Crohn's Disease is a form of inflammatory bowel disease characterized by a genetically determined, uncontrolled aberrant immune reaction to an unknown environmental trigger, causing transmural inflammation that typically contains granulomata. The long-term goal of this research is to determine whether there is a bacterial or viral infectious etiology of CD. The hypothesis to be evaluated is that the environmental trigger is an infectious agent present within the granulomata. This hypothesis has been evaluated by many investigators. A number of bacterial and viral organisms have been detected in specimens from CD patients, but the question has never satisfactorily or conclusively been answered, because it is not known whether the detected organisms contribute to the development of CD or simply are intestinal colonizers due to fecal contamination of the specimen. The investigator will use a novel combination of molecular and pathological techniques to address this question.
The specific aims of this study are to identify and characterize bacteria in CD tissue and to identify and characterize viruses in CD tissue. To accomplish these goals, an approach to isolate pathological tissue free of surface contamination and a broadly based strategy to screen for all bacteria and viruses that could be etiologic agents in CD will be employed. To identify a bacterial agent, using light microscopy, granulomas will be dissected from paraffin-embedded CD tissue blocks to obtain pathological tissue free of either mucosal or serosal contamination. After deparaffinization of the granuloma, DNA will be extracted and used as a template to amplify bacterial DNA using consensus eubacterial rDNA primers. To identify a viral agent, a serosal window will be opened on fresh ileectomy/colectomy specimens resected from CD patients, and a biopsy of the muscularis will be obtained through the window. The biopsy will be divided into two parts for extraction of DNA and total RNA, respectively, and the remaining biopsy will be submitted for histological examination. The DNA recovered will be compared with that obtained from peripheral blood/normal tissue at the margin of resection by the previously described representational difference analysis to identify the presence of exogenous DNA. The RNA recovered will be used to construct a cDNA library and the library screened using sera obtained from patients with active CD to identify clones expressing foreign antigens due to the presence of viral RNA. Foreign DNA or RNA identified will be sequenced and analyzed for phylogenetic relationships with known bacteria and viruses. This strategy will unambiguously determine whether patients with CD have bacterial or viral genetic materials located within the lesions, which will be an important step toward clarifying whether infection with the organisms(s) causes the disease. Understanding of the etiology could enable eventual development of new diagnostic methods and specific prevention and treatment strategies for CD, and clarify backgrounds for genetic studies.
| Pei, Zhiheng; Yang, Liying; Peek, Richard M et al. (2005) Bacterial biota in reflux esophagitis and Barrett's esophagus. World J Gastroenterol 11:7277-83 |
