Infectious Agents in Pediatric Crohn's

Inflammatory bowel disease (IBD), which includes CD and ulcerative colitis (UC), is estimated to affect approximately 1 - 2 million Americans. Recent studies strongly suggest that these disorders are increasing in prevalence in developed and developing countries, and in first generation immigrants to industrialized nations. Changing epidemiology of IBD implies that, in addition to a genetic susceptibility for disease, an environmental trigger or exposure likely impacts disease expression. However, the specific pathobiology and etiology of IBD remain unclear. Knowledge of the host genetic basis of IBD has advanced, including characterization of a novel susceptibility gene in adult and adolescent CD (NOD2/CARD-15) located on the pericentromeric region of chromosome 16. Recent studies not only implicate NOD2/CARD-15 in susceptibility to CD, but also suggest a link between the innate immune response to bacterial components and development of disease. Thus, well-designed investigations of infectious etiologies of CD, with sufficient power to detect differences in infection between IBD and non-IBD patients, are critically needed. Yet, deficiencies in the methods and tools for such studies still exist.

Both infectious and noninfectious mechanisms of pathogenesis remain plausible but unconfirmed. To date, scientific evidence cannot conclude whether one or more infectious pathogens triggers or determines the development and outcomes of CD or UC. Persistent exposure to "normal resident flora" in an abnormally permeable gut might also conceivably determine disease. Thus carefully designed investigations that apply systematically evaluated, reliable and reproducible detection assays are needed to confirm or disprove infectious hypotheses of causation. The first step is a pilot study that validates study design and complementary assays (morphology-, molecular- and culture-based) and uniformly applies these to target patient and control tissues (i.e., pediatric endoscopic biopsies). It must focus on newly diagnosed CD (surrogate for "incident" CD) in a well-characterized group of patients and appropriate, well-selected controls who meet uniform case and control definitions. It must develop reproducible methods to ensure that results from different studies can be compared and appropriate clinical interpretations made in order to translate research results into treatment and prevention strategies that decrease the burden of CD. At this time, deficiencies in both methodology and laboratory technology impede successful conduct and interpretation of the needed studies on causality.

Based on age of onset, newly diagnosed children with IBD, may be closer to triggering immunopathogenic events that initiate the inflammatory response than adults. They may also represent a unique population for the study of environmental factors (e.g., infection), i.e., younger age usually brings shorter and less complicated exposure histories to confound analyses. It is reasonable to postulate that a single agent or polymicrobial insults, in the genetically susceptible host, may determine the development or course of CD and it may be easier to identify some infectious determinants in childhood CD. We hypothesize that adequate collection of intestinal tissue biopsies from uniformly defined pediatric CD cases and controls and validation of sensitive and specific laboratory tools to detect potential infectious triggers of CD in these particular specimen types are needed to test causality for infection in CD. The Pedi IBDC, 6 large, geographically diverse U.S. centers represents an excellent platform to investigate potential infectious factors in CD, annually evaluating and recording clinical and epidemiologic data on ~288 newly diagnosed pediatric CD cases in a comprehensive data repository. The Pedi IBDC has a system to assess the safety and efficacy of its research. The Consortium also provides a pool of cases and controls in which to implement standardized specimen collection techniques under a well-conceived algorithm. The R03 assembled research team draws on a wide range of expertise and institutional resources in epidemiology, infectious disease pathology, molecular microbiology, mycobacteriology and virology, and the conduct of clinical trials. The multi-disciplinary expertise of this proposal can test and validate in small endoscopic biopsies the histopathology and molecular microbial detection assays previously validated in alternative paraffin-embedded or frozen tissues.

The R03 funding mechanism will facilitate tissue collection and banking techniques and evaluations of a panel of assays to detect select infectious agents ranging from those thought not to cause CD to those speculated as etiologic, from non-pathogenic commensals to known and undetermined pathogens. Therefore, our specific study aims are to:

1. standardize the collection (e.g., anatomic site of biopsy origin, biopsy number and collection technique), processing and banking of gastrointestinal tissue biopsies obtained during clinically-indicated endoscopy of well-characterized children with CD followed by the Pedi IBDC;
2. verify and standardize the definition of appropriate controls, evaluating both "non-diseased" biopsies from cases and clinically-indicated biopsies from children without IBD (e.g., juvenile polyposis, Hirschsprung's disease) to improve the design of future studies;
3. standardize and validate in pediatric biopsies detection methods to identify potential infectious risk factors for CD through parallel, paired and complementary pathology-based (IHC), broad range amplification-based molecular (PCR) and organism-specific (e.g., MAP) molecular analysis, and primary culture of tissue specimens taken from well-defined cases and controls.

Additionally, molecular characterization of microbiota in the feces from cases and controls will be compared to microbial detection in their respective tissue biopsies. Successful completion of these goals will provide a platform for imminent comprehensive investigations that characterize clinically relevant microbial risk factors for CD in the child and quite possibly the adult patient.