APPENDIX I: NIAID REPORT

CRITICAL NEED -- SUBSTANTIAL RESEARCH FUNDING
TO COMPLETE NATIONAL INSTITUTES OF HEALTH'S
NEW RESEARCH PLAN FOR CROHN'S DISEASE

Sequence of Events:

December 1998 -- The National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH/NIAID) hosts a critically important workshop entitled, "Crohn's Disease: Is there a Microbial Etiology?"

The NIH/NIAID Workshop, held in Bethesda, MD, convened the most prominent researchers from all over the world to discuss the rapidly mounting scientific evidence linking an infectious agent or agents -- with the primary suspect being a mycobacterium known as Mycobacterium avium subspecies paratuberculosis (MAP) -- to the devastating condition known as "Crohn's disease."

May 1999 -- Subsequent to the Workshop and collaborations with the top experts in the field of Crohn's Disease research, the NIH/NIAID makes a swift and astounding move to publish an entirely new and comprehensive Crohn's Disease Research Agenda -- a research agenda which targets an infectious cause for Crohn's disease.

The focus of the new research agenda reflects a dramatic shift away from the recent path of research which has heretofore sought, unsuccessfully, to implicate mysterious "immune system dysfunctions or defects" as the cause of Crohn's Disease. Instead, the NIH/NIAID has now moved to rapidly target research to determine whether the mycobacterium known as Mycobacterium avium subspecies paratuberculosis (MAP), and/or other bacterial infections cause Crohn's disease.

Just as stomach ulcers were recently proven to be caused by a bacterial infection, and not stress, it appears now that Crohn's disease may also be proven to be caused by a bacterial infection rather than being the result of an incurable and baffling "autoimmune" disease.

What is Critically Needed at This Time

Significant new funding specifically earmarked to complete the NIH/NIAID Research Recommendations (copy attached) is critically needed at this time to complete the necessary research set forth by the NIH/NIAID.

Meetings with top experts in the field disclosed that funding approximating $500 million is needed over the next three years to complete the NIH/NIAID Research Agenda.

Research Priorities

To bring an end to the suffering as soon as possible, PARA urgently requests specific prioritization of diagnostic tests and drug susceptibility testing coupled with multi-center controlled trials in completion of the NIH/NIAID Research Agenda.

Below we have provided the Goals of the NIH/NIAID workshop and the actual critical NIH/NIAID Research Recommendations developed from the workshop. The full text of the NIH/NIAID document can be found at http://www.niaid.nih.gov/dmid/crohns.htm

National Institute of Allergy and Infectious Diseases
"Crohn's Disease -- Is There a Microbial Etiology?
Recommendations for a Research Agenda"

December 14, 1998

Chair: Dr. Patrick Brennan, Ph.D.

Sponsored by: NIAID, NIDDK

Prepared by: Dennis Lang, Ph.D., NIAID

Goals of the Workshop

This conference was held in the Natcher Conference Center on the NIH campus in Bethesda, Maryland on December 14^th, 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.

Research Recommendations

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.

Clinical Studies:

1. 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
2. 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".
3. 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.
4. 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.
5. 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.
6. 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 1) CD case definition should be developed by participating investigators with the help of NIH and should be consistently applied in various clinical protocols. 2) Cases should be stratified into aggressive (perforating) and contained (non-perforating) pathology as well as to stage (active or quiescent) and duration of disease. 3) 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. 4) PCR and serology pre-, during and post-treatment in conjunction with culture studies should determine Map status. 5) Follow-up should be planned to determine the incidence of reinfection or disease recurrence. 6) 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.

Basic Studies:

If Map is established as a likely etiologic agent by clinical studies (see #1 above), 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.

1. 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?
2. 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.
3. 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.
4. 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.
5. 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).
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.

      List of Presenters

      Advisory Panel