MAP & Crohn's Disease research
NIAID: Recommendations for a Research Agenda
On behalf of Crohn's patients everywhere, PARA further offers its highest
commendation to the National Institutes of Health (NIH), and specifically to the
National Institute of Allergy and Infectious Diseases (NIAID) of the NIH, for
the very significant actions the Institute has recently taken relative to the
determination of a potential infectious etiology of Crohn's disease.
Summary of NIAID/NIH actions
In the summer of 1998, PARA began a series of pleas to the NIH/NIAID, to
investigate the relationship between Crohn's disease and the bacterium known as
Mycobacterium avium subspecies paratuberculosis(MAP). The
National Institute of Allergy and Infectious Diseases (NIAID) of the NIH took
the decisive lead in this matter and moved swiftly forward with the initiative
to thoroughly research PARA's concerns. In October 1998, the NIAID stated to
PARA its intent to host a formal workshop to further explore these concerns. On
December 14, 1998, the NIAID hosted a groundbreaking workshop bringing together
top researchers from all over the United States and the world to discuss a
potential infectious etiology of Crohn's disease and to devise new research
approaches to address this critical concern.
Subsequent to the NIAID workshop, the NIAID diligently continued its efforts
relative to this matter, and recently the NIAID/NIH took very significant action
to address PARA's concerns. Specifically, in June of 1999, the NIAID published
its "Research Recommendations" as part of its workshop report entitled,
Disease - Is there a microbial etiology? Recommendations for a research agenda."
The significance and importance of the NIAID actions cannot be overstated.
Perhaps Dr. Patrick J. Brennan, who chaired the NIAID Workshop, best summed up
the actions of the NIAID in his statement:
"The highly satisfactory outcome of the NIH Workshop "Crohn's Disease - Is there
a microbial etiology? Recommendations for a research agenda" demonstrates the
power of patient advocacy and excellent government response. The scientific
meeting itself was unique in the assemblage of people afflicted with Crohn's
disease, their loved ones, the best of the scientific community conducting
research on the various aspects of Crohn's disease and the affiliated Johne's
disease, and the best minds at the National Institute of Allergy and Infectious
Diseases, NIH. All shared their personal and scientific experiences and the
outcome was excellent, resulting in a document that has as its underpinning the
human affliction and stresses the need to define the potential infectious
etiology, characterize the host immune and inflammatory responses and conduct
crucial epidemiological and familial genetic research. On behalf of the
scientific research community, I would like to thank PARA for raising our
consciousness of Crohn's disease and its consequences, and the NIAID, notably
Dr. Dennis Lang, for the production of an historic document that will set the
research agenda into this disease for years to come."
PARA vigorously agrees with Dr. Brennan. Indeed, the NIAID's published report
and "Research Recommendations" is a very significant "historic document" - a
document which brings the search for an infectious cause for Crohn's disease
into the forefront of Crohn's research and which will, indeed,
research agenda into this disease for years to come." The
NIAID's historic "Research Recommendations" document has been reproduced in its entirety
at the end of this web page.
Additionally, in mid 2002, NIAID and NIDDK
funded significant rsearch into MAP/Crohn's. These are the studies
recently funded based upon the RFA resulting from NIAID's December 1998
Workshop on an infectious cause of Crohn's disease:
National Institute of Allergy and
Infectious Diseases (NIAID) Research:
- Julia Inamine, R01 A151283-01, "Molecular definition of Mycobacterium
Paratuberculosis," Colorado State University, $313,753
- Saleh Naser, R01 A151251-01, "Mycobacterium avium subspecies in Crohn's
Disease," University of Central Florida, $317,800
- Andrew Neish, R01 A151282-01A, "Epithelial responses to enteric
organisms," Emory University, $362,840
- Norman Pace, R01 A151298-01, "Molecular analysis of microbes in chronic
bowel diseases," University of Colorado, $335,250
- David Relman, R01 A151259-01, "Crohn's disease - microflora analysis and
immune response," Stanford University, $319,000
National Institute of Diabetes Digestive
and Kidney Diseases (NIDDK) Research:
- Fouad El-Zaatari, RO1 DK063092, "The role of Mycobacteria in Crohn's
disease," Baylor College of Medicine, $225,750
The NIAID is extremely well placed to execute this research, being the force responsible for the creation of many of the groundbreaking molecular techniques in use in infectious disease research around the world today. The
NIAID has the knowledge and the expertise, as well as the access to the
technology and the experience necessary, to deliver on its research agenda and
Again, on behalf of Crohn's patients everywhere, PARA extends its highest commendation for the care, diligence and professionalism of the
NIH/NIAID, and Dr. Dennis Lang in particular, for rigorously addressing this highly complex area of research and for preparing these immediately critical "Research Recommendations." Most certainly, it is responsiveness coupled with significant and groundbreaking actions such as these that have earned the
NIH its reputation for excellence and have made the NIH the most highly regarded health organization in the world!
Crohn's disease:- Is there a microbial etiology? Recommendations for a research agenda.
This conference was held in the Natcher Conference Center on the NIH campus in Bethesda, Maryland on December 14th, 1998. The purpose of the conference was to review the current state of knowledge relevant to a microbial etiology of Crohn's disease (CD), a serious, debilitating, inflammatory bowel disease. In particular, we set out to review evidence for and against the hypothesis that the bacterium, Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of CD, and to define needed research that could shed light on the etiology and pathogenesis of this chronic disease.
One of the major objectives of the conference was to have a productive scientific exchange between researchers with differing views on whether MAP is a human pathogen and if it is involved in the pathology of CD. There has been little agreement on this point in the scientific community. Much exciting research conducted in the 1980's on this organism failed to provide conclusive evidence that it was the cause of CD. The difficulty stems from the fact that the organism is extremely difficult to grow in the laboratory and to detect or recover from patients. Additionally, it has been difficult to demonstrate an immune response to MAP in patients with CD. In spite of this, there exist a number of respected clinicians who treat Crohn's patients with antibiotics effective against mycobacteria, and who believe that there is an improvement in their condition. Moreover, a number of patients and their advocates support the use of antibiotics, and gave personal testimony at the conference to the improvement of their disease symptoms following such treatment. They advocate the conduct of controlled antibiotic treatment trials to resolve this issue. A major objective of the workshop was to define new research opportunities and to explore possible mechanisms for stimulating additional research.
- There is insufficient evidence to prove or disprove that MAP is a human pathogen or that it is the cause of Crohn's disease. Considerable controversy continues to exist in the scientific community on this point.
- The difficulty in detecting and growing the organism, or being able to demonstrate a consistent immune response to it in CD patients, continues to frustrate researchers.
- Newer methodologies including the detection of microbial DNA sequences and functional genomics are available and should be applied in the search for a microbial etiology of Crohn's disease. A wide net should be cast. Characterization of microbial "communities" associated with this disease should be sought. Comparison with the completed genome sequences of the related species M. tuberculosis, M. leprae, and M. avium, may provide clues to the genetic basis of the pathogenesis of MAP.
- Good animal models for CD do not exist and should be sought. The development of additional genetically engineered mouse strains or primate models may be worthwhile, since current models do not develop much of the pathology associated with CD. Large animals, including sheep and cattle that get Johne's disease from MAP, should be treated with vigorous antimicrobial, anti-inflammatory, and immunosuppressive treatment (typical of what is used in humans) in conjunction with molecular characterization of associated microbial populations and strains.
- If MAP is proven to be a human pathogen, there is the potential for an enormous impact on human health due to the prevalence of this organism on the farm and in water. Further study of MAP as a food and/or waterborne pathogen should be conducted. Viable MAP should be sought in commercial milk and other dairy products as well as in meat. Conclusive studies of the effectiveness of pasteurization using commercial equipment and process rather than laboratory simulations should be performed. In order to conduct the above, standard methods for the concentration, detection, and in vitro culture of MAP should be developed and used by participating researchers. Federal agencies with regulatory authority over the food supply should consider conducting such research in cooperation with relevant food production industries and academic researchers.
Basic and clinical research should be aimed at answering the following fundamental question: Does MAP, or other microbial pathogen(s), cause CD? Answering this question requires addressing the following additional questions: Do affected tissue samples from Crohn's patients consistently contain MAP or any other pathogen? Can we detect specific immune reactions to a CD associated pathogen? What is such a pathogen's phenotype and genotype? Can we make the disease better by using appropriate antimicrobial drugs?
Workshop participants identified the following specific research needs.
- Determine potential infectious etiologies of CD by collecting and studying biopsy tissues from the intestines of Crohn's patients (stratified into perforated and contained lesions) and controls, and using sensitive diagnostic methods to enumerate any microbial flora associated with the disease. The use of anti-inflammatory drugs before obtaining biopsies may serve to close lesions/ulcers so that there is less contamination with normal gut flora or foodborne organisms. Ribosomal RNA typing (ribotyping) and other newer methodologies (such as subtractive hybridization) should be applied to tissues, as well as more traditional microbial culture and diagnostic techniques. Patients should be clinically well defined in terms of stage (quiescent or active) and duration (recent or long term) of disease, and tissues should be collected under defined standardized protocols. Such a search should not look exclusively for MAP, but should cast a wide net, seeking perhaps a "suite of organisms".
- Define the host immune response in Crohn's disease. What are the factors that contribute to the continuing inflammatory cascade observed in Crohn's disease? Normal flora, pathogens, diet, and stress have all been suggested as contributors to disease. Is initial infection with MAP or another organism acting to "prime" the immune system to respond to other stimuli in an abnormal, pathologic way? Will elimination of an underlying chronic infection allow the immune system to behave more normally? Immune cells in CD and control biopsy tissues should be analyzed and compared. If there is a microbial etiology, definition of the antimicrobial immune response will be important.
- Conduct epidemiological research to elucidate risk factors for human infection. Studies of farm workers and their families should be performed using modern diagnostic methods. Evidence of occupational or farm-life exposure to domestic animals should be sought in recently "emergent" clusters of CD. Studies should include prospective surveillance of young children to see if and when they may be infected with MAP (seroconversion to P35 and P36 or other antigens). Clinical specimens, if obtained, should be probed for the IS900 repetitive element or any other repetitive element identified in MAP. Evidence should be sought for the presence of MAP in dairy products, meat, and domestic water sources.
- Conduct genetic studies of families with a history of CD. Linkages have been tentatively assigned to chromosomes 1P, 4Q, 3 and 16, and 12. Are there others? What are these loci? What are the genes and what role do they play in CD? If a better animal model of CD were available, such genetic analysis might be facilitated.
- Antimicrobial treatment of CD. The use of anti-mycobacterial chemotherapy in the context of Crohn's disease is controversial. Many clinical studies employing empirical antimicrobial chemotherapy have been performed and investigators have reached different conclusions regarding the role of MAP in CD. NIH is supportive of finding resolution of this issue and would welcome the opportunity to work with clinical investigators on case definition, experimental design, and tissue collection protocols that would permit meaningful molecular and microbiological studies as part of future antimicrobial treatment protocols. NIH-supported investigators and available laboratory facilities may be helpful and could provide expertise and support in the conduct of studies to determine if there is a microbial etiology of CD. Some of the approaches that should be taken are described elsewhere in this document and may be conducted as part of future clinical strategies not requiring definitive blinded trials. Recommendations for study design of treatment protocols include
- CD case definition should be developed by participating investigators with the help of NIH and should be consistently applied in various clinical protocols.
- Cases should be stratified into aggressive (perforating) and contained (non-perforating) pathology as well as to stage (active or quiescent) and duration of disease.
- If MAP is the target of antimycobacterial therapy, minimal inhibitory concentrations (MIC's) of the antibiotics proposed for use should be determined prior to start of the trial employing clinical isolates of MAP (as opposed to lab strains) to insure that effective therapy is delivered.
- PCR and serology pre-, during and post-treatment in conjunction with culture studies should determine MAP status.
- Follow-up should be planned to determine the incidence of reinfection or disease recurrence.
- Clinical specimens should be obtained which would be suitable for ribotyping, PCR, subtractive hybridization, or other sensitive methods as discussed elsewhere in this report. Properly obtained samples will be invaluable for the purpose of defining the microbial flora associated with CD lesions. For this reason, consent documents should indicate that tissue samples and sera will be stored and used for research purposes.
Because evidence linking MAP to CD is not conclusive, the conduct of large, multi-center, blinded, placebo controlled trials of anti-mycobacterial drug therapy may be premature at this time. Such treatment protocols are complicated by the lack of sensitive and specific diagnostic tests for MAP and the difficulty in culturing the organism from clinical specimens, making stratification of cases based on MAP status difficult. When evidence is available to better support this, or any other microbial etiology, blinded antimicrobial trials of appropriate drugs at effective doses should be considered. Such evidence can be obtained by cooperative efforts between clinicians and basic scientists, and NIH can assist in this effort.
If MAP is established as a likely etiologic agent by clinical studies (as described under "Clinical Research"), basic investigations of MAP pathogenesis should be performed. If another pathogen(s) is/are identified as playing a role in CD pathogenesis, similar studies of such pathogen(s) should be performed.
- Establish cell or organ culture models of infection focusing on growth characteristics and gene expression of MAP in cell culture (ex. macrophages, intestinal epithelial cells). Does ex vivo growth of MAP (in organ or cell culture, for example) affect pathogenicity in an animal model?
- Establish new animal models of MAP infection. Clinical isolates of MAP should be used. A small animal model would be ideal, but has been elusive. Genetically engineered knock out mice or rats may be useful. Primate models may be helpful, but would be costly. Characterize the virulence and host preference of different MAP strains obtained from humans or animals. Determine the minimal infectious dose for MAP in an animal. Determine whether the infectious dose varies in animals of different ages.
- Develop an improved large animal model of CD. Treat animals with Johne's disease for extended periods with antibiotics and/or immunosuppressive drugs (including thalidomide?) in an attempt to develop a better animal model of Crohn's disease and to see if Johne's disease can be cured (long term follow-up). Determine the effect of such treatment on inflammation and on the levels of cytokines and other immunomodulators.
- Perform in vivo expression technology (IVET) studies in animals susceptible to Johne's disease to identify bacterial genes uniquely expressed in vivo. Such studies may be instructive of what to look for in other animal models and might provide valuable new information on the importance and role of new virulence factors in human disease.
- Compare MAP DNA sequences to available genome sequences of other mycobacteria. These comparisons may yield clues to pathogenicity. Is there a role for genetic insertion elements such as the MAP IS900 in pathogenesis? Gene expression arrays developed for other sequenced mycobacteria may be useful in determining if there are analogous virulence genes expressed in MAP, for example. Are there genes in MAP or other mycobacteria that may be homologous to virulence genes in other intracellular pathogens (Salmonella, Shigella, Listeria, and Chlamydia for example).
- Identify and optimize diagnostic MAP antigens that can be isolated or produced by recombinant technology or other means and made widely available to researchers. Purified peptide, carbohydrate, and lipid epitopes should be sought.
- Adapt antibiotic susceptibility testing methods to deal with a species that grows even more slowly than the so-called "slow growing mycobacterial pathogens". Studies should be expanded to look at combinations of drugs and to look at their efficacy against intracellular organisms and spheroplast forms. Drugs that are effective in vitro should be examined for efficacy against MAP infection in cell culture, in animals and eventually in humans.
- Determine the relationship between MAP and the M. avium complex, whether from Crohn's disease or Johne's disease. Molecular techniques including ribotyping, multi-locus enzyme electrophoresis, and DNA fingerprinting could be used to characterize and distinguish species. The MAP specific IS900 element has proven valuable in this regard. Comparative difference sequencing could identify other candidate markers and may lead to more useful diagnostic reagents and methods.
- Develop a high-density array of ribosomal DNA or RNA on a chip that can be used to more completely define the organisms associated with Crohn's disease. Use such a chip to examine tissues from patients with differing disease severity and duration.
- Apply subtractive hybridization techniques to look at the difference between CD tissues obtained by intestinal biopsy, tissues from a non-involved area of the intestine from the same CD patient, and normal tissues from controls. Tissues from early apthous or focal lesions as seen in post-operative recurrence models or in areas adjacent to grossly involved areas should be studied.