state of the art technology to monitor medication adherence, we will subsequently determine the primary clinical outcome of clinical remission at one year on mesalamine only without the concurrent use of steroid medications or the need for more potent immunosuppressive medications or surgery. Biospecimens including blood, stool, and colonic tissue will be obtained at diagnosis and during follow-up, and pre-specified clinical, genetic, environmental, and immune factors will be determined and tested for their impact upon the primary clinical outcome as well as mucosal healing at one year. When possible patients will be followed beyond one year and up to 2-5 years depending upon time of entry into the study. The collective results of the PROTECT study will have a significant and sustained impact upon the field by: 1) providing data regarding response to standardized therapy maximizing the likelihood of response to mesalamine, 2) identifying novel biologic pathways associated with treatment response, and 3) establishing a repository of clinical, genetic, and immune data, as well as biospecimens, that can be used by for future ancillary studies. Ultimately, results of PROTECT will be integrated with those of an ongoing prospective study of 1100 pediatric Crohn's Disease (CD) patients sponsored by the Crohn's and Colitis Foundation of America (CCFA), the RISK study, to provide a powerful new platform for discovering mechanisms which drive pathogenesis and clinical outcomes in pediatric IBD. PUBLIC HEALTH RELEVANCE: The PROTECT Study will, for the first time; describe the course of children newly diagnosed with ulcerative colitis treated with standardized therapy that has been devised to minimize exposure to more toxic drugs. By concomitantly obtaining biospecimens at diagnosis and during the first year following therapy PROTECT will facilitate the understanding of inter-patient variability in response to therapy and provide insights into the pathways that sustain colonic inflammation. This effort will not only improve the health of children newly diagnosed with ulcerative colitis, but will also lessen the likelihood of medication toxicity and the need for colectomy.

DESCRIPTION (provided by applicant): Anti-microbial sero-reactivity (AMS) and chronic intestinal inflammation similar to Crohn's Disease (CD) during the first decade of life in children with inherited disorders of phagocyte function suggests that loss-of-function in neutrophil antimicrobial pathways is likely to be a fundamental mechanism of pediatric CD pathogenesis. GWAS in CD have accounted for only a portion of the heritability and have rarely identified few loci of large effect. Rare variants, which GWAS are underpowered to detect, have been hypothesized to explain a substantial fraction of complex disorders like CD, so gene discovery efforts have now shifted to characterization of deleterious / loss of function mutations. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is required for priming of neutrophil antimicrobial function, and bioinformatic analysis of genomic studies has suggested a central role in CD pathogenesis. We discovered that older pediatric (early onset, EO, age 10-17) and adult patients exhibit an acquired basis for neutrophil dysfunction, GM-CSF auto-antibodies (GM-CSF Ab), which increase in titer with increasing age. GM-CSF Ab carriage is associated with reduced neutrophil STAT5 activation, phagocytic capacity, and bacterial killing, and high rates of AMS and stricturing behavior. To test the significance of these exciting developments, we have established a prospective clinical database and biobank for 1130 pediatric CD patients enrolled at diagnosis (the RISK study). We found that neutrophil phagocytosis and bacterial killing is reduced in a subset of patients. VEO patients have exhibited rare coding region mutations in genes predicted to affect GM-CSF priming of bacterial killing (CSF2RB & ITGAM/CD11b) and neutrophil oxidative burst (CYBA/p22phox), while EO patients have exhibited increasing titers of GM-CSF Ab. We hypothesize that genetic variants and GM-CSF Ab cause neutrophil dysfunction and thereby contribute to disease pathogenesis in an age-dependent manner in pediatric CD. Aim 1: Identify all coding genetic mutations in 127 genes likely to disrupt GM-CSF signaling and/or neutrophil function in 500 very early onset pediatric CD patients. Aim 2: Test the functional consequences of genetic mutations upon neutrophil GM-CSF signaling and bacterial killing. Peripheral blood samples will be collected from RISK patients carrying genetic mutations predicted to affect GM-CSF priming and neutrophil function including neutrophil candidate protein expression/localization, GM- CSF signaling, CD64 activation, adhesion, chemotaxis, oxidative burst, phagocytosis, and bacterial killing. We will use state-of-the-art genomic and immune approaches to define for the first time the causes and consequences of neutrophil dysfunction in the largest pediatric CD inception cohort in North America. Collectively, our studies will have direct implications for novel therapeutic approaches designed to modulate this critical host defense pathway in patients who experience the worst outcomes with current approaches.

PROJECT SUMMARY The overall goal of the Enrichment Program of the Digestive Health Center (DHC) is to advance digestive disease research by creating a forum where investigators will present new research findings, learn about new technologies, and pursue new collaborations. The Director of the Program and DHC leaders pursue this goal with four complementary aims. In the first aim ?to promote knowledge sharing through a weekly seminar and workshop series,? the Program sponsors a series of highly interactive seminars and presentations by investigators from the academic health center and extramural scientists who are leaders in digestive disease research. These seminars also include technology-centered workshops that are given by Core directors and staff. Presenters receive timely feedback and suggestions on their experimental approaches and emerging technologies. Seminars and workshops represent a nidus of collaboration and knowledge-sharing among participants. In the second aim ?to increase collaboration with other academic units by joint educational programs,? the Program Director seeks opportunities to partner with focus groups or study groups to fertilize ongoing interests in the field, with three recurring meetings with: 1) the ?Fluxes and Barriers? group of intestinal physiologists; 2) the ?Endoderm Club,? which focuses on organoid systems to model human disease; and 3) the ?Distinguished Speaker Program? with the Division of Developmental Biology to expose DHC investigators and trainees to the most successful scientists in the field. In the third aim ?to hold an annual scientific symposium to highlight digestive disease discoveries,? the DHC combines the annual visit of the External Advisory Board with a day devoted to digestive disease research via a highly subscribed symposium featuring a distinguished speaker and a scientific forum for the presentation of posters reporting novel findings by trainees and junior faculty. And in the fourth aim, ?to increase visibility for digestive disease research in the Academic Health Center? the Program publishes a quarterly Newsletter that is circulated throughout the academic health center to share information on upcoming seminars, accomplishments and awards of DHC investigators, and updates of Core services.

The Inflammatory Bowel Diseases (IBD), Crohn Disease (CD) and Ulcerative Colitis (UC), are chronic and debilitating disorders with peak incidence in the second and third decades of life. While considerable progress has been made in optimizing medications to achieve remission, relapse is common and unpredictable. Altered microbiota likely drive gut inflammation and clinical relapses. Microbiota-accessible dietary carbohydrates with beneficial health effects, known as ?prebiotics,? hold promise for restoring healthy gut microbiota in IBD and preventing clinical relapse. Here, we propose the first studies of the prebiotic human milk oligosaccharide, 2?-fucosyllactose (2?-FL), for maintaining remission in IBD. Our overarching hypothesis is that 2?-FL supplementation in IBD will be safe and well tolerated, while increasing fecal Bifidobacterium abundance and butyrate in a dose dependent manner. We will test this hypothesis by conducting a randomized, placebo- controlled dose-ranging study and completing the following Aims: Aim 1. Define dose dependent safety and tolerability of 2?-FL as a dietary supplement in IBD. We will test 1, 5, or 10 gm 2?-FL compared to 2 gm glucose placebo as a daily dietary supplement in pediatric and young adult IBD patients in stable remission receiving infliximab or adalimumab anti-TNF therapy. Safety and tolerability will be assessed using validated clinical disease activity indices, a novel electronic symptom tracker, and fecal calprotectin. Aim 2. Define dose dependent efficacy of 2?-FL as a dietary supplement in IBD. We will utilize our established fecal metagenomic, metatranscriptomic, and metabolite assays to test the effect of a range of 2?FL doses upon the gut microbial community and associated metabolic functions with a focus upon butyrate production. Efficacy will be assessed by determining the dose dependent effect of 2?-FL upon increased fecal Bifidobacterium and butyrate abundance. We will account for FUT2 secretor status in the analysis. These studies will have a high impact in the field by providing critical phase I/IIa safety and efficacy data in support of a phase III RCT using our NIH-supported IBD clinical research network to test the efficacy of 2?-FL in directly modulating beneficial microbiota and thereby enhancing sustained clinical remission. Ultimately the proposed studies will promote a fundamental shift in clinical practice towards personalized microbial therapeutic interventions.

Project Summary/Abstract Therapeutic goals in pediatric Crohn?s Disease (CD) have shifted from clinical improvement or remission to endoscopic healing (EH) by ileocolonoscopy and transmural healing (TH) by magnetic resonance enterography (MRE). This shift happened because patients who achieve complete healing (CH, TH and EH) experience lower rates of subsequent hospitalization, therapy escalation, or surgery than those with EH alone or no healing. We hypothesize that specific pre-treatment clinical, radiologic, genomic, and microbial factors along with attainment of targeted anti-TNF biologic exposure will be associated with the primary endpoint, CH, and the major secondary endpoints, EH and TH, 52 weeks after anti-TNF start.. We will test this hypothesis in a prospective cohort study of 570 newly diagnosed pediatric-onset CD subjects who initiate treatment with anti-TNF medication within 6 months of diagnosis. We will administer personalized anti-TNF biologic therapy guided by therapeutic drug monitoring (TDM) using a novel dosing algorithm which we developed. Aim 1. Evaluate putative associations and explore novel associations between CH and baseline measures of clinical and radiologic severity. We hypothesize that pre-treatment nutritional status, antimicrobial serologies, and MRE findings will be associated with CH 52 weeks after anti-TNF start. Formal hypothesis tests will be carried out to confirm the predictive power of a set of pre-specified measures using a logistic regression model. We will also conduct exploratory analyses of novel predictors, identified via machine learning methods, to assess their relationship with CH after adjusting for the confirmed primary predictors. Aim 2. Evaluate putative associations and explore novel associations between CH and baseline host and microbial genomic and metabolic factors. We hypothesize that pre-treatment gene expression signatures and microbial factors will be associated with year 1 CH. We will characterize the host genotype, longitudinal microbial taxonomic and metabolomic profiles, and ileal and colon host epigenome and transcriptome at baseline and at 52 weeks after anti-TNF start. Aim 3. Use a k-fold cross-validation procedure to determine the optimal predictive model of year 1 CH. We hypothesize that a model which includes host gene signatures and microbes will improve prediction of CH beyond one based on clinical and imaging factors alone. The model will include significant clinical and imaging predictors from Aim 1 and the subset of baseline host and microbial characteristics found to be potentially explanatory in Aim 2. Impact. The proposed inception cohort study, CAMEO, will be unique in providing a robust, novel platform to study factors that contribute to healing in pediatric CD that can then be immediately translated into clinical practice, as well as guiding future therapies targeting the microbiota and host immune responses in patients unlikely to achieve healing with current approaches.

The long-term goal of our research program is to define genomic and microbial mechanisms driving disease complications in Crohn's Disease (CD). Persistent rates of stricturing complications requiring surgery despite increased use of anti-TNF? therapy increase the urgency of this work. Interactions between intestinal epithelial cells, myeloid cells, and myofibroblasts regulate the balance between wound healing and fibrosis in CD. The overall objective of this project will be to develop a novel human intestinal organoid (HIO) model system to test genetic mechanisms regulating tissue inflammatory responses and collagen production. Our preliminary data suggests a critical role for myofibroblast extrinsic reactive oxygen species (ROS) production by the intestinal epithelial DUOX2 and neutrophil NOX2 NADPH oxidases in regulating production of epithelial and myeloid inflammatory cytokines including TGF? which promote myofibroblast activation and extra-cellular matrix production. We found that loss-of-function genetic variants in DUOX2 were associated with three-fold lower rates of stricturing in CD, while loss-of-function genetic variants in the CYBA, NCF1, NCF2, and NCF4 genes comprising the NOX2 complex were associated with three-fold higher rates of stricturing. Analysis of CD patient ileal biopsies defined a gene expression program encoding epithelial and myeloid inflammatory signals driving myofibroblast activation and collagen production in those who later developed strictures. A novel perturbagen bioinformatics approach prioritized small molecules likely to reverse the pro-fibrotic gene expression signature. We have established inducible pluripotent stem cell (iPSC)-derived human intestinal organoids and neutrophil- like cells from CD patients with and without DUOX2 and NCF1 mutations, and confirmed variation in ROS production. This will provide a novel model system to test genetic regulation of tissue fibrosis. We hypothesize that genetic variation in NADPH oxidase function regulates ROS production and epithelial and myeloid inflammatory signals driving myofibroblast activation and extra-cellular matrix production. We will test this hypothesis in the following Aims: In Aim 1 we will differentiate isogenic iPSC with and without DUOX2 mutations into human intestinal organoids. We will test the effects of microbial products upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using gene expression and collagen content data derived from CD patient ileal biopsy and surgical resection samples. In Aim 2 we will differentiate isogenic iPSC with and without NCF1 mutations into neutrophil-like cells and human intestinal organoids. We will determine the effects of neutrophil products and small molecules upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using CD patient neutrophils and ileal samples. These studies will address the goals of this Program Announcement to utilize identifiable human specimens to delineate candidate gene functions in disease states, and to more precisely define the pathophysiologic mechanisms that lead to end-organ injury.