Appendix I

CRITICAL NEED -- SUBSTANTIAL FUNDING TO COMPLETE NATIONAL INSTITUTES OF HEALTH'S AND CENTERS FOR DISEASE CONTROL'S NEW RESEARCH AGENDAS 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.

May 2000 -- Centers for Disease Control issues its Working Document on Potential Infectious Etiologies of Crohn's disease, with emphasis on identification of questions remaining to be answered and the technologies and techniques required to answer these questions.

What is Critically Needed at This Time

Significant new funding specifically earmarked to complete the NIH/NIAID Research Recommendations and to answer the critical questions set forth in CDC's Working Document (copies attached) is critically needed at this time. 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 and CDC Research Agendas.

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 Research Agendas.

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 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.

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 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".
2. 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.
3. 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.
4. 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.
5. 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

THE CDC'S WORKING DOCUMENT

The CDC document is broken into three sections:

1. Introduction. This section introduces the general topic of infectious causes of chronic diseases, the specific topic of an infectious cause of Crohn's disease.
2. Unanswered Questions. The CDC recognizes the fact that there is a wide range of important questions in relation to Mycobacterium avium subspecies paratuberculosis and Crohn's disease, to which the answers are, unfortunately, currently unknown.
3. Potential NCID Response. In this section, the National Center for Infectious Diseases outlines the response required from it to deal with this serious and complex problem. The response, as is appropriate for dealing with any widespread infection problem, is broken into short, medium and long term responses.

DEPARTMENT OF HEALTH & HUMAN SERVICES

Public Health Service
Centers for Disease Control and Prevention
Atlanta GA 30333

Infectious Causes of Chronic Diseases

Potential Infectious Causes of Crohn's Disease

Recently, scientists have recognized the importance of infectious diseases as causes of chronic diseases. Many of these conditions were long-considered to be caused solely by genetic or environmental factors. The Centers for Disease Control and Prevention recognizes the potential public health impact of these developments, and has designated Infectious Causes of Chronic Diseases as a Target Area within its prevention plan Preventing Emerging Infectious Diseases: A Strategy for the 21st Century. (available from http://www.cdc.gov/ncidod)

Crohn's disease, one of several Inflammatory Bowel Diseases, causes substantial suffering in affected individuals, and seriously impacts their families and society. Usually Crohn's disease first appears in people between the ages of 20 and 40 years, and it can last a lifetime. Conservative estimates suggest that 400,000-500,000 people in the United States suffer from Crohn's disease. Infectious agents may play a role in this chronic illness. Recent scientific developments in the field underscore a basic need to define potential links between microbes and Crohn's disease.

Many questions regarding the relationship between microbes and Crohn's disease remain unanswered today. Currently, CDC is working to better understand the epidemiology of Crohn's disease in the U.S. and to define the current knowledge of physicians concerning its diagnosis and treatment.

The potential causal role of infectious agents in Inflammatory Bowel Disease and numerous other chronic diseases will only be understood with more intense and collaborative applied research throughout the public health, scientific, and medical communities. Increased understanding and answered questions are certain to create new prevention opportunities that benefit many people.

Potential Infectious Etiologies of Crohn's Disease

Introduction

Infections have recently come to be recognized as important causes of chronic disease. Many of these conditions were long-considered to be "non-communicable". The Centers for Disease Control and Prevention recognizes the potential public health impact of these developments, and has designated Infectious Causes of Chronic Diseases as a Target Area within its plan for Preventing Emerging Infectious Diseases.

Crohn's disease, one of several Inflammatory Bowel Diseases (IBD), imposes substantial suffering and burden upon affected individuals, their families, and society. Usually beginning between the ages of 20 and 40 years, it can last a lifetime. Conservatively, Crohn's disease (CD) afflicts an estimated 400,000-500,000 persons in the United States. Infectious agents may cause some portion of this chronic illness. Recent scientific developments in the field underscore a basic need to define potential links between microbes (e.g., Mycobacterium paratuberculosis and others) and Crohn's disease through application of sound scientific methods and technologies.

Unanswered Questions

(This is not a prioritization or description of necessary studies, but a summary of many related questions.)

In response to the illness and disability caused by this chronic disease, and to evolving scientific data, the Center for Disease Control and Prevention has outlined a number of gaps in the knowledge and understanding of potential causal relationships between infectious agents and Crohn's disease:

1. What are the true incidence and prevalence of Crohn's disease in the U.S. population?

• across geographic regions
• across racial/ethnic groups

2. What are the current knowledge and practices of U.S. physicians surrounding its potential etiologies?

• familiarity with scientific data linking microbes (e.g., M. paratuberculosis) with Crohn's disease
• clinical use of antimicrobial agents, including anti-mycobacterial regimens

3. If one or more microbes are causal in Crohn's disease:

• which infectious agents are responsible ---; individually or in synergy?
• is the microbe(s) a trigger for Crohn's disease, or does infection exacerbate established disease, determine its severity, or both?
• who is susceptible to infection-related disease ---; due to genetics, other environmental factors?

4. Are current diagnostic technology and study designs sufficient to confirm a causal relationship between microbes (including M. paratuberculosis) and Crohn's disease? i.e., (1) sensitive, specific and standardized assays and culture; (2) applied to well-defined, standardized case definitions and pathologic specimens; (3) with comparable study designs and reproducible results:

• can laboratory tools and epidemiology be improved?
• how can tools be validated for use in determining the attributable fraction of disease?

5. If one or more microbes are causal in Crohn's disease:

• what is the fraction of disease attributable to infection?
• what is the fraction of disease attributable to each implicated agent?
• what determines disease and its severity ---; any exposure, repeat exposures, or infectious load?

6. If infection is causal in Crohn's disease:

• can regimens that include antimicrobial agents change the course of disease?
• does outcome differ if antimicrobial (including anti-mycobacterial) treatment is administered early vs. later in the disease course?
• does outcome after antimicrobial therapy differ from that of immune modulating therapies?
• what specific changes in gut microflora occur with antimicrobial therapy vs. regimens that do not include antimicrobial agents (e.g., immune modulating) ---; does treatment eradicate or suppress certain microbes, and/or alter inflammation?

7. With respect to M. paratuberculosis, one potential pathogen in Crohn's disease:

• are current laboratory diagnostic assays and culture methods sufficiently sensitive, specific, and standardized to confirm or refute a causal association between M. paratuberculosis (M. para) and Crohn's disease?
• have diagnostic tools been validated sufficiently to determine a M. paratuberculosis attributable fraction of disease?
• are preceding and current experimental and epidemiologic studies sufficiently comparable and reproducible, using standardized case definitions and pathologic specimens, to confirm or refute a causal association between M. para and Crohn's disease?
• what validations have been performed for the diagnosis of human M. para infection ---; including translation of ruminant assays to human subjects?
• if validated tests become available, what are the U.S. incidence and prevalence of human M. para infection, and is infection persistent?
  
  
  • across geographic regions
  • in different racial/ethnic or cultural groups
    
    
• is M. para pathologic to humans and, if so, how is it transmitted to immunocompetent and to immunocompromised hosts?
  
  
  • is the organism found in food products?
  • do differences in U.S. and European milk pasteurization processes alter/minimize risk of human M. para infection?
  • if M. para enters ground or surface water, do U.S. water treatment processes minimize risk of human infection?
  • if it is present in soil, can agricultural and urban ecological practices minimize risk of human infection?
    
    

Potential NCID Response

(Intramural and extramural activities are dependent upon new and sustained sources of funding, excepting the first two Short-term Response items.)

To address the public health implications and the individual suffering caused by IBD, specifically Crohn's disease, the CDC/NCID is developing a research agenda designed to define these unresolved issues. These intra- and extramural NCID research activities, coordinated with those of NIH, other federal agencies, and external investigators, would advance our understanding of the potential portion of Crohn's disease due to infection. Implementing this research could notably advance the prevention or treatment of Crohn's disease.

Programs would draw upon the expertise of other agencies and outside researchers, as well as that of CDC. Proposed activities must be flexible and may change as the field evolves. This document represents an internal, working outline of the potential response.

Short-term Response:

• Completed first- of multi-stage Emerging Infections Network Query (with feedback education component) to assess current knowledge of U.S. physicians on the potential role of infection (including M. paratuberculosis) in Crohn's disease (CD), and current use of antimicrobial agents for the treatment of CD
• Completed first- of multi-level analysis in FoodNet database to determine and compare CD prevalence in each regional site and across racial/ethnic lines, with special attention to variations in prevalence that may be tied to specific exposures
• Advise on pilot epidemiologic studies designed to correlate food, environmental and habitation exposures, along with age and duration of exposure, to prevalent CD
• Analyze comprehensive patient data bases (e.g., HMO network database) to determine the true incidence and prevalence of CD on national, regional and racial/ethnic levels
• Developing a transparent network through which CDC may offer technical advice to ongoing epidemiologic and laboratory investigations on potential infection-CD links

Intermediate-range Response:

• Lead development and use of innovative diagnostic assays able to confirm or refute a causal and temporal (infection precedes disease) relationship between specific microbes and Crohn's disease (e.g., microarray host gene expression, microarray microbial gene expression)
• Promote use of validated and standardized assays to define the routes of human infection with proposed agents
• Lead cooperative development and collection of well-defined case definitions and pathology specimens, particularly from incident cases and from sterile sites less likely to harbor nonpathogens or commensals
• Lead cooperative validation and standardization of improved, sensitive and specific diagnostic tests for human M. paratuberculosis infection
• Extend epidemiologic studies (e.g., pilots, FoodNet network or HMO network) to investigation of incident CD and its correlation with exposures, including childhood and local environmental exposures (e.g., regional milk supply, food consumption habits, water treatment processes)
• Advise and provide technical assistance for well-designed clinical studies that assess the effects of antimicrobial/anti-mycobacterial treatment regimens upon Crohn's disease symptoms and tissue pathology (e.g., initial remission, persistent remission, "cure")
• If an association between infectious agents and Crohn's disease is confirmed, lead cooperative studies that define the true fraction of Crohn's disease attributable to one or more microbes, and the population/persons susceptible to infection-induced disease
• Provide technical assistance for research that compares changes in gut microflora or microbial "load" after certain antimicrobial regimens, immunotherapy and anti-inflammatory agents
• Lead appropriate public health prevention/intervention response to validated associations

Long-term Response:

• Assist in prospective evaluation of family members of Crohn's disease patients for asymptomatic infection with implicated agents
• Extend intermediate-range responses to define the distribution of risk for exposure to confirmed causal agents, identifying strategies that minimize these exposures
• Monitor changes in incident and prevalent Crohn's disease through FoodNet or HMO networks ---; particularly as measures to prevent or treat infection with implicated agents are implemented
• Define risk co-factors (e.g., genetics, co-infections or other environmental factors) for proven infection-related Crohn's disease through large-scale studies that integrate laboratory and behavioral analyses
• Target appropriate public prevention and intervention strategies towards high-risk populations