United States

Chairman

Dr. Jerrold J. Ellner
(1990-   , Member 1988-1990)
Chief, Division of Infectious Diseases
Case Western Reserve University
School of Medicine, W113
10900 Euclid Avenue
Cleveland, Ohio 44106-4984

Japan

Chairman

Dr. Izuo Tsuyuguchi
(1994-   , Member 1988-1994)
Chief
Department of Internal Medicine
Osaka Prefectural Habikino Hospital
3-7-1 Habikino, Habikino
Osaka 583, Japan

Panel Members

Dr. Philip Hopewell (1992-1995)
Chest Service, Room 5K1
San Francisco General Hospital
University of California
1001 Potrero Avenue
San Francisco, California 94110

Dr. Marcus Horwitz (1992-1995)
Chief, Division of Infectious Diseases
Department of Medicine, CHS 37-121
University of California at Los Angeles
10833 Le Conte Avenue
Los Angeles, California 90024

Dr. David N. McMurray (1990-   )
Texas A&M University
Medical Microbiology and Immunology Department
Mail Stop 1114
College Station, Texas 77843-1114

Dr. Thomas M. Shinnick (1990-   )
Chief, Hansen Disease Laboratory
Division of Bacterial Diseases
Centers for Disease Control and Prevention
1600 Clifton Road, NE, MS G-35
Atlanta, Georgia 30333

Dr. Chiyoji Abe (1991-   )
Chief
Department of Bacteriology
Research Institute of Tuberculosis
Japan Anti-Tuberculosis Association
3-1-24 Matsuyama, Kiyose
Tokyo 204, Japan

Dr. Ichiro Azuma (1994-   )
(Chairman 1988-1994, Member 1981-1988)
Director General
Institute of Immunological Science
Hokkaido University
N-15, W-7, Kita-ku, Sapporo
Hokkaido 060, Japan

Dr. Fumiyuki Kuze (1994-   )
Professor
Chest Disease Research Institute
Kyoto University
53 Kawaramachi, Shogoin, Sakyo-ku
Kyoto 606, Japan

Dr. Masao Mitsuyama (1991-   )
Professor
Faculty of Medicine
Niigata University
1-757 Asahimachi-dori
Niigata 951, Japan

Guidelines

Tuberculosis Panels USJCMSP

Tuberculosis research is concerned primarily with projects relating to the pathogenesis and immunological aspects of the disease. Subjects of special interest are immunity and hypersensitivity, the analysis of cell substances of tubercle bacilli that might be related to immunity, and experimental models for the study of tuberculoimmunity:

1. Biology of mycobacteria

   • Fractionation and identification of biologically and immunologically active constituents of tubercle bacilli
   • Genetics and physiology
   • Mechanisms of drug action and development of resistance

2. Pathogenesis of tuberculosis and other mycobacterioses
   
   • In man and experimental models
   • Host-parasite relationship
   • Environmental and nutritional factors

3. Immunology of tuberculosis
   
   • Mechanisms of hypersensitivity, anergy, and acquired resistance
   • Immunoglobulin response to infection and immunization
   • Activation of nonspecific effectors mechanisms of mycobacterial and other products
   • Adjuvant properties of mycobacterial and other products
   • Immunoprophylaxis

   • Live vaccines
   • Killed vaccines
   • Natural and synthesized mycobacterial fractions

To facilitate these studies, special reagents are made available only for research purposes to qualified investigators. These reagents are lyophilized PPD tuberculin, characterized mycobacterial cultures of research interest, a culture filtrate reference antigen preparation, and polyvalent reference antiserum with which investigators can standardize laboratory produced material.

Five-Year Summary

Broad Goals

The last 5 years have seen remarkable and unprecedented expansion of the research agenda of the Tuberculosis Panels. The basis for this growth can be traced to the resurgence of tuberculosis in certain areas of the world, the association between infection with M. tuberculosis and the human immunodeficiency virus (HIV), the emergence and spread of multidrug-resistant (MDR) mycobacteria, and increased scientific opportunity. The incidence of tuberculosis in the United States and Japan has increased during the last 5 years. These realities require an expanded scientific agenda focusing on areas that can impact the clinical and public health problems of tuberculosis in the 1990's.

The Tuberculosis Panels identified the following mutual goals:

1. Application of molecular genetics to elucidate the virulence and pathogenicity of M. tuberculosis
2. Understanding the mechanisms of action and resistance to drugs as the scientific foundation for new drug development
3. Development of improved animal and cell culture models of protective immunity to identify protective antigens
4. Characterization of the immunopathogenesis of tuberculosis in the normal and HIV-infected host
5. Development of new approaches for the early diagnosis of tuberculosis, particularly for drug resistant disease
6. Definition of the epidemiology and optimal therapy of M. tuberculosis and disease caused by M. avium complex.

Progress/Accomplishments

The increasing challenge of tuberculosis in its resistant forms and in the AIDS era is matched by the scientific opportunities. The last 5 years have been a time of unprecedented scientific progress. Many of the advances in fundamental understanding in this period have ready or potential application in the clinical and public health arenas.

Here are some of the major accomplishments of this period. The genes of M. tuberculosis were expressed in E. coli, allowing characterization of some of the major protein constituents. Several of the antigens identified by immunizing mice with killed M. tuberculosis were shown to be heat shock protein (HSP) with homology to HSP of gram negative bacteria. These and other genes have been used to construct primers so that the polymerase chain reaction (PCR) could be applied to the diagnosis of tuberculosis. The precise role of this technology in the diagnosis of tuberculosis in developed and developing countries should be determined in the next few years. Description of the IS 6110 repetitive element provided the cornerstone for identifying strains of mycobacteria by restriction-fragment-length polymorphism (RFLP), or DNA fingerprinting. RFLP analysis demonstrated the differences between the Tokyo and Paris strains of M. bovis BCG. RFLP also opened the door to molecular epidemiology, which has proved to be an essential tool in determining the epidemiology of nosocomial tuberculosis and for demonstrating the importance and relative frequency of progressive primary tuberculosis and reinfection tuberculosis in patients with HIV-disease. Genetic systems for the study of mycobacteria have found many applications. Perhaps none is more dramatic than cloning of the inhA gene. Mutations in this gene are associated with clinically significant resistance to isoniazid (INH) as well as ethionamide INH. The mutations in the rpoB gene responsible for rifampin resistance also have been mapped. A reporter mycobacteriophage construct expressing the luciferase gene provides the basis for the rapid identification of drug-resistant strains. PCR based assays also are being developed for the rapid identification of rifampin resistant strains. These are particularly important clinically, as rifampin resistance is a reliable marker for MDR strains that require different approaches for treatment, and infection produced by such strains is difficult to cure.

Major advances in immunology have come from the use of genetically manipulated mice to characterize lymphocyte populations and cytokines involved in protective immunity. Progressive areactive tuberculosis developed in interferon-y knockout mice. ß2 microglobulin knockout mice showed increased susceptibility to tuberculosis, supporting other studies implicating the role of CD8 positive T-lymphocytes in protective immunity. Advances in basic immunology found ready application to tuberculosis with respect to the role of (/* T-cells. This T-cell population is expanded by M. tuberculosis antigens including cord factor and low molecular weight constituents and appears to play a role in the initial/innate immune response to M. tuberculosis. Great progress has been made in exploring the regulation of the human immune response during tuberculosis and the identification of potential immunosuppressive mediators (IL-10, transforming growth factor ß). Of interest is the finding that MDR forms of M. tuberculosis are more potent stimulators for IL-10 production than drug-sensitive mycobacteria. Sophisticated immunologic systems have been developed and used to identify potential vaccine candidates of which the 10 kDa and the 30 kDa antigens are leading contenders. Viable, but not killed, M. bovis BCG promote the functional differentiation of IFN-y producing, protective Th1 cells in vivo. Mycobacterial protein antigens and lipoarabinomannan have been shown to stimulate mononuclear phagocyte production of tumor necrosis factor-alpha (TNF-alpha), a key mediator in granuloma formation. A new element to immunopatho-genesis emerges from considering individuals infected with both M. tuberculosis and HIV-1. Although TNF-alpha may play a role in the protective immune response against M. tuberculosis, this cytokine may promote HIV replication in dually-infected persons. The latter notion already has fostered clinical trials of TNF-alpha inhibitors, pentoxifylline, and thalidomide. Progress has been made in elucidating the metabolic pathways involved in mycolic acid synthesis and considering these as potential targets for new drugs. Several newly synthesized rifamycin derivatives were demonstrated in vitro and in vivo in mice to provide more potent antituberculous activity than original rifamycin, suggesting their promising use for MDR-TB and MAC infection.

Future Goals

Major priorities of tuberculosis research involve the development of better vaccines and the understanding and intervention of MDR tuberculosis and tuberculosis in the HIV infected. Subjects of special interest are the application of molecular genetics to understand the virulence and pathogenicity, drug targets, and mechanisms of resistance of M. tuberculosis; improved animal and human models of protective immunity; and the analysis of constituents of tubercle bacilli that induce cytokines related to immunity and pathogenesis. Research on diseases caused by nontuber-culous mycobacteria, particularly M. avium, is of comparable importance.

The future goals of the combined Panels are to progress in the following areas:

1. Molecular genetics of M. tuberculosis
• The molecular basis for virulence and pathogenicity
• Mechanisms of drug action and drug resistance
• Mapping and sequencing of the genome of M. tuberculosis.
2. Immunobiology and pathogenesis of tuberculosis
   
   • Cells and cytokines involved in pathogenesis and protective immunity
   • Antigen(s) in mycobacteria that evoke protection
   • Mycobacterial products that induce cytokine production and possess adjuvant properties
   • HIV-infection and tuberculosis.

3. Development of improved animal and cell culture models


• Models resembling human infection and natural history
• Genetic manipulation of experimental animal models for the study of immunity
• Improved in vitro models for studying interactions of host lymphocytes and macrophages with mycobacteria.
4. Clinical application of basic technology
   
   • Early diagnosis of tuberculosis and identification of drug resistant disease
   • Immunological intervention for intractable tuberculosis and TB-HIV
   • Molecular epidemiology of tuberculosis
   • Potential targets for new drug development.

Selected References

United States

1. Toossi Z, Gogate P, Shiratsuchi H, Young TZ, Ellner JJ. Enhanced production of TGF-ß by blood monocytes from patients with active tuberculosis and presence of TGF-8 in tuberculous granulomatous lung lesions. J Immunol 1995; 154:465-73.

2. Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, Collins D, de Lisle G, Jacobs Jr WR. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994; 263:227-30.
3. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon-y gene-disrupted mice. J Exp Med 1993; 178:2243-7.
4. King C II, Sathish M, Crawford JT, Shinnick TM. Expression of contact-dependent cytolytic activity by Mycobacterium tuberculosis and isolation of the genomic locus that encodes the activity. Infect Immun 1993; 61:2708-12.
5. Jacobs Jr WR, Tuckman M, Bloom BR. Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature 1987; 327:532-6.

Japan

1. Inoue T, Yoshikai Y, Matsuzaki G, Nomoto K. Early appearing (/*-bearing T cells during infection with Calmette Guerin bacillus. J Immunol 1991; 146:2754-62.

2. Tominaga A, Takaki S, Koyama N, Katoh S, Matsumoto, R, Migita M, Hiroshi Y, Hosoya Y, Yamauchi S, Kanai Y, Miyazaki J, Usuku G, Yamamura K, Takatsu K. Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production. J Exp Med 1991; 173:429-37.
3. Tsuyuguchi I, Kawasumi H, Ueta C, Yano I, Kishimoto S. Increase of T cell receptor (/*-bearing T cells in cord blood of newborn babies obtained by in vitro stimulation with mycobacterial cord factor. Infect Immun 1991; 59:3053-9.
4. Kawamura I, Tsukada H, Yoshikawa H, Fujita M, Nomoto K, Mitsuyama M. IFN-( producing ability as a possible marker for the protective T cells against Mycobacterium bovis BCG in mice. J Immunol 1992; 148:2887-93.
5. Maekura R, Nakagawa M, Nakamura Y, Hiraga N, Yamamura Y, Ito M, Ueda E, Yano S, He H, Oka S, Kashima K, Yano I. Clinical evaluation of rapid serodiagnosis of pulmonary tuberculosis by ELISA with cord factor (trehalose 6, 6'-dimycolate) as antigen purified from Mycobacterium tuberculosis. Am Rev Respir Dis 1993; 148:997-1001.