Scientific Abstract
Proposal No. IBD-0115
Principal Investigator: Robert L. Clancy, M.D., Ph.D.
Applicant Organization: The University of Newcastle Research Associates (TUNRA) Limited (Callaghan, New South Wales, Australia)
Project Title: Mycobacterium avium paratuberculosis (MAP) plays a critical role in the pathogenesis of Crohn’s disease in a substantial proportion of subjects
Period of Award: April 19, 2004 – April 30, 2006
Currently, those interested in the pathogenesis of Crohn’s disease (CD) fall into one of two camps that hold either:
a. that transmural inflammation in CD reflects genetically determined defects in mucosal barrier function and/or mucosal immune regulation, or
b. that mucosal infection with MAP is the critical event leading to disease.
The hypothesis being tested in this proposal combines these viewpoints. It is proposed that MAP initiates the inflammation in a framework that includes genetic defects in mucosal immunoregulation and the mucosal barrier. Three specific questions are asked:
1. Is MAP detectable in CD and is such detection of clinical value?
2. Is there defective handling of, and/or an inappropriate immune response to MAP in CD?
3. Does anti-MAP therapy reverse parameters of mucosal inflammation?
The experimental design includes (i) a cross-sectional study of subjects with CD (and normal and inflammation control groups) to address questions 1 and 2, and (ii) a prospective controlled study of anti-MAP therapy over six months, to address question 3.
Detection of MAP by nested PCR will address issues raised to account for the considerable differences in positivity that have been published – initial processing ‘on site’ to minimize degradation and the use of a ribolyser to extract MAP DNA. Additional markers of infection will be MGIT long-term cultures and antibody to p35 and p36 antigens (Dr D. Graham, USA). The antigen processing-T cell activation ‘unit’ in CD will be assessed in two ways –
(i) ‘pulsing’ antigen presenting cells with MAP antigens and co-culturing with autologous CD4+ T cells, with secreted cytokines (IL-4, IL-10, IL-12, INF-γ and TNF-α) measured in culture supernatant as ‘read outs’, and (ii) detecting cytokines (above) secreted from colonic mucosal and whole blood cultures (methodologies developed to study gastric mucosal responses to H.pylori infection).
The randomized controlled prospective study of 30 subjects with CD will include Rifabutin, Clarithromycin, and Clofazimine in the treatment arm, over six months. Blood and colon biopsy assays (as above) will monitor the effect of anti-MAP therapy on microbiological and immunological parameters.
Innovative aspects of this study include an integration of MAP infection into the ‘traditional’ framework of genetic defects in mucosal physiology, the direct examination of MAP processing and activation of lymphoid cells in CD, and correlation of clinical and immunological events following anti-MAP therapy. The significance of this study is that it aims particularly to identify defective MAP ‘handling’ in CD, which if present, could have significant impact on future therapy options. In more general terms, the outcome of these studies could contribute to resolving the current conundrum of MAP infection in CD.