TUBERCULOSIS A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R E FERENCES
J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS
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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright 2003 by ICON Group International, Inc. Copyright 2003 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1
Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Tuberculosis: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-597-83644-2 1. Tuberculosis-Popular works. I. Title.
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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.
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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on tuberculosis. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.
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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.
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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes & Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health
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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON TUBERCULOSIS .......................................................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Tuberculosis .................................................................................. 9 E-Journals: PubMed Central ..................................................................................................... 133 The National Library of Medicine: PubMed .............................................................................. 176 CHAPTER 2. NUTRITION AND TUBERCULOSIS .............................................................................. 315 Overview.................................................................................................................................... 315 Finding Nutrition Studies on Tuberculosis............................................................................... 315 Federal Resources on Nutrition ................................................................................................. 323 Additional Web Resources ......................................................................................................... 324 CHAPTER 3. ALTERNATIVE MEDICINE AND TUBERCULOSIS ........................................................ 327 Overview.................................................................................................................................... 327 National Center for Complementary and Alternative Medicine................................................ 327 Additional Web Resources ......................................................................................................... 345 General References ..................................................................................................................... 350 CHAPTER 4. DISSERTATIONS ON TUBERCULOSIS .......................................................................... 353 Overview.................................................................................................................................... 353 Dissertations on Tuberculosis.................................................................................................... 353 Keeping Current ........................................................................................................................ 360 CHAPTER 5. CLINICAL TRIALS AND TUBERCULOSIS..................................................................... 361 Overview.................................................................................................................................... 361 Recent Trials on Tuberculosis.................................................................................................... 361 Keeping Current on Clinical Trials ........................................................................................... 377 CHAPTER 6. PATENTS ON TUBERCULOSIS ..................................................................................... 379 Overview.................................................................................................................................... 379 Patents on Tuberculosis ............................................................................................................. 379 Patent Applications on Tuberculosis ......................................................................................... 432 Keeping Current ........................................................................................................................ 465 CHAPTER 7. BOOKS ON TUBERCULOSIS ........................................................................................ 467 Overview.................................................................................................................................... 467 Book Summaries: Federal Agencies............................................................................................ 467 Book Summaries: Online Booksellers......................................................................................... 482 The National Library of Medicine Book Index ........................................................................... 496 Chapters on Tuberculosis........................................................................................................... 498 CHAPTER 8. MULTIMEDIA ON TUBERCULOSIS.............................................................................. 501 Overview.................................................................................................................................... 501 Video Recordings ....................................................................................................................... 501 Audio Recordings....................................................................................................................... 507 Bibliography: Multimedia on Tuberculosis................................................................................ 508 CHAPTER 9. PERIODICALS AND NEWS ON TUBERCULOSIS........................................................... 511 Overview.................................................................................................................................... 511 News Services and Press Releases.............................................................................................. 511 Newsletter Articles .................................................................................................................... 513 Academic Periodicals covering Tuberculosis ............................................................................. 514 CHAPTER 10. RESEARCHING MEDICATIONS................................................................................. 515 Overview.................................................................................................................................... 515 U.S. Pharmacopeia..................................................................................................................... 515 Commercial Databases ............................................................................................................... 517 Researching Orphan Drugs ....................................................................................................... 517
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APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 521 Overview.................................................................................................................................... 521 NIH Guidelines.......................................................................................................................... 521 NIH Databases........................................................................................................................... 523 Other Commercial Databases..................................................................................................... 551 The Genome Project and Tuberculosis....................................................................................... 551 APPENDIX B. PATIENT RESOURCES ............................................................................................... 555 Overview.................................................................................................................................... 555 Patient Guideline Sources.......................................................................................................... 555 Associations and Tuberculosis ................................................................................................... 590 Finding Associations.................................................................................................................. 592 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 595 Overview.................................................................................................................................... 595 Preparation................................................................................................................................. 595 Finding a Local Medical Library................................................................................................ 595 Medical Libraries in the U.S. and Canada ................................................................................. 595 ONLINE GLOSSARIES................................................................................................................ 601 Online Dictionary Directories ................................................................................................... 602 TUBERCULOSIS DICTIONARY................................................................................................ 603 INDEX .............................................................................................................................................. 691
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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with tuberculosis is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about tuberculosis, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to tuberculosis, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on tuberculosis. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to tuberculosis, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on tuberculosis. The Editors
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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.
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CHAPTER 1. STUDIES ON TUBERCULOSIS Overview In this chapter, we will show you how to locate peer-reviewed references and studies on tuberculosis.
The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and tuberculosis, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “tuberculosis” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •
Preventing and Controlling Tuberculosis Along the US-Mexico Border : Work Group Report Source: MMWR Morbidity and Mortality Weekly Report 2001; 50(RR-1):1-28. Contact: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for Health Statistics, Office of the Morbidity and Mortality Weekly Report Series, 1600 Clifton Rd NE M/S C-08, Atlanta, GA, 30333, (404) 639-2100, http://www.cdc.gov/mmwr. US Government Printing Office, PO Box 371954, Pittsburgh, PA, 15250-7954, (202) 512-1800, http://www.access.gpo.gov. Summary: This report, for health professionals, organizations, and government agencies, provides recommendations for the improvement of tuberculosis (TB) infection
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control and prevention along the border between the United States (US) and Mexico. It addresses the challenges created by border-crossing populations, for example, Mexico's higher TB rate, low socioeconomic status and limited access to health care, frequent border crossing and travel to the United States, and a lack of coordinated care across health jurisdictions in Mexico and the US. Surveillance needs, case management and therapy completion, performance indicators and program evaluation, and research needs are discussed. •
Tuberculosis Treatment Source: City Health Information June 1999;18 (2):1-8. Contact: New York City Department of Health, Bureau of Tuberculosis Control, 125 Worth St Rm 216 Box 74, New York, NY, 10013, (212) 788-4155, http://www.ci.nyc.ny.us/nyclink/html/doh/html/tb/tb.html. Summary: This newsletter provides healthcare professionals information on tuberculosis (TB) treatment. It presents ten basics on the diagnosis, treatment, and prevention of TB and information on countries and areas with an estimated or reported high incidence of TB, a therapy timeline for previously untreated TB patients with drugsusceptible active disease, medications used in the treatment of TB, recommended antituberculosis treatment regimens for HIV-infected persons with drug-susceptible TB disease, and the use of antituberculosis medications in special situations, such as pregnancy, TB meningitis, and renal failure. The newsletter lists contact information for resources in New York City providing TB-related services.
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Testing and Treatment for Latent Tuberculosis Infection Source: City Health Information October 2000;19(3):1-8. Contact: New York City Department of Health and Mental Hygiene, Bureau of Tuberculosis Control, PO Box 74, New York, NY, 10013-0061, (212) 788-4155, http://www.ci.nyc.ny.us/nyclink/html/doh/html/tb/tb.html. Summary: This newsletter provides healthcare professionals with information on the testing and treatment of latent tuberculosis infection (LTBI). It summarizes and synthesizes the Centers for Disease Control and Prevention's and the American Thoracic Society's treatment recommendations, offers ten basic steps in testing and treatment, and provides a pullout guide on fundamental aspects of the testing and treatment of LTBI, including when and how to administer therapy, how to interpret the tuberculin skin test in BCG-vaccinated individuals, and how to treat HIV-positive individuals who are taking antiretroviral agents. The newsletter lists contact information for resources in New York City providing TB-related services.
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Treatment of Tuberculosis and Tuberculosis Infection in Adults and Children Source: American Journal of Respiratory and Critical Care Medicine 1994 (May);149(5):1359-1374. Contact: American Lung Association of Maryland, 11720 Beltsville Drive 3rd FL, Beltsville, MD, 20705, (301) 572-3205. CDC National Prevention Information Network, PO Box 6003, Rockville, MD, 20849-6003, (800) 458-5231, http://cdcnpin.org. Summary: This is an official statement of the American Thoracic Society on the treatment of tuberculosis (TB) and TB infection in adults and children. This paper discusses the following topics: treatment of TB, drugs in current use, potentially
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effective drugs, initial treatment regimens, monitoring adverse reactions, evaluation of response to treatment, management of patients whose treatment has failed or who have relapsed, management of patients who have drug-resistant disease, TB in children and adolescents, special considerations in treatment, treatment of TB infection, persons for whom preventive therapy is recommended, screening procedures, administration of isoniazid preventive therapy, monitoring preventive therapy, and alternative forms of TB prevention. •
Controlling the Resurgent Tuberculosis Epidemic: A 50 - State Survey of TB Statutes and Proposals for Reform Source: Journal of the American Medical Association; Vol. 269, No. 2. Contact: American Society of Law Medicine and Ethics, 765 Commonwealth Ave Ste 1634, Boston, MA, 02215-1401, (617) 262-4990, http://www.aslme.org. Summary: This article reports on a 50-state survey of tuberculosis (TB) laws, based on an examination of both TB-specific statutes and more general communicable disease statutes. The survey found that many statutes still in force were enacted at the turn of the century or in the early to mid-1900s, before the development of modern concepts of constitutional law and the concept of the need to protect public health. The article also recommends the development of an individualized treatment plan for all persons diagnosed with the disease, the balancing of constitutional rights against public health risk, the maintainence of confidentiality and antidiscrimination, and the bringing of communicable disease statutes into conformity with the American With Disabilities Act (ADA).
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Return of a Plague: The Perils of Tuberculosis in the 90's Source: The Exchange; Issue 21, July 1993. Contact: National Lawyers Guild AIDS Network, 558 Capp St, San Francisco, CA, 94110, (415) 824-8884. Summary: The re-emergence of the tuberculosis (TB) epidemic with its associated medical and legal problems, especially in relation to AIDS, is examined in this journal article. TB, its treatment, and the problem of incomplete treatment are described. The definition of AIDS has been expanded to include pulmonary TB. The article suggests that social reform is required to control TB, including the restructuring of the public health and social service systems. The interdependent reforms include public funding for TB education, emphasizing Directly Observed Therapy (DOT) to use in fighting incomplete treatment, funding research in diagnostics and treatments of TB, and developing guidelines for the legal issues involved. These legal concerns include civil rights with respect to state-mandated quarantine, confidentiality, and employment practices where infection control, not discrimination, prevents disease.
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Tuberculose et Infection par le VIH. [Tuberculosis and HIV Infection.] Contact: Agence Nationale de Recherches Sur le SIDA, 101 rue de Tolbiac, Paris. Summary: This factsheet explains epidemiology and treatment of tuberculosis (TB) in Persons with AIDS (PWA's). Since 1988, the number of French TB cases has risen, especially among PWA's. Active tuberculosis makes its appearance in one of two ways: a previous infection resurfaces, or a fresh infection is transmitted by another infected individual. Although PWA's experience more serious side effects, drugs are very effective against the tuberculosis bacterium. The drug-resistant strain of TB which has
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been observed in the United States is very rare in France. The factsheet also outlines the stages of TB and suggests medications for each stage. A short bibliography is appended. •
An Outbreak of Tuberculosis With Accelerated Progression Among Persons Infected With the Human Immunodeficiency Virus: An Analysis Using Restriction - Fragment - Length Polymorphisms Source: New England Journal of Medicine; Vol. 326, no. 4. Contact: San Francisco General Hospital, Department of Medicine, Chest Service, Pulmonary Complications of AIDS Study, SFGH-5K1, 1001 Potrero Ave, San Francisco, CA, 94110, (415) 821-8313. Louisiana State University Medical Center, Delta Region AIDS Education and Training Center, 136 S Roman St 3rd Fl, New Orleans, LA, 70112, (504) 568-3855, http://www.lsumc.edu. Summary: This reprint of a journal article analyzes an outbreak of tuberculosis (TB) that took place among residents of a housing facility for persons with HIV infection. As background information, the article points out that TB can develop from either a reactivation of a latent infection or from a primary infection. It then reviews the outbreak, which took place between December 1990 and April 1991, during which time 12 cases of TB were diagnosed. Two patients under treatment for TB had been admitted in the previous six months. Organisms isolated from 11 culture-positive residents had similar patterns, but isolates from the two patients treated prior to the outbreak showed different strains. The article says that this implicates the first of the 12 patients to come down with TB as the source of the outbreak. It concludes that newly acquired TB infection in HIV-positive patients can spread readily and progress rapidly to active disease. There should be heightened TB surveillance in facilities where HIV-positive persons live.
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Prevention and Control of Tuberculosis in Facilities Providing Long-Term Care to the Elderly : Recommendations of the Advisory Committee for Elimination of Tuberculosis Source: Morbidity and Mortality Weekly Report (MMWR). 39 (RR-10): 7-20; July 13, 1990. Contact: US Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Center for Health Statistics, Office of the Morbidity and Mortality Weekly Report Series, 1600 Clifton Rd NE M/S C-08, Atlanta, GA, 30333, (404) 332-4555, http://www.cdc.gov. US Government Printing Office, PO Box 371954, Pittsburgh, PA, 15250-7954, (202) 512-1800, http://www.access.gpo.gov. Summary: This report discusses managing and preventing tuberculosis (TB) in facilities that provide long-term care for the elderly. It provides a brief epidemiological history of TB among elderly people in the United States, including those in nursing homes. General guidelines for prevention and control of TB in the areas of surveillance, containment, assessment, and education are provided. Recommendations for surveillance include a tuberculin skin testing program for all new residents and employees to detect TB infection and active TB, case reporting, and encouraging HIV counseling and testing. Containment recommendations include information about appropriate isolation and treatment of the elderly resident, and an overview of contact investigations for residents and staff and appropriate preventive therapy. A recordkeeping system that includes specific data for annual review and assessment is recommended. The report concludes with the role of the local health department in the management and prevention of TB in long-term care facilities for the elderly.
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Spinal Tuberculosis Source: Current Opinion in Orthopedics. 11(3): 196-201. June 2000. Summary: This journal article provides health professionals with information on the diagnosis and management of spinal tuberculosis. Tuberculosis of the spine is still a common disease in some endemic regions and is returning to developed countries. The diagnosis is usually made with a high index of suspicion in endemic areas in the presence of pain and appropriate clinical symptoms and signs of a systemic infection. Typical plain radiographic changes include destruction of two adjacent vertebral bodies, narrowing of the intervening disk, scalloping of the anterior vertebrae, and a fusiform paravertebral abscess shadow. The development of the polymerase chain reaction technique and magnetic resonance imaging (MRI) in the past decade has greatly improved diagnostic accuracy. MRI is useful in diagnosing early or multicentric lesions before plain radiographic changes become obvious. The aims of treatment in spinal tuberculosis are eradication of the infection, preservation or restoration of neurologic integrity, and prevention or correction of spinal deformity. Combination chemotherapy remains the most important and effective treatment, whereas surgical intervention can help in achieving earlier fusion and less late deformity. Early results of spinal instrumentation with or without fusion appear to be promising in further enhancing good outcome. 1 figure, 2 tables, and 54 references. (AA-M).
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Primary Pancreatic Tuberculosis Source: Missouri Medicine. 88(11): 766-768. November 1991. Summary: This article describes the first reported case that fulfills the diagnostic criteria for primary pancreatic tuberculosis. After a detailed case presentation, the authors discuss extrapulmonary tuberculosis in general, the clinical presentation, and the diagnostic testing required to determine pancreatic tuberculosis. They also briefly review the literature covering this condition. Diagnostic tests considered include radiographic studies, ultrasound, CT guided needle aspiration of pancreatic masses, and exploratory laparotomy. 1 figure. 14 references.
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Tuberculosis and the Kidney Source: JASN. Journal of the American Society of Nephrology. 12(6): 1307-1314. June 2001. Contact: Available from Lippincott Williams and Wilkins. 12107 Insurance Way, Hagerstown, MD 21740. (800) 638-6423. Website: www.jasn.org. Summary: Tuberculosis of the kidney and urinary tract is, like other forms of the disease, caused by members of the Mycobacterium tuberculosis complex. This article reviews the clinical features of classical renal (kidney) tuberculosis, tuberculous interstitial nephritis, tuberculosis and glomerular disease, end stage renal disease (ESRD) caused by tuberculosis, tuberculosis developing in patients on hemodialysis and peritoneal dialysis, tuberculosis in transplant patients, genital tuberculosis, and hypercalcemia in dialysis patients. The authors also discuss laboratory diagnosis, pathology, the role of immunodeficiency in genitourinary tuberculosis, and treatment options. In developed nations, tuberculosis is relatively uncommon, but the risk of acquiring the disease is increased in immunosuppressed individuals, including patients on dialysis and recipients of kidney transplants. The signs and symptoms of renal tuberculosis mimic those of other infections of the kidney, so diagnostic awareness may prevent unnecessary morbidity (illness or complications). Tuberculosis may involve the
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kidney as part of generalized infection throughout the body or as localized genitourinary disease. The morphology (shape and appearance) of the lesions depends on the site of infection, the virulence of the organism, and the immune status of the patient. Modern short course antituberculosis drug regimens are effective in all forms of tuberculosis. However, special considerations apply to the treatment of tuberculosis in patients with impaired renal (kidney) function, as some drugs may not be metabolized properly. Surgical intervention is indicated in cases of advanced unilateral (involving one kidney) disease complicated by pain or hemorrhage and for bladder augmentation. 5 figures. 49 references. •
When to Consider Tuberculosis in Evaluating Hematuria Source: Consultant. 39(12): 3225. December 1999. Contact: Available from Cliggott Publishing Company. 55 Holly Hill Lane, Box 4010, Greenwich, CT 06831-0010. Summary: Tuberculosis of the urinary tract is rare enough to justify the selective use of tuberculosis cultures when evaluating hematuria. Nonpulmonary (not in the lungs) tuberculosis accounts for only about 2 percent of diagnosed cases of tuberculosis. This brief article, one from a regular column that answers clinical questions, addresses the need to consider tuberculosis when evaluating a patient with hematuria (blood in the urine). The author recommends that physicians consider obtaining tuberculosis urine cultures for patients who are at increased risk for genitourinary tuberculosis. Such patients include immunocompromised persons and those with active pulmonary tuberculosis or a history of tuberculosis. A history of travel in or immigration from an area of high tuberculosis activity should also raise the index of suspicion. Readers are also encouraged to seek the advice of an infectious diseases consultant.
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Tuberculosis Remains an Important Factor in the Morbidity and Mortality of Hemodialysis Patients Source: Transplantation Proceedings. 30(3): 846-847. May 1998. Contact: Available from Appleton and Lange. P.O. Box 86, Congers, NY 10920-0086. (203) 406-4623. Summary: Patients with end-stage renal disease (ESRD) are considered moderately immunocompromised and are susceptible to mycobacterial infection, including tuberculosis. This article explores the incidence of tuberculosis, organ involvement, diagnostic parameters, and response to tuberculosis treatment in the hemodialysis population. Based on data from 3 years, the incidence of tuberculosis at the authors hemodialysis unit is 2 percent (23 of 1140 patients). Diagnosis of tuberculosis in these patients is difficult, and biopsy, an invasive procedure, was required for diagnosis in 14 cases in this study. Extrapulmonary involvement (in organs other than the lungs) was a frequent finding in this population. The authors stress that, in spite of treatment, the high mortality rate due to this disease underlines tuberculosis as a persistent and significant problem that cannot be underestimated for hemodialysis patients. Because of this, all possible diagnostic methods need to be used in this group, including RT-PCR studies and tissue biopsy. 1 figure. 1 table. 5 references.
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Renal Tuberculosis Presenting as a Large Perinephric Mass Source: Infections in Urology. 10(6): 171-172, 175. November-December 1997.
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Contact: Available from SCP Communications, Inc. 134 West 29th Street, New York, NY 10001-5399. (212) 631-1600. Fax (212) 629-3760. Website: http://www.scp.com. Summary: Tuberculosis (TB) infections most commonly affect the respiratory and genitourinary systems. In industrialized countries, between 8 and 10 percent of patients with pulmonary TB develop renal (kidney) TB, and 15 to 20 percent of the population in developing countries have Mycobacterium tuberculosis cultured from the urine. This article reports a case of a patient who exhibited an unusual case of renal TB presenting as a perinephric mass. A 56 year old man with a 3 year history of untreated hypertension presented with a 2 week history of low grade fever, 10 pound weight loss, and acute onset of right hemiparesis (muscular weakness on one side of the body). The patient had no history of TB or urinary symptoms. Muscle strength in the right arm and right leg were diminished. A 15 by 20 cm, well defined, round, nontender, mobile mass was found in the left upper quadrant and flank. Other findings on abdominal and chest exam were normal. Urinalysis revealed many white blood cells per high power field, but no bacteria. Intravenous pyelogram was performed: the upper and mid poles of the left kidney were absent on nephrogram, and delayed excretion of contrast from the lower pole was observed. The right kidney was normal. The patient underwent an uncomplicated left radical nephrectomy for suspected liposarcoma. The gross pathology revealed a hard, fibrotic mass measuring 24 by 18 by 12 cm, filled with yellow purulent material surrounding a dilated collecting system and a 2 cm calculus. The renal tissue culture grew M. tuberculosis. The patient did well postoperatively and was discharged on the fourth day after the operation. In this patient, computed tomography (CT scan) provided the first clue to the diagnosis by suggesting the inflammatory nature of the renal mass; the diagnosis was confirmed by culture. 2 figures. 12 references.
Federally Funded Research on Tuberculosis The U.S. Government supports a variety of research studies relating to tuberculosis. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to tuberculosis. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore tuberculosis. The following is typical of the type of information found when searching the CRISP database for tuberculosis:
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Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).
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Project Title: A CENTRE FOR TUBERCULOSIS RESEARCH AND EDUCATION AT THE* Principal Investigator & Institution: Beyers, Nulda; University of Stellenbosch Stellenbosch, South Africa Stellenbosch, Timing: Fiscal Year 2002; Project Start 22-SEP-2002; Project End 21-AUG-2003 Summary: (provided by applicant): Southern Africa has the highest tuberculosis incidence in the world and South Africa is one of the 22 countries in the world that account for 80% of the global TB burden. The Western Cape province of South Africa has one of the highest reported tuberculosis incidences in the world at 500/100,000 population/year. TB, particularly in association with HIV/AIDS, is one of the major problems confronting sub-Sahara Africa where TB is the leading killer of people living with HIV. The Stellenbosch University Faculty of Health Sciences recently chartered a Centre for Tuberculosis Research and Education (CENTRE) with the mission of significantly contributing to the control of tuberculosis through purposeful interdepartmental and multidisciplinary research, education and training in collaboration with other national and international institutions. The CENTRE aims among others are 1. to enhance the collaboration and communication among TB and TB/HIV/AIDS researchers within the University as well as with other research collaborators in the Western Cape, South Africa and southern Africa, and 2. to improve the structured and unstructured education and training of graduate and post-graduate (Masters, Ph.D. and post-doctorate) students and young researchers who investigate adult and pediatric TB and TB/HIV/AIDS in southern Africa. The CENTRE is expected to have a significant impact on the training of researchers with a specific focus of understanding the dynamics of the rapidly spreading HIV/AIDS epidemic in southern Africa and its effect on the TB epidemic. The grant funds are intended to fund the work of a Steering Committee consisting of members from the CENTRE, the Harvard School of Public Health, and other potential co-operating partners. The Steering Committee with the CENTRE Director as Chairman will lead the development of a 2003 Strategic Plan for Graduate & Post-graduate Education in TB/HIV/AIDS Research. By the end of one year the CENTRE Director will prepare and assemble a Phase II Comprehensive Agreement application to fund the subsequent implementation of the 2003 Strategic Plan. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: A NEW TARGET FOR ANTI-TUBERCULOSIS AGENTS, FAS 1 Principal Investigator & Institution: Welch, John T. Chemistry; State University of New York at Albany 1400 Washington Ave Albany, NY 12222 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2004 Summary: (provided by applicant): As a sentinel disease for the diagnosis of AIDS, early reports of tuberculosis complicating AIDS seemed to indicate the disease was a result of reactivation of a previous infection, however today the picture of tuberculosis as a complication of AIDS has changed dramatically. The development of multidrug resistant strains of M. tuberculosis and the prospect of nosocomial transmission make the development of new, better tolerated therapies ever more important. It is the inclusion of pyrazinamide (PZA), an agent with a unique sterilizing activity, with isoniazid and rifampin (RIF) in current treatment regimens that constitutes the basis for 6-month short course therapy for M. tuberculosis (Mtb).l Surprisingly for a clinical agent, a detailed understanding of the mechanism of action of PZA continues to elude researchers. Recently the surprising observation was made that an analog of PZA, 5-
Studies 11
chloropyrazinamide (5-CI-PZA), inhibits fatty acid synthetase I, (FAS 1) in Mycobacterium tuberculosis. This observation has been confirmed independently in other laboratories. In prokaryotes, the synthetase is typically composed of at least seven peptides that represent the individual enzyme components and are generally classified as Type II synthases. However in mammals and mycobacteria, the synthase activity is carried out by single high-molecular weight, multifunctional peptide chains which are known as Type I synthases. In cell free extracts of Saccharomyces cerevisiae not only was the activity of 5-CI PZA confirmed to be very comparable with that of the wellknown inhibitor cerulenin in blocking the activity of FAS 1 it was also found that other pyrazinamide analogs also apparently inhibit FAS 1. The hypotheses to be tested in this proposal are that characterization of the mechanism of 5-CI-PZA on the inhibition of FAS 1 can lead to the development of better anti-mycobacterial agents and, secondly, that this inhibition process will be sensitive to structural variations of the pyrazinoic or nicotinic acid structure. The utility of the observed anti FAS activity can be enhanced by developing an understanding of the nature of the FAS 1 inhibition process so that this activity can be employed in the design of novel anti-tuberculous agents. As it is known that treatment of microbes with type I FAS inhibitors increases the sensitivity of those microbes to downstream inhibitors in a synergetic fashion, the development of antimycobacterial agents that act on FAS 1 and that can be used in combination with the aforementioned downstream agents may have a profound effect on improving tuberculosis treatment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: A NEW TUBERCULIN FOR THE DIAGNOSIS OF TUBERCULOSIS Principal Investigator & Institution: Einck, Leo; Sequella, Inc. 9610 Medical Center Dr, Ste 200 Rockville, MD 20850 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 29-SEP-2003 Summary: (provided by applicant):Accurate diagnosis of tuberculosis (TB) infection is one of the pillars of effective TB control programs. The current tool to diagnose infection with Mycobacterium tuberculosis is the tuberculin skin test (TST). Because of the crossreactive nature of purified protein derivative (PPD) of tuberculin, TST currently does not discriminate between infection with M. tuberculosis, vaccination with M. bovis BCG and sensitization with non-tuberculous mycobacteria found in the environment. The recent achievements in the field of bacterial genomics enable us to undertake a new approach to formulate a M. tuberculosis-specific tuberculin and to overcome poor TST specificity. Comparative genomic analyses show that the M. tuberculosis genome includes regions that do not exist in M. bovis BCG (RD regions). Thus, we can tailor a new, M. tuberculosis-specific tuberculin by selecting from the pool encoded by the RD regions those antigens that elicit TB-specific, cell-mediated immune responses. Our specific aims are 1) Identification of candidate antigens. Gene selection will be based on published results of comparative genomic analyses, BLAST protein homology searches, and Southern blot analyses. 2) Antigen screening in guinea pigs. Antigens will be selected that induce skin test reactions in tuberculous guinea pigs but not in control animals sensitized with M. bovis BCG and with non-tuberculous mycobacteria. 3) Antigen evaluation in human TB. Antigens selected in guinea pig studies will be evaluated for the ability to induce in vitro lymphoproliferation and cytokine secretion by peripheral blood mononuclear cells (PBMC) of M. tuberculosis-infected persons but not by PBMC of negative control individuals (BCG vaccinees, patients having nontuberculous mycobacterioses, and healthy, PPD-negative individuals). Antigens selected in the proposed Phase I studies will be used to formulate a multi-antigen tuberculin to
12 Tuberculosis
be taken to human clinical trials during Phase II work. PROPOSED COMMERCIAL APPLICATION: 2.1 Billion people harbor a TB infection. UNICEF reports that tuberculosis poses a serious risk to Asia's sustained socioeconomic development. In a recent National Intelligence Estimate, the Central Intelligence Agency singled out drugresistant TB-and especially its incidence among immigrants-as a potential threat to national security. Salomon and Murray report that world expenditure on TB therapy and diagnosis was $4.1 Billion (USD) in 1998. Diagnosis of TB worldwide is by sputum smear, with 50% accuracy. This new diagnostic is desperately needed. Short term markets will be in developing countries where the test will replace the current skin test as an adjunct diagnostic. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: VACCINE
A
PLANT-DERIVED,
MULTI-COMPONENT
TUBERCULOSIS
Principal Investigator & Institution: Walmsley, Amanda; Plant Biology; Arizona State University P.O. Box 873503 Tempe, AZ 852873503 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): Tuberculosis is the leading cause of death due to a single infectious agent among adults in the world. The HIV-AIDS pandemic has had a strong influence on the incidence of tuberculosis in the developing world, greatly increasing the annual risk of developing tuberculosis in HIV positive individuals and contributing to the spread of tuberculosis in the community. The current tuberculosis vaccine, bacillus Calmette-Guerin (BCG) has a variable protective efficacy and can cause serious, even fatal disease in immunocompromised patients. An effective, inexpensive, easily distributed and administered subunit vaccine is required for the control of tuberculosis. The goal of this project is to investigate the ability of plant-derived, tuberculosis antigens to induce immune responses appropriate for the protection of mice against tuberculosis. The success of this project will be the first step in our overall goal of developing an inexpensive, subunit vaccine that will enable control of tuberculosis (TB) in humans including immunocompromised individuals. Investigations will include the ability of plants to correctly process and express protective antigens against Mycobacterium tuberculosis; verification of the mucosal immunogenicity of the resulting plant-derived antigens; characterization of the induced immune response(s) in mice and guinea pigs and the ability of the plant-derived antigens to protect animals from challenge with M. tuberculosis. Synthetic, plant optimized coding regions have been constructed for the tuberculosis antigens Ag85B and ESAT-6. These synthetic coding regions have been fused to the B and A2 subunits of the heat labile enterotoxin (LT) of enterotoxigenic Escherichia coil (ETEC) to promote targeting to mucosal lymphoid tissues. The resulting fused coding sequences were cloned into plant expression vectors for future Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv "TA234). Expression, correct folding and concentration of the antigens in plant materials will be verified through Western analysis and enzyme linked immunosorbent assays (ELISA). Plant lines displaying high antigen expression, or elite plants, will be cloned, and transferred to the TB Research Materials and Vaccine Testing Contract at Colorado State University to be tested for mucosal immunogenicity and protective ability in mice trials. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 13
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Project Title: A RAPID SERODIAGNOSTIC TEST FOR TUBERCULOSIS Principal Investigator & Institution: Lyashchenko, Konstantin P.; Chembio Diagnostic Systems, Inc. 3661 Horseblock Rd Medford, NY 11763 Timing: Fiscal Year 2001; Project Start 15-JUL-1998; Project End 30-NOV-2002 Summary: (Adapted from Applicant's Abstract): Control of tuberculosis (TB), a disease that causes 3 million deaths a year worldwide, requires rapid methods for timely diagnosis. The goal of the proposed work is to develop a simple, rapid, accurate and inexpensive serodiagnostic assay for active TB using a thin-layer immunochromatography (TLIC) format and multiple recombinant antigens of Mycobacterium tuberculosis. Serodiagnostic tests for TB require multiantigen cocktails because of patient-to-patient variation in recognition of antigens by serum antibodies. Phase I studies have already identified eight antigens suitable for cocktail development and demonstrated that TLIC methodology is applicable to multiantigen-based TB serodiagnostics. Work proposed for Phase II is expected 1) to advance test development in terms of antigen composition and assay conditions, 2) to validate the diagnostic performance of selected cocktails in different TB cohorts and control groups, and 3) to establish methodologies for batch production of antigens. The goal of Phase II studies is to develop a test having a specificity close to 100% and a sensitivity of 90% in smearpositive pulmonary TB. The commercial opportunities for a rapid serodiagnostic TB test having the proposed specifications are immense. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ADJUNCT VITAMIN A THERAPY FOR TUBERCULOSIS AND HIV/AIDS Principal Investigator & Institution: Semba, Richard D. Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2003; Project Start 15-SEP-1998; Project End 28-FEB-2006 Summary: (provided by applicant): Tuberculosis is one of the leading causes of mortality worldwide, accounting for an estimated three million deaths annually. Micronutrient deficiencies are common during HIV infection and during pulmonary tuberculosis, and micronutrient deficiencies are associated with impaired immunity and increased morbidity and mortality. Despite adequate chemotherapy for tuberculosis, the morbidity and mortality of HIV-infected adults with pulmonary tuberculosis remains extremely high. The goal of this ongoing Phase III randomized, double-blind, controlled clinical trial is to determine whether a daily multivitamin (vitamins and minerals) supplement can reduce morbidity and mortality of HIVinfected adults with pulmonary tuberculosis in Malawi. All participants receive standard tuberculosis chemotherapy for eight months as per the Malawi National Tuberculosis Control Programme, and the total duration of follow-up in the trial is twenty-four months. The trial has a Community Advisory Board which consists of local community members and participants. The original sample size of the trial was reduced to 500 HIV-infected adults in order to complete enrollment within the study period and deal with a budget reduction. Interim analyses conducted for the NIAID HIV Therapeutic Clinical Trials Data and Safety Monitoring Board (DSMB) at NIH show that the trial has been conducted with steady enrollment, good adherence to supplementation, and high follow-up. The DSMB has recommended that the sample size of this trial be increased to have at least 80% power to detect a 20% reduction in mortality. To meet this recommendation, we propose to increase the sample size to 840
14 Tuberculosis
HIV-infected adults with pulmonary tuberculosis. The trial also includes 300 HIVnegative adults with pulmonary tuberculosis in order to reduce stigmatization. If this study demonstrates that a multivitamin supplement can reduce morbidity and mortality of HIV-infected adults with pulmonary tuberculosis, this appropriate, low cost intervention could be integrated with tuberculosis treatment to improve health and survival of HIV-infected individuals in developing countries. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AGING AND IMMUNITY IN TUBERCULOSIS Principal Investigator & Institution: Orme, Ian M. Professor; Microbiology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2001; Project Start 01-SEP-1986; Project End 30-SEP-2002 Summary: (Adapted from the applicant's abstract): Tuberculosis is still relatively common in the elderly, probably reflecting the very high incidence of skin test positivity of the population in the first third of the century, and the fact that many of these individuals are still alive and generally healthy at the present time. As they age, however, such individuals run the risk of developing recrudescence of latent tuberculosis, while nonsensitized individuals run the risk [especially if institutionalized] of contracting a primary tuberculosis infection. The basis of the susceptibility of the aged to tuberculosis infection or recrudescent disease is still poorly understood, but is believed to reflect age-associated changes in the cell-mediated immune response to the organism. Work specifically in the investigator's laboratory supports this contention by showing a series of defects in the CD4 TH1 IFN-secreting T cell pathway associated with protection and memory immunity to the disease. Hence, using a realistic low dose aerosol infection model in aging mice, the investigators have identified specific lesions such as the low expression of adhesion markers on CD4 T cells, and the absence of a pulmonary IL-12 response needed to enhance IFN secretion by such cells. In the proposed study, they intend to continue to define the cellular response in the old lung to tuberculosis, including the use of new markers such as CD49E and CD95. In addition, they intend to resolve the nature of the recrudescent event, framed on new evidence that the capacity of a given M.tuberculosis isolate to promote local TNF and chemokine production may directly correlate with the nature of the granuloma response and its potential breakdown as the mouse ages. Further issues to be addressed include the role of the predominant TH2/IL-4 environment in old mice [an environment that includes unusual IL-4 secreting CD4+NK1. 1+ and CD8+ T cell populations] in potential interference with TH1 responses, which they will investigate in part by using aging IL-4 gene disrupted mice, and the potential reversal or delay of endogenous reactivation of disease in the old lung by administration of a sub-unit vaccine preparation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ALCOHOL, TB AND AIDS Principal Investigator & Institution: Mason, Carol M. Medicine; Louisiana State Univ Hsc New Orleans New Orleans, LA 70112 Timing: Fiscal Year 2001; Project Start 25-SEP-1997; Project End 31-AUG-2003 Summary: APPLICANT'S ABSTRACT: During the late 1980's, the previous decline in tuberculosis ceased and rates began to rise, heralding a resurgence of disease. Human immunodeficiency virus infection is largely responsible for the excess number of cases. A co-factor for TB infection is alcohol consumption. Alcohol is immunosuppressive, and
Studies 15
facilitates acquisition of pulmonary infections through malnutrition, greater propensity for indulging in high risk behaviors, and alteration of normal host defenses. Defense alterations include changes in immune effector cell populations and down regulation of cytokine production and/or function, which are critical components in the containment of intracellular pathogens, such as Mycobacterium tuberculosis. We hypothesize that alcohol adversely alters pulmonary host defense mechanisms against Mycobacterium tuberculosis by disrupting early cytokine interactions [tumor necrosis factor (TNF), interleukin (IL)-12, and interferon (IFN)-gamma] which are critical in the orchestration of effective Th1 CD4+ lymphocyte cell-mediated responses to contain the mycobacteria. We will test this hypothesis in a murine model of acute and chronic alcohol consumption and M. tuberculosis infection, initially employing mycobacterial lipoarabinomannan (LAM; a cell wall component) and nonviable organisms as probes, and subsequently utilizing an in vivo model of M. tuberculosis infection. To achieve this, we propose the following: Specific Aim 1: To test the prediction that alcohol consumption suppresses the pulmonary production of early cytokines in response to mycobacterial antigens in mice. Specific Aim 2: To test the theory that alcohol, through disruption of early cytokine pathways, impairs the development of Th1-directed cellmediated immunity to viable M. tuberculosis infection. Specific Aim 3: To demonstrate that defective antimycobacterial cell-mediated host defenses can be enhanced with IFNgamma organ specific cytokine gene therapy in an animal model. Our goal is to elucidate the mechanisms by which alcohol consumption further diminishes host resistance to M. tuberculosis infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AN EFFICIENT BACTERIAL VECTOR-BASED TUBERCULOSIS VACCINE Principal Investigator & Institution: Szumanski, Maria Bw.; Veterinary Technologies Corporation 1872 Pratt Dr Blacksburg, VA 24060 Timing: Fiscal Year 2001; Project Start 01-MAY-2001; Project End 31-MAY-2002 Summary: (Adapted from Applicant's Abstract) Tuberculosis (TB) still remains a major global health threat; worldwide, it is responsible for over 3 million deaths annually. In lieu of the emerging multi-drug resistant Mycobacterium tuberculosis strains, control of TB through an effective vaccination is of a paramount importance. Although Mycobacterium bovis strain BCG is widely used for TB prophylaxis, its protective effect has recently been shown to be far below the necessary level to protect populations efficiently. A strong ThI type of cell mediated immune response is crucial for protection against M. tuberculosis infections. Several proteins of M. tuberculosis that are involved in stimulating a protective immune response have been identified and their genes have been cloned and sequenced. The overall objective of the proposed research project is to develop a highly efficacious and safe vaccine for TB. Specifically, the company proposes to prepare an effective vaccine against tuberculosis by expressing previously identified protective protein(s) of M. tuberculosis in Brucella abortus strain RB51, a bacterial vector with unique adjuvant properties and can stimulate a strong Th1 type of immune response. In the phase I part of the project, the company intends to i) construct recombinant RB51 strains that express 85A and ESAT-6 antigens of M. tuberculosis and ii) immunize mice with irradiated recombinant RB51 vaccines and characterize their antigen-specific antibody and cell-mediated immune responses to confirm that a Th1 type of immune response is induced. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
16 Tuberculosis
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Project Title: ANTI TUBERCULOSIS CANDIDATE VACCINE TESTING IN RHESUS MONKEYS Principal Investigator & Institution: Gormus, Bobby J.; Tulane University of Louisiana New Orleans, LA 70118 Timing: Fiscal Year 2001 Summary: Eight normal RM were inoculated with MTB via fiberoptic bronchoscope. Four received the less virulent H37Rv strain and four received the highly virulent Erdman strain via fiberoptic bronchoscope. Both monkeys that received the high dosage of H37Rv (6 X 106 cfu) developed extensive multifocal expansile and coalescing granulomas with broad central zones of necrosis, neutrophilic infiltration, and mineralization in the inoculated right lobe of the lung by 5 and 11 weeks (wk) postchallenge. Monkeys receiving the low dose of H37Rv (30 cfu) had granulomatous lesions in the bronchial lymph nodes 18 wk after infection. Lesions suggest that low doses of M. tuberculosis H37Rv are controlled, at least in the short term, by natural defenses in the simian lung and that challenge with higher doses of bacteria produces significant tissue response without evidence of septicemic spread. Two high dose (150 cfu) Erdman recipient monkeys developed extensive granulomatous pneumonia, pleuritis, and bronchial node necrosis with microgranulomas in the liver, kidney, and other sites in 7-9 weeks. The appearance of pneumonia and time course were roughly comparable to disease produced by high dose (>106) H37Rv. The 2 low dose Erdmaninoculated monkeys (15 cfu) remained clinically normal 9 weeks post-challenge and at 19 wk had minimal clinical manifestations, but fibrotic granulomatous pulmonary lesions with spread to other tissues. We conclude that chronic disease can be established by low dose Erdman inoculation with a wider distribution of lesions being produced compared to the H37Rv strain. FUNDING Base Grant, Venture Research PUBLICATIONS Didier PJ, Blanchard JL and Gormus BJ. Pulmonary Tuberculosis in Normal Rhesus Monkeys Produced by M. tuberculosis H37Rv. Am J Soc Trop Med Hyg 57:156, 1997 [Abstract]. Didier PJ, Blanchard JL and Gormus BJ. Chronic Tuberculosis Produced by Low Dosage of M. tuberculosis (Erdman) in Rhesus Monkeys. Am J Soc Trop Med Hyg 59:361, 1998. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ANTIMICROBIAL PEPTIDES IN RESISTANCE TO TUBERCULOSIS Principal Investigator & Institution: Kisich, Kevin O.; National Jewish Medical & Res Ctr and Research Center Denver, CO 80206 Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2004 Summary: (provided by applicant): Infections with drug resistant strains of Mycobacterium tuberculosis and most strains of Mycobacterium avium are challenging to treat chemotherapeutically. As HIV infection and the AIDS epidemic spreads, adequate treatment of mycobacterial infections in both immunocompetent and immunocompromised patients becomes increasingly difficult. Additional understanding of innate and immune mechanisms of resistance as well as new antibiotics will be important for designing new, more effective treatment regimens for mycobacterial infection. We hypothesize that small, cationic human antimicrobial peptides (AMP), defensins and cathelicidin stored in granules of human polymorphonuclear leukocytes (PMN), and secreted from epithelia play important roles in resistance to mycobacterial infection, and may be adaptable for chemotherapeutic use. Human neutrophil peptides 1,2, and 3 (HNP 1,2, and 3) have shown to be antimycobacterial for laboratory and clinical isolates of M. tuberculosis and M. avium in
Studies 17
vitro, and we have confirmed this in our laboratory. We propose to extend these observations by fully characterizing the anti-mycobacterial activity of human AMP, examining the relationship between binding of AMP to mycobacteria and antimycobacterial activity, and evaluating the relationship between resistance to human AMP and pathogenicity. We will also examine pharmacological interactions between human AMP and first-line anti-tuberculosis drugs for mycobacteria in liquid and macrophage cultures. The proposed studies are designed to fill an important gap in our knowledge of how mycobacteria evade the innate immune system. Understanding of the relative sensitivities of mycobacteria will help us understand how mycobacteria survive their first encounter with the antimicrobial peptides in pulmonary secretions and in responding neutrophils. Correlation of AMP sensitivity with binding of the peptides will help determine whether and how AMP sensitivity is related to pathogenicity. In addition, the proposed studies will define the potential of AMP to participate in the anti-mycobacterial response of human macrophages, or whether intracellular mycobacteria are protected from exposure to AMP. These studies will further our understanding of how endogenous anti-mycobacterial substances may enhance resistance to infection, and will also reveal whether these substances may be adaptable to chemotherapeutic use for treatment of mycobacterial infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BCG + HSP, A NEW TUBERCULOSIS VACCINE Principal Investigator & Institution: Nikonenko, Boris; Sequella, Inc. 9610 Medical Center Dr, Ste 200 Rockville, MD 20850 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 30-JUN-2004 Summary: (provided by applicant): This proposal describes research that will examine the safety and efficacy of a novel strain of BCG in which the heat shock repressor gene has been deleted, resulting in the overexpression of a heat shock protein (hsp70). Heat shock proteins show a good deal of promise in the protection against and treatment of tuberculosis, and this novel BCG strain produces a T-cell response indicative of a more protective immune response. The Specific Aims of this Phase I proposal are: 1. In a guinea pig model of tuberculosis (TB), we will compare the efficacies of (1) intradermally administered (i.d.) BCGdeltahspR; (2) i.d. parent BCG; (3) subcutaneously administered (s.c.) BCGdeltahspR; (4) s.c. parent BCG; and (5) no vaccination (control). 2. In a mouse model of tuberculosis, we will compare the efficacies of (1) BCGdeltahspR administered subcutaneously; (2) parent BCG, administered subcutaneously; and (3) no vaccination (control). We believe it is important to test this vaccine in several different strains of mice that represent a spectrum of susceptibility to TB, as it is not known which strain might be the best model of TB in humans. Therefore, we will test the efficacy of rBCGdeltahspR in C57BI6, DBA/2, and CBA. 3. We will examine the safety of this modified BCG as compared to the parent BCG. We will infect mice and guinea pigs with each BCG strain and examine lungs for pathology, and count the CFU of BCG still present. We will also examine the antibiotic sensitivity of this rBCGdeltahspR to 10 commonly used TB drugs to ensure that the genetic manipulations performed have not made it less sensitive to drug treatment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: BINDING AND PRESENTATION OF LIPID ANTIGENS BY CD1B Principal Investigator & Institution: Porcelli, Steven A. Associate Professor; Microbiology and Immunology; Yeshiva University 500 W 185Th St New York, NY 10033
18 Tuberculosis
Timing: Fiscal Year 2001; Project Start 01-JUL-1999; Project End 30-JUN-2004 Summary: (Adapted from Investigator's Abstract): Recent studies have identified the CD1 family of cell surface glycoproteins as novel antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. Identification of naturally occurring antigens presented by CD1 has revealed the surprising finding that these prominently include a range of foreign lipids and glycolipids, such as several that are known to exist in the cell walls and membranes of pathogenic mycobacteria. Among the currently known CD1-presented antigens, the best structurally characterized are the mycolic acids and their monoglucosylated analogue, glucose monomycolate (GMM). These compounds have a lipid structure that is unlike any found in mammalian tissues, and occur abundantly as a major outer cell membrane component in a variety of pathogenic bacteria, including Mycobacterium tuberculosis. Recognition of these lipids appears to be a frequent feature of CD1b-restricted M. tuberculosis-specific T cells in humans, suggesting that T cell responses to mycolic acids and GMM may play a significant role in the protective host response to this important pathogen. Studies in the current proposal will assess whether mycolic acids or GMM are among the immunodominant T cell antigens of M. tuberculosis in humans infected with this bacterium, and will seek to clarify the molecular basis for the presentation and recognition of these novel T cell antigens. A combination of preparative and synthetic chemistry will be used to generate a range of structural analogues of mycolic acids and GMM. These will be studied for their ability to be recognized by CD1b-restricted T cells, and for their ability to bind directly to CD1b proteins. In addition, the structural basis for the binding of these antigens to the CD1b protein will be analyzed by carrying out site directed mutagenesis of residues forming the predicted ligand binding groove of CD1b. These studies will lead to a detailed understanding of the fundamental rules governing the interaction of a newly recognized class of T cell antigens with their antigen presenting molecule. The identification and detailed analysis of this newly recognized pathway for T cell antigen recognition is likely to be important for understanding the human immune response to M. tuberculosis and related pathogens, and has potential implications for future vaccine development efforts. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BIOSYNTHESIS OF THE MYCOBACTERIAL PEPTIDOGLYCAN Principal Investigator & Institution: Pavelka, Martin S. Microbiology and Immunology; University of Rochester Orpa - Rc Box 270140 Rochester, NY 14627 Timing: Fiscal Year 2001; Project Start 30-SEP-2000; Project End 31-JUL-2005 Summary: (Adapted from the Applicant's Abstract): Tuberculosis is the leading cause of death in adults due to an infectious organism. By the end of this century there may be as many as 90 million new cases of tuberculosis resulting in up to 30 million deaths. The failure of antimicrobial therapy and the dangerous association between tuberculosis and AIDS have brought renewed interest in studying M. tuberculosis, the organism responsible for this disease. A better understanding of the basic biology of the organism and the development of new anti-mycobacterial drugs are important goals of mycobacterial research. The cell envelope is an outstanding characteristic if the mycobacteria, consisting of a variety of polysaccharides such as arabinogalactan, lipoarabinomannan, and peptidoglycan, along with several different types of lipids including various glycolipids and the mycolic acids. The peptidoglycan layer of the cell envelope serves as the anchor for the principal components of the cell envelope and provides shape and structural integrity to the cell. The long-term goal of this proposal is a deeper understanding of the biosynthesis and assembly of the mycobacterial cell
Studies 19
envelope. The specific goal of this proposal is to understand more about the genetics and biosynthesis of the peptidoglycan layer of the envelope. The specific aims of this proposal are: 1) Determining the significance of N-glycolylation of the mycobacterial peptidoglycan. 2) Investigating the architecture of the peptidoglycan and the role it plays in the organization of the cell envelope. 3) Using beta-lactam antibiotics as tools to probe mycobacterial peptidoglycan biosynthesis. For the aims of this proposal, the Pi will study M. tuberculosis and M. smegmatis as a model organism using the techniques of classical bacterial genetics, molecular biology and biochemistry. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ACTIVATION
CATALASE-PEROXIDASE
CATALYSIS
IN
ANTIBIOTIC
Principal Investigator & Institution: Magliozzo, Richard S. Chemistry; Brooklyn College 2900 Bedford Ave New York, NY 11210 Timing: Fiscal Year 2001; Project Start 15-JUN-1998; Project End 31-MAY-2003 Summary: (adapted from applicant's abstract) The major role of the propose research is to explain the catalytic function of Mycobacterium Tuberculosis catalase-peroxidase, a heme-enzyme, in the activation of the antimycobacterial antibiotic, isoniazid (isonicotinic acid hydrazide). The specific aims include identification of the proximal ligand to heme iron in catalase-peroxidase, characterization of the spin state and coordination number of the heme iron in the resting enzyme, identification of hypervalent enzyme intermediates, and kinetic analysis of the reaction of these intermediates with isoniazid. Of special interest is the potential catalytic competence of oxy-ferrous catalase- peroxidase, and the potential for peroxynitrite to activate the enzyme. The role of selected amino acid residues in the catalytic mechanism an in isoniazid binding will be evaluated through examination of the properties of two mutant catalase-peroxidase enzymes identified from clinically isolated, isoniazid resistant M. tuberculosis strains. Inhibition of another M. tuberculosis enzyme, a fatty acyl enoyl reductase (the inhA protein) thought to be a target of drug action, by isoniazid activated catalase-peroxidase, will also be investigated. Other aims address the Mn(II)-peroxidase activity of catalase-peroxidase considered important because Mn(III) is an efficient single electron oxidant of isoniazid. The techniques of optical stoppedflow spectroscopy, resonance Raman, and electron paramagnetic resonance spectroscopies will be applied in the experimental protocols. The results of the proposed studies will advance a detailed understanding of the action of a first line antibiotic in current use to treat tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CD1-TARGETED VACCINATION AGAINST TUBERCULOSIS Principal Investigator & Institution: Modlin, Robert L. Professor; Medicine; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JAN-2008 Summary: (provided by applicant): The long-range goal of this project is to develop and validate the CD1-based vaccine technology platform to immunize humans against microbial pathogens. This approach is based on the knowledge that several of the CD1 proteins have specific targeting sequences that direct their traffic into endosomal compartments, structures also critical for antigen presentation by MHC class II molecules to CD4+ T cells. We will determine whether this novel vaccine strategy can induce protective immunity against Mycobacterium tuberculosis, a worldwide killer,
20 Tuberculosis
and now because of multi-drug resistant tuberculosis, a potential agent of a bioterrorist attack. We propose to determine the trafficking pattern of CD1 chimers in antigen presenting cells including monocytes and dendritic cells. The ability of CD1 targeting to be used in immunization in humans will be assessed by studying the immune response to an M. tuberculosis antigen ESAT-6. In addition, the ability of ESAT-6/CD1 fusion constructs to immunize human T cell responses in vitro will be investigated. To determine whether ESAT-6/CD1 chimers can induce protective immunity, ESAT6/CD1 fusion constructs will be used to immunize mice, which will be subsequently challenged with virulent M. tuberculosis. The studies proposed will help develop and test the efficacy of CD1-based DNA vaccines for the prevention of infectious disease, including those from natural pathogens and bioterrorist attacks. Specifically, it should be possible to develop a new approach to the prevention of tuberculosis, including multidrug resistant tuberculosis, in humans. Finally, the CD1-based vaccine technology should prove useful in the vaccination against microbial pathogens in which MHC class II-restricted presentation of antigen to CD4+ T cells is required for host defense. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CD8 T CELLS AND IMMUNITY TO TUBERCULOSIS IN OLD MICE Principal Investigator & Institution: Turner, Joanne; Assistant Professor; Microbiology, Immunology & Pathology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 30-JUN-2003 Summary: (provided by applicant): The elderly are more susceptible to many infectious diseases, and yet vaccinating this population is less effective when vaccines that are designed for young individuals are used. To design a vaccine that specifically targets an aging immune system it is first necessary to understand how the aging immune response differs from younger individuals when it encounters a pathogen. Using the aging mouse model of tuberculosis we have found that old mice express a transient early resistance to infection that correlates with the rapid presence of CD8 T cells within the lungs. This identifies a previously unrecognized novel immune mechanism in old mice that is clearly absent from the lungs of young mice. Using the low-dose aerosol infection model of tuberculosis we will characterize this CD8 T cell population further by determining the signals that rapidly recruit it to the lungs, and the mechanism by which it mediates early resistance to infection. Finally, we will use vaccine strategies known to elicit antigen specific CD8 T cell responses to mycobacterial antigens to see if these cells can then be detected within the CD8 cell population recruited into the lungs of old mice after infection with M. tuberculosis. Studies will be carried out in a new BSL-3 facility at Colorado State University and will use old wild type and genedisrupted mice from our existing in-house aging mouse colonies. The technical approaches in the proposed studies will use a combination of flow cytometry, FACS cell sorting, immunohistochemical staining, and real-time PCR, to address the proposed Aims. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CD8+ T CELL RESPONSE IN RESISTANCE TO TUBERCULOSIS Principal Investigator & Institution: Bushkin, Yuri; Associate Member; Public Health Research Institute 225 Warren St Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 30-SEP-1997; Project End 31-AUG-2003 Summary: Tuberculosis (TB) is a chronic infectious disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. Hence, cell- mediated immunity rather
Studies 21
than antibodies is the critical protective immune response against M. tb. Studies with animal models of TB suggest that the major histocompatibility complex (MHC) class Irestricted CD8+ T-cell response complements host defenses mediated by activated macrophages and CD4+ T cells, and has an important role in successful resolution of mycobacterial infections. However, in humans the role of CD8+ T cells has not been fully addressed. We have developed an in vitro assay that is based on a novel computerdriven algorithm predicting MHC class I ligands (peptides) with a proven high accuracy. Our assay can measure the presentation of predicted peptides by proper MHC class I alleles, and thus allows for identification of the epitopes recognized by CD8+ T cells. Thus, the overall objective of these studies is the analysis of the role of CD8+ T-cell response in resistance to TB. First, secreted mycobacterial proteins with known sequences will be analyzed by the computer program for the presence of potential CD8+ T-cell epitopes. Presentation of these predicted M. tb. -derived peptides by MHC class I will be determined in our peptide-binding assay with an antigen transporter-deficient cell line expressing HLA alleles of interest. This analysis will be extended to HLA alleles predominant in populations with high incidences of TB and HIV infection. Second, this methodology will be used in quantitative and qualitative analysis of the MHC class Irestricted CD8+ T-cell immune responses to M. tb. -derived peptides in PPD+ healthy donors and TB patients. The frequencies of memory CD8+ T cells in the peripheral blood and activated cytotoxic T lymphocytes in the bronchio-alveolar lavage will be measured. Comparison of CD8+ T-cell effector functions in TB patients vs. PPD+ healthy donors, and also in PPD+/HIV-infected and active TB/HIV- infected patients will determine the correlation between the CD8+ T-cell responses and resistance to M. tb. infection in these patients. Finally, M. tb. -specific CD8+ T cell clones will be isolated and functionally characterized. These T cell clones will be used to study the requirements for MHC class I-restricted presentation of M. tb. antigens by macrophages infected in vitro with viable bacilli. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CD8+ T CELLS AND MYCOPLASMA TUBERCULOSIS Principal Investigator & Institution: Canaday, David H. Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 01-JUL-1999; Project End 30-JUN-2004 Summary: This application for a mentored scientist award (KO-8) seeks 5 years of funding for research training in the cellular immunology and cell biology of M. tuberculosis infection for David H. Canaday, M.D. Research training will be provided by W. Henry Boom, M.D. and Clifford V. Harding, M.D.-Ph.D. in the Division of Infectious Diseases and the Department of Medicine at Case Western Reserve University. The research proposal which will provide the focus for Dr. Canaday's training is outlined below. M. tuberculosis is spread from person to person by inhalation of aerosolized mycobacteria. Most healthy people do not develop clinical tuberculosis. Instead, cellular immune responses become activated and are able to successfully control the active infection. T cells play a crucial role in regulating the cellular immune response. T cell subsets(CD4+, CD8+, gammadelta+), are activated by mycobacterial antigens, yet little is known about the roles and function of the different T cell subsets in the protective immune response to M. tuberculosis. While CD4+ T cells have been the focus of many studies, CD8+ T cells are an important accessory T cell subset in the protective immune response to M. tuberculosis. Recent studies by us and others have demonstrated that human CD8+ T cells serve as CTL for M. tuberculosis infected macrophages, produce IFN-gamma and are activated by mycobacterial
22 Tuberculosis
antigens. The broad goal of the current studies is to determine the repertoire of mycobacterial proteins which stimulate human CD8+ T cells, to examine the antigen processing mechanism the macrophages use to present M. tuberculosis antigens on MHC class I molecules, and to determine the function of CD8+ T cells in patients with active tuberculosis. The Aims are: Aim 1. To determine the mycobacterial proteins and peptides recognized by human alphabeta TCR+ CD8+ T cells. Aim 2. To determine the mechanism(s) used by M. tuberculosis infected macrophages to process and present mycobacterial proteins by MHC class I molecules. Aim 3. To characterize the functional CD8+ T cell responses to specific proteins and peptides from in patients with active M. tuberculosis infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CELL ENVELOPE BIOSYNTHESIS IN M TUBERCULOSIS Principal Investigator & Institution: Glickman, Michael S. Medicine; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2001; Project Start 01-JUL-1998; Project End 07-DEC-2001 Summary: This proposal outlines a career development plan that consists of intensive research experience and didactic training in bacterial pathogenesis. The institutional environment is scientifically rich and well suited for the training of young physicianscientists. The ultimate goal of this period of intensive research training is to train the candidate as an independent investigator on a medical school faculty. Tuberculosis, caused by Mycobacterium tuberculosis, is the single leading cause of death from an infectious disease in the world today. Three million die each year from Tuberculosis and one third of the world's population is infected with M. tuberculosis MTB. The cell envelope of MTB is a complex structure composed of hydrophobic lipids, mycolic acids, cord factors, and Lipoarabinomannan and is thought to contribute to the virulence of the organism. Despite substantial investigation into the ultrastructure and chemical composition of the mycobacterial cell envelope, the contribution of specific cell walls constituents to mycobacterial virulence remains poorly defined. The importance of the cell envelope to the viability of the mycobacterial cell is emphasized by the observation that several antimycobacterial drugs including Isoniazid are thought to exert their bacteriacidal effect through inhibition of cell envelope biosynthesis. This proposal outlines a series of experiments designed to investigate the role of the cell envelope of MTB in pathogenesis through the generation and characterization of mycobacterial mutants deficient in the synthesis of cell envelope constituents. Using a recently developed transposon mutagenesis system, a series of insertional mutants will be generated and screened for alterations in their cell envelope composition. Morphologic, immunologic, and functional approaches to the isolation and characterization of cell envelope altered mutants will be used. Isolation and characterization of such mutants will likely yield important insights into the role of the mycobacterial cell envelope in the pathogenesis of Tuberculosis and the interaction of the tubercle bacillus with host cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CELL MIGRATION IN TUBERCULOSIS INFECTION Principal Investigator & Institution: Chan, John R. Associate Professor; Medicine; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-MAY-2007 Summary: (provided by applicant): The granuloma plays an important role in host defense against M. tuberculosis. The mechanisms that regulate the formation and
Studies 23
maintenance of the tuberculous granuloma are, however, poorly understood. Chemokines and chemokine receptors play an essential role in cell migration in both physiological and pathophysiological states. Emerging evidence suggests a role for chemokine and chemokine receptors in regulating the granulomatous response during infection. M. tuberculosis has the ability to modulate chemokine and chemokine receptor expression in both in vitro and in vivo systems. Tumor necrosis factor-alpha (TNF-alpha) is essential for the control of tuberculosis, and is a potent regulator of chemokine expression and leukocyte trafficking. We have shown that neutralizing TNFalpha in mice with persistent tuberculosis results in disease recrudescence associated with granuloma disorganization and diffuse cellular infiltration in the lungs. Based on these observations, we propose to test the hypotheses that: i) chemokines and chemokine receptors play an important role in orchestrating cell migration and granuloma formation in tuberculosis; and ii) TNF-alpha regulates the granulomatous response by directing the trafficking of immune cells at the site of infection via regulation of specific chemokines and chemokine receptors. Because of the importance of Type 1 T cells in host defense against M. tuberculosis, efforts will be focused on examining a subset of chemokines and receptors that can modulate migration of these T lymphocytes. Murine tuberculosis models, as well as immunohistochemical, laser microdissection, and realtime PCR techniques will be used to characterize the expression of these specific chemokines and receptors during tuberculous infection. Mice with disruption of specific chemokine receptor genes and ligand neutralizing reagents will be exploited to dissect specific chemokine network. Similar techniques, in conjunction with in vitro cell migration assays and the TNF-alpha neutralization model of murine reactivation tuberculosis, will be employed to evaluate the effects of TNFalpha on the expression of specific chemokines and receptors, as well as on migration of T cells and monocytes during tuberculous infection. These studies should yield valuable information that will shed light on the roles of chemokines and receptors on cell migration, granuloma formation, and host defense in tuberculous infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CELL WALL BIOGENESIS-TARGET FOR NEW ANTI-TB DRUGS Principal Investigator & Institution: Mcneil, Michael R. Professor; Microbiology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2002; Project Start 30-SEP-1992; Project End 31-AUG-2007 Summary: (Provided by applicant): In this competing continuation, mycobacterial cell wall metabolism is targeted as a route to new tuberculosis drugs. In one approach, essential cell wall biosynthetic enzymes are targetted. It is proposed to prepare M. tuberculosis luciferase reporter strains that respond specifically to inhibitors of the following essential enzymes: rhamnosyl transferase (WbbL), galactofuranosyl transferase (GlfT), dTDP-Rha formation enzymes (RmIB-D), and the UDP-Galf formation enzyme (Glf). In addition, direct enzyme assays amenable to screening for inhibitors will be developed for WbbL and GlfT (such assays are already in place for RmIB-D and Glf). It is further proposed to identify the genes encoding the three enzymes that form the arabinosyl donor, decaprenylphosphoryl-D-arabinose, show that their corresponding enzymes are essential, and prepare M. tuberculosis luciferase reporter strains and enzyme assays for use in finding inhibitors of them. In the second approach it is hypothesized that compounds that activate or deregulate the cell wall degrading arabinases and peptidoglycan hydrolyases can act more quickly than inhibition based drugs and thereby shorten tuberculosis therapy. Thus newly discovered M. tuberculosis endogenous arabinanases (of unknown function) that cleave
24 Tuberculosis
cell wall arabinan will be purified and their genes identified. The effect of overexpressing these enzymes in M. tuberculosis will be tested; it is hoped the effect will be lethal. Reporter strains of M. tuberculosis to detect small molecules that up regulate the expression of the arabinanases will be prepared. The presence of a cell wall metabolic enzyme complex containing these arabinases (which can potentially be disrupted for therapy) will be searched for using a novel cross-linking reagent, cyanogen complexes of peptidoglycan synthetic and degradative enzymes, after covalent crosslinking with cyanogen, will be isolated. The identity of all proteins present in the complex will be determined by LC/MS and how individual proteins interact in the complex will be determined for the long-term goal of disrupting the complex to release the peptidoglycan cleaving enzymes. We will test M. tuberculosis for increased arabinase activity after exposure to novel highly active ethambutol like compounds (the presence of endogenous arabinase was first recognized during ethambutol treatment). Finally, large numbers of novel Betalactams will be tested for their action against M. tuberculosis with and without ethambutol like compounds anticipating that some will strongly stimulate cell wail degradation and bacterial death. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CELLULAR TUBERCULOSIS
NECROSIS
INDUCED
BY
MYCOBACTERIUM
Principal Investigator & Institution: King, C Harold. Medicine; Emory University 1784 North Decatur Road Atlanta, GA 30322 Timing: Fiscal Year 2002; Project Start 15-SEP-2002; Project End 14-SEP-2003 Summary: (provided by applicant): A key step in the pathogenesis of M tuberculosis is its ability to cause caseating necrosis, parenchymal lung destruction, and cavity formation, which develop into the characteristic necrotizing bronchointerstitial pneumonia and bronchiolitis of tuberculosis. M tuberculosis is cytotoxic to epithelial cells in vitro, and we have shown that this cytotoxicity is associated with cell membrane permeation to lactose dehydrogenase and is mediated by necrosis of lung epithelial cells after infection with virulent mycobacteria (Dobos, K. M., Quinn, F. D. and King, C. H. 2000, Infect. Immun. 68:6300-6310). Interestingly, the attenuated M bovis BCG does not induce necrosis in this epithelial cell model suggesting that necrosis is related to the virulence of mycobacteria. Our working hypothesis is that M tuberculosis possesses factors that cause necrosis. We intend to identify the genes that encode or synthesize such factors and determine their functions. Towards this goal, we have been successful in isolating two such (necrosis-deficient) mutants with insertions into genes that have no known function by screening a transposon library of the Erdman strain of M tuberculosis (TN5370) for mutants that have lost their ability to cause cell membrane permeation and necrosis. Both nec mutants possess extremely interesting phenotypes when grown in mice. The first mutant (necA) appears to be highly attenuated for growth and virulence in SCID mice. This is an important result as it suggests that we have identified a gene whose product either directly causes necrosis or induces necrosis and thus should enhance our understanding of tuberculosis pathogenesis. Interestingly, the second mutant (necB) appears to kill SCID mice more rapidly than the parental strain. We intend to characterize these mutants, characterize the functions of the gene products, and extend this mutant isolation strategy to identify a large battery of mutants defective for necrosis of host cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 25
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Project Title: CHARACTERIZATION OF ACID INDUCED PROMOTERS OF M. TB Principal Investigator & Institution: Saviola, Beatrice J. Basic Sciences; Western University of Health Sciences Health Sciences Pomona, CA 91766 Timing: Fiscal Year 2003; Project Start 15-MAY-2003; Project End 30-APR-2005 Summary: (provided by applicant): Tuberculosis exacts an enormous burden in morbidity and mortality on the global population. With the advent of the HIV epidemic and multiple drug resistances, disease due to tuberculosis has increased leading to a need for research into the basic mechanisms of pathogenesis. The long-range goal of this proposal is to understand how Mycobacterium tuberculosis, the causative agent of tuberculosis, can sense environmental stresses, up regulate critical genes, and survive in the hostile environment of the host. When M. tuberculosis invades a host it infects human macrophages. M. tuberculosis can also be found within host granulomas that have been shown to have an acidic pH. The putative promoter regions of the M. tuberculosis genes lipF and Rv0834c have been identified to be upregulated in response to acidic pH. The specific aims of this proposal are to: 1) characterize the putative promoter regions of lipF and Rv0834c to better understand how acid induced promoters are regulated 2) determine if lipF and Rv0834c are required for mycobacteria to resist acidic stress and identify additional stresses which may upregulate these genes. The expectation is that this work will result in the characterization of acid responsive promoter regions and provide the groundwork for the eventual identification of a general mechanism by which M. tuberculosis can resist environmental stresses such as acidic stress. This work is significant because it will contribute to a greater understanding of mycobacterial stress response and ultimately survival and persistence in the host during infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CHARACTERIZATION OF MYCOBACTERIAL AUTOLYSINS Principal Investigator & Institution: Deng, Lingyi; Medicine; Boston University Medical Campus 715 Albany St, 560 Boston, MA 02118 Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2002 Summary: (Adapted from the Applicant's Abstract): Mycobacterium tuberculosis remains the bacterial leading cause of death worldwide and multidrug-resistant strains have emerged as a new problem in both developed and developing countries. The cell wall of M. tuberculosis has a unique, complex structure and is extraordinarily thick, rigid, and hydrophobic. Because of these characteristics, it is highly impermeable to ordinary antimicrobial agents. Bacterial autolysins are enzymes which are capable of hydrolyzing the bacterial cell wall and are associated with normal bacterial cell division, growth, and autolysis. Little is known about the autolysins of mycobacteria. The central hypothesis of this proposal is that mycobacterial cell wall autolysins are essential for the growth and survival of the organisms and can be exploited as new targets for antimycobacterial agents. In preliminary studies, the investigators have (i) prepared a mycobacterial cell lysate that hydrolyzed the cell wall polysaccharide; (ii) observed that ethambutol treated mycobacteria have increased cell wall hydrolysis; and (iii) identified an open reading frame (ORF) in the M. tuberculosis genome that is highly homologous to a Bacillus subtilis cell wall hydrolases, i.e., an N-acetylmuranoyl-L-alanine amidase. The investigators now propose the following: AIM1. The putative M. tuberculosis amidase gene noted above will be amplified by PCR, cloned and expressed. If the protein is confirmed to have the predicted enzymatic activity, then its functional and biochemical characteristics will be determined and the expression properties of the gene
26 Tuberculosis
will be analyzed. AIM 2. (a) Develop rapid and sensitive in situ phenotypic assays and apply these to screening for cloned mycobacterial autolysins expressed in E. coli. (b) Develop improved zymography and cell-free enzymatic assays and apply these to the isolation and purification of putative hydrolases. Purified enzymes will be analyzed for structural information (e.g. N-terminal sequence, quantitative protein mass as determined by MALDI/TOF mass spectroscopy), which will be used to identify the encoding gene in the published M. tuberculosis genome sequences. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHARACTERIZATION OF MYCOBACTERIAL SIGMA FACTORS Principal Investigator & Institution: Husson, Robert N.; Children's Hospital (Boston) Boston, MA 021155737 Timing: Fiscal Year 2001; Project Start 01-JUN-1995; Project End 28-FEB-2005 Summary: (provided by the applicant): The long-term goal of this research is to investigate the role of the alternative sigma factors SigD, SigE, SigH and SigM in the regulation of Mycobacterium tuberculosis gene expression and virulence. The underlying hypothesis of this research is that extracytoplasmic function (ECF) sigma factors of M tuberculosis play an important role in the regulation of gene expression during the interaction of this organism with the host during infection, and that this regulation is important for the virulence of this organism. This proposal has four complementary specific aims. The first aim is to complete construction of sigma factor mutant strains of M tuberculosis and to compare the survival of these mutants vs. the parental strain H37Rv following in vitro oxidative and nitrosative stresses that may be relevant to infection. The second aim is to compare the virulence of these mutants vs. H37Rv in macrophage and mouse models of infection. Mutant strains will be complemented to verify that any phenotypes observed in these experiments result from the disruption of the sigma factor gene that was mutated. The third aim is to investigate the role of these sigma factors in regulating M tuberculosis gene expression, focusing on the use of microarray technology, supplemented with bioinformatic and traditional molecular genetic methods. The fourth aim is to investigate the role of specific sigma factor-regulated genes in M tuberculosis virulence, by examining their expression and by constructing and testing M tuberculosis strains with mutations in these genes. These investigations are expected to identify regulatory networks and specific genes that are important in M tuberculosis virulence. This research thus has the potential to generate new insights into the pathogenesis of M tuberculosis infection, and may provide the basis for new approaches to the control of tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CHARACTERIZATION TUBERCULOSIS
OF
THE
SIGE
REGULON
OF
M
Principal Investigator & Institution: Posey, James E.; U.S. Centers for Disease Control & Prev Control and Prevention Atlanta, GA 30333 Timing: Fiscal Year 2001; Project Start 01-APR-2001 Summary: (provided by the applicant): The experiments outlined in this proposal are designed to elucidate the sigE regulon of M. tuberculosis and the role it plays in the infection process. Recent data demonstrate the induction of sigE when M. tuberculosis is phagocytized by the macrophage. The in vivo signal and the genes regulated by SigE are not known. The objectives of this proposal are to: (1) determine if SigE is required for intracellular survival in macrophage, (2) identify genes regulated by SigE, and (3)
Studies 27
characterize the regulation of sigE at the transcriptional and post-translational level. A delta sigE strain of M. tuberculosis will be constructed, and its ability to survive and replicate in the macrophage will be assessed. This strain will also be used to identify genes that are regulated by SigE. The expression of sigE will be controlled in the strain by cloning this gene downstream of an acetamide inducible promoter located in a shuttle vector. The protein expression profiles of strains harboring this plasmid in the presence and absence of acetamide will be compared to identify genes regulated by SigE. To assess the regulation of sigE, a delta orf1 strain of M. tuberculosis will be constructed, and the levels of SigE in wild type and this strain will be compared by western blot. The transcript levels of sigE in both strains also will be examined. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CLINICAL INVESTIGATIONS IN LUNG HOST DEFENSES Principal Investigator & Institution: Schluger, Neil W. Associate Professor of Medicine & Public; Medicine; Columbia University Health Sciences New York, NY 10032 Timing: Fiscal Year 2001; Project Start 20-SEP-1999; Project End 31-JUL-2004 Summary: Tuberculosis remains the world's leading infectious cause of death, and the rise of multidrug resistant tuberculosis and the interaction of the TB and AIDS epidemics worldwide are creating a public health threat of massive proportions. In recent years, we have pursued studies aimed at elucidating ways in which the host immune response to tuberculosis may offer protection from the disease, as well as ways in which inflammatory responses can be modified to improve outcomes in patients with multidrug resistant tuberculosis. We have demonstrated that the cytokine interferon (IFN) gamma plays a major role in human host defense against tuberculosis; a detailed understanding of immune mechanisms upstream and downstream from IFN's action in patients should provide significant insight into protection against tuberculosis infection and disease. To that end, three specific aims are planned: Specific aim 1: To assess, by determining the types and levels of chemokine present in bronchoalveolar lavage fluid of patients with tuberculosis, the signals in the lung which may cause recruitment of immune effector cells associated with an effective host response. Specific aim 2: To assess the state of activation of antigen presenting cell as well as immune effector cells in the lungs of patients with tuberculosis compared to controls. Specific aim 3: To assess changes in the pulmonary host response after administration of interferon gamma by aerosol to clearly define the mechanisms by which IFN causes clinical improvement in tuberculosis patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CLINICAL RESEARCH IN TUBERCULOSIS Principal Investigator & Institution: Chaisson, Richard E. Professor; Medicine; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-SEP-1999; Project End 31-AUG-2004 Summary: This application for a Midcareer Investigator Award in Patient-Oriented Research is submitted by Richard E. Chaisson, M.D, Professor of Medicine, Epidemiology and International Health at the Johns Hopkins University. The applicant completed subspecialty training in infectious diseases and clinical epidemiology in 1987. Since that time, he has built a successful and productive research program within the Johns Hopkins AIDS Service, a multidisciplinary clinical care enterprise for adults with HIV infection. In addition, he has organized and led the clinical and research efforts of the Baltimore City Health Department Tuberculosis Clinic, and has undertaken field
28 Tuberculosis
trials in HIV-related tuberculosis in Haiti and South Africa. In 1998 Dr. Chaisson was promoted to Professor and began a refocusing of his academic activities and commitments. This application is intended to support this effort. Dr. Chaisson has relinquished the directorship of the AIDS Service and has appointed a replacement director of the Tuberculosis Clinic in order to devote his full attention to clinical research in tuberculosis and HIV infection. Dr. Chaisson has founded the Johns Hopkins University Center for Tuberculosis Research, an interdivisional, multidisciplinary program dedicated to improving global tuberculosis control through innovations in diagnosis, treatment and prevention. The centerpiece of this initiative is field trial comparing alternative tuberculosis control strategies in communities within high-incidence countries, funded under the US Agency for International Development's Infectious Disease Initiative. This trial will be conducted in several countries and will randomly assign communities to one of three tuberculosis control strategies: i) directly observed therapy (DOT) for active cases; ii) DOT plus screening of household contacts for active disease; and iii) DOT plus screening and preventive therapy for household contacts. This trial will provide important insight into effective strategies for global tuberculosis control. The applicant is also Principal Investigator of the Baltimore Tuberculosis Trials Consortium site, and a funded investigator in other studies of tuberculosis epidemiology and control. With support under this award, he will develop an integrate program in tuberculosis research utilizing the clinical and laboratory resources of the Baltimore Tuberculosis Clinic, the Johns Hopkins University and sites in developing countries with high tuberculosis incidence. Moreover, he will serve as mentor to junior faculty in infectious diseases and related fields who are beginning academic careers in patient-oriented research. A mentoring program organized to enhance professional development and independent scholarly activity will be undertaken with two recent faculty recruits. An award under this program will substantially enhance the research and mentoring capabilities of the applicant, and will contribute to the generation of new knowledge essential for the control of tuberculosis worldwide. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CONJUGATION AND RECOMBINATION IN MYCOBACTERIA Principal Investigator & Institution: Derbyshire, Keith M.; Wadsworth Center Empire State Plaza Albany, NY 12237 Timing: Fiscal Year 2001; Project Start 01-FEB-1999; Project End 31-JAN-2004 Summary: Mycobacterium tuberculosis accounts for more deaths worldwide than any other infectious agent. Recent increases in the number of cases have been associated with the spread of the AIDS virus and the appearance of drug-resistant strains of M. tuberculosis. The development of new treatments for mycobacteria requires an understanding of the biology of these bacteria and the ability to manipulate their genomes to determine the genetic basis of pathogenesis and drug resistance. In M. tuberculosis, studies have been hindered by inefficient methods for the introduction of DNA into and between strains, and by the low frequency of targeted recombination. In particular, the ability to make site-directed changes within the M. tuberculosis genome is crucial for understanding the role of particular genes in virulence. We propose to characterize a novel conjugation system that can transfer chromosomal DNA between strains of Mycobacterium smegmatis and use it as a tool for the analysis and transfer of DNA between mycobacteria. This naturally occurring system could offer an efficient method for the introduction of DNA into the slow-growing pathogenic mycobacteria. Furthermore, by examining conjugation between different species of mycobacteria we
Studies 29
will assess its promiscuity and possible role in the spread of drug resistance. Finally, we have identified three M. tuberculosis genes that are highly homologous to those encoding the RecBCD pathway of recombination in Escherichia coli, which is the major pathway for conjugal recombination and double-strand break repair. We intend to characterize these genes to facilitate the development of a defined homologous recombination system in M. tuberculosis. These goals will be achieved by: 1. Cloning and characterizing the cis-acting sequences necessary for conjugal DNA transfer. 2. Identifying the trans-acting protein functions necessary for transfer. 3. Monitoring conjugal transfer from M. smegmatis into other mycobacterial species including the slow-growing pathogenic species. 4. Developing a defined allele-exchange system by characterizing the RecBCD pathway of M. tuberculosis Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CONTACT-MEDIATED HOST RESISTANCE TO M TUBERCULOSIS Principal Investigator & Institution: Silver, Richard F. Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 30-SEP-1997; Project End 31-AUG-2003 Summary: Tuberculosis remains an international health problem of immense proportions, as one-third of the world's population is currently infected with the causative organism, Mycobacterium tuberculosis. Cell-mediated immunity, involving the interaction of sensitized lymphocytes with M. tuberculosis-infected mononuclear phagocytes, can contain the organism, resulting in the finding that the great majority of otherwise healthy individuals do not develop active tuberculosis following infection. HIV-positive individuals have greatly increased susceptibility to the development of active disease following infection with M. tuberculosis. Furthermore, tuberculosis is often an early complication of HIV infection which precedes marked declines in CD4+ T-cell counts. In contrast to studies of other intracellular pathogens, in vitro addition of activating cytokines does not mediate significant reduction in the intracellular growth of M. tuberculosis. Utilizing a method of quantitative infection of human mononuclear phagocytes with virulent M. tuberculosis in which the interactions of lymphocytes with these infected cells can be characterized, preliminary studies indicate that addition of lymphocytes to cultures of M. tuberculosis-infected monocytes is more effective at limiting intracellular growth than is transfer of the supernatants of those cultures. It is hypothesized that cell-to-cell contact involving the interactions of specific cell surface molecules is necessary to maximally activate human mononuclear phagocytes to contain intracellular growth of M. tuberculosis. This hypothesis will be addressed in the following Specific Aims: 1) To determine the contact-dependent mechanisms by which antigen-specific CD4+ T-cells activate bactericidal functions of M. tuberculosis-infected mononuclear phagocytes; 2) To determine whether maximal CD4+ T cell-mediated activation of M. tuberculosis-infected mononuclear phagocytes requires sequential or interactive effects of cell contact and cytokine production; and 3) To determine whether CD8+ T-cells, gammadelta T-cells, and NK cells mediate contact-dependent activation of M. tuberculosis-infected mononuclear phagocytes, to compare the mechanisms used by these effector populations to those of CD4+ T cells, and to determine the role of Fas/FasL-mediated cytotoxic effector mechanisms on killing of M. tuberculosis within human mononuclear phagocytes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
30 Tuberculosis
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Project Title: CONTROL OF BACTERIAL TOXINS BY VIRUSES AND PLASMIDS Principal Investigator & Institution: Holmes, Randall K. Professor and Chair; Microbiology; University of Colorado Hlth Sciences Ctr Uchsc at Fitzsimons Aurora, CO 800450508 Timing: Fiscal Year 2001; Project Start 30-SEP-1976; Project End 31-MAR-2005 Summary: (Adapted from the Applicant's Abstract): The long term goals of this project are to determine molecular mechanisms for virulence regulation in pathogenic bacteria and to develop new methods to treat bacterial infections. The investigators will study Corynebacterium diphtheriae, a paradigm for toxin-mediated bacterial infections, and Mycobacterium tuberculosis, a prototype for intracellular bacterial infections. These very different bacterial pathogens produce closely related, iron-activated, global regulatory proteins that govern virulence: the diphtheria toxin repressor (DtxR) and the iron-dependent regulator (IdeR), respectively. The investigators will determine the molecular basis for function of DtxR, IdeR and the homologous regulator SirR from Staphylococcus epidermidis. The investigators will use structure-based design to develop new antimicrobial drugs called "super-activators" that will target DtxR, IdeR or their homologs; activate them by iron-independent mechanisms; and inhibit production of virulence factors that are negatively regulated by iron- and DtxR-related repressors. The development of IdeR as a novel target for antimicrobial therapy could address the urgent global need for improved treatment of tuberculosis. The investigators will characterize the genes and gene products that are iron-regulated and under control of DtxR and IdeR, both to provide new insights into the pathogenesis of diphtheria and tuberculosis and for development of additional classes of antimicrobial agents. Specific Aim I will analyze structure and function of DtxR, IdeR and SirR. The investigators will investigate the molecular basis for repressor-operator interactions, for iron-independent super-repressor activity, and for domain function in biological activity of these regulatory proteins. Specific Aim 2 will characterize the DtxR and IdeR regulons in C. diphtheriae and M. tuberculosis. The investigators will develop an allelic exchange system for C. diphtheriae, characterize the DtxR and IdeR regulons by proteomic and molecular genetic methods, assess physiological functions of DtxR and IdeR domain 3, and investigate atypical phenotypes among clinical isolates of C. diphtheriae. Specific Aim 3 will develop super-activators of DtxR and IdeR by structure-based design. The investigators will design combinatorial peptide libraries, test them for super-activator function, identify individual peptides with activity, determine the structural basis for that activity, and develop better super-activators by iterative application of these methods. The investigators will also use molecular genetic methods to identify novel mechanisms for super-repressor activity and new lead compounds for development as tools against these bacterial infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CORE--INTERNATIONAL Principal Investigator & Institution: Remick, Scot; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001 Summary: Since 1988, an interactive and multi-disciplinary team of investigators at the Case Western Reserve University and the Tuberculosis Research Unit have effectively and creatively fostered the development of scientific, clinical, laboratory, and education programs in the area of infectious disease (tuberculosis, HIV-AIDS) with investigators at the Makerere University (MU) in Kampala, Uganda and the AIDS Control Program of
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the Ugandan Ministry of Health. The intent of this application is to continue to build on this program by expanding expertise within the center for AIDS research at CWRU and create an International Clinical Coordinating Center (ICCC). The core facility will provide an administrative structure with which to coordinate and considerably expand upon the international collaborative endeavors of CWRU and to foster realistic and meaningful clinical advances to clinicians, scientists, and other healthcare providers in Uganda with the emphasis on certain aspects of the HIV/AIDS pandemic. The creation of the ICCC core facility represents an unprecedented and exciting opportunity to join basic and clinical researchers from six-NIH designated centers at CWRU inclusive of: The Tuberculosis Research Unit, Dr. J.J. Ellner, Director; University Hospitals Ireland Cancer Center (UHICC), Dr. James Wilson, Director; Skin Disease Research Center (SDRC), Dr. Craig Elmets, Director; AIDS Clinical Trials Unit (ACTU), Dr. Michael Lederman, Director; Fogarty AIDS International Training and Research Program, Dr. Christopher Whalen, Director; and the Center for AIDS Research (CFAR), Dr. Stuart Le Grice, Director. The specific aims of this proposal arena to enhance interaction between Ugandan and CWRU clinicians and scientists in the HIV/AIDS are inclusive of tuberculosis, medical mycology, AIDS- related malignancies, and primary HIV vaccine development; to coordinate research activities in the field, clinic, and hospital settings in Kampala, Uganda and research programs/laboratories at CWRU; and to create an environment in Uganda for collaborative research suitable for realistic and meaningful technology transfer. Key research projects that would be funded by the International Core are DNA metabolizing enzyme targeted therapy (CA62502, J. Wilson), Viral etiology and cellular signals in AAKS (CA43703, J. Wilson), preparation for AIDS vaccine evaluation AI35173/CPV1, J.J. Ellner); Preventative therapy for TB in Uganda(CCU50671605, J.J. Ellner); Tuberculosis prevention and control unit (AI45244, J.J. Ellner); Impact of TB on HIV infection in Uganda (AI32414, C. Whalen). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: COST EFFECTIVE CONTROL OF TUBERCULOSIS IN THE US Principal Investigator & Institution: Brewer, Timothy F. Assistant Professor of Medicine; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 01-JUL-1997; Project End 30-JUN-2003 Summary: In the mid-1980's, the long-standing historical decline in tuberculosis cases in the United States reversed itself and case rates began to rise. With the tuberculosis epidemic came the recognition that this disease was becoming increasingly concentrated in defined segments of the overall population. Though total case rates are falling again, groups in the US at significantly higher risk for tuberculosis than the general population exist. It is unknown whether the current recommendations for the control of tuberculosis in the US general population are as effective for these groups. To address this issue, a desktop Computer based mathematical model has been developed to simultaneously project tuberculosis cases and deaths over a l 0 year period using the best available epidemiological data. By adapting this model for US groups at increased risk for tuberculosis, this project will evaluate tuberculosis control strategies for four high risk groups. The groups are persons infected with human immunodeficiency virus, health care workers, immigrants, and the homeless. For each group, a database of inputs based on the epidemiology of tuberculosis within that group will be created. These databases will draw on the published literature and available government information such as that provided by the Centers for Disease Control and Prevention revised report of verified case of tuberculosis. The following tuberculosis control strategies will be evaluated singly and in combinations: increased coverage and improved efficacy of
32 Tuberculosis
preventive therapy, increased coverage and improved efficacy of treatment, increased effectiveness of contact tracing, and the introduction of bacille Calmette-Guerin vaccination. The robustness of the model results will be assessed in sensitivity analyses by simultaneously varying all inputs using Latin hypercube sampling. The combination of mathematical modeling and epidemiology provides the most complete evaluation of TB control measures among these high risk groups to date, and forms a basis for improving approaches to control this epidemic among these populations. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CRYSTALLIZATION OF THE GALACTOSYLTRANSFERASE FROM MTB Principal Investigator & Institution: Reynolds, Robert C.; Southern Research Institute Birmingham, AL 35205 Timing: Fiscal Year 2002; Project Start 15-SEP-2002; Project End 31-AUG-2004 Summary: (provided by applicant): This application is in response to PAS-02-031 for a supplement to our current NIAID-funded program AI45317 entitled Glycosyltransferases as Drug Targets in Mycobacteria. This supplemental program will involve the expression, purification, and crystallization of the galactosyltransferase (Rv3808c) from Mycobacterium tuberculosis. The galactosyltransferase adds galactofuranose units to the growing galactan, a polysaccharide that anchors the mycolylarabinan superstructure and is critical to mycobacterial cell wall integrity in the harsh environment of the human macrophage. This effort is responsive to the announcement in that it supplements our current program by applying modern structural biology techniques to obtain a structure of a protein targeted for drug development under our current grant. This protein was not available at the time of our application, and the information provided through the application of the requested supplemental funds will give us a specific picture of the protein active site allowing more rapid development of drugs to target this critical mycobacterial enzyme. As well, this grant is responsive to the PA in that it addresses a new drug target in tuberculosis, and may have implications in the treatment of multi-drug resistant tuberculosis (MDRTB), a critical health problem worldwide. Tuberculosis and its drug resistant forms are the focus of two active NIAID Program Announcements (PA-99-124 & PA-01-113) and is considered a potential biological weapon by the Department of Defense. This application involves modern structural biology that may not be considered as innovative as recent techniques in bioinformatics, genomics, and microarray technology. This effort, however, is innovative in the choice of target and the impact that structural biology can have on our funded program. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CYTOTOXIC RESPONSE TO HISTOCOMPATIBILITY ANTIGENS Principal Investigator & Institution: Bevan, Michael J. Professor; Immunology; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2003; Project Start 01-JUN-1990; Project End 31-DEC-2007 Summary: provided by applicant): Our goal is to investigate the CD4 and CD8 T cell response to pathogenic infectious agents to uncover basic principles of T cell biology in controlling these infections. Two contrasting models of intracellular bacterial pathogens are chosen to ask fundamental questions about how the immune system perceives, responds, contains and eliminates these pathogens. One model to be studied is virulent Mycobacterium tuberculosis delivered via aerosol to the lungs of mice. This organism
Studies 33
can survive inside vacuoles within macrophages and set up a chronic infection which can be contained, but not eliminated, by adaptive CD4+T cell immunity. The role of CD8+ T cells in controlling tuberculosis is controversial. Recombinant organisms producing a model antigen containing well-studied epitopes for CD4 and CD8 T cells will allow a study of the tempo of the T cell response, and provide new information on protection against what has been called the world's most successful pathogen. M. tuberculosis may infect one third of the world's population. The second intracellular pathogen we plan to use is a common food-borne pathogen, Listeria monocytogenes. Excellent mouse models exist already for the study of this Gram positive pathogen, where it appears that adaptive CD8+ T cell immunity is the critical force in eliminating the infection. L. monocytogenes is a candidate vaccine vector for the induction of CD8 immunity, for example, against tumor antigens. The massive response of CD8+ T cells to acute infection with Listeria and the generation of long-lived protective immunity may also require CD4+ T cell help. The proposed studies are aimed at a greater understanding of the development of long-lived, protective immunity induced by this pathogen. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DEVELOPMENT AND TESTING OF NEW TUBERCULOSIS VACCINES Principal Investigator & Institution: Horwitz, Marcus A. Medicine; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024 Timing: Fiscal Year 2001; Project Start 01-APR-1991; Project End 31-AUG-2005 Summary: (Adapted from Applicant's Abstract) Mycobacterium tuberculosis, the primary agent of tuberculosis, infects one-third of the world's population and kills 3 million people annually, making it the world's leading cause of death from a single infectious agent. It is a leading cause of disease and death in AIDS patients, particularly in the developing nations of the world. The rapid global emergence of strains resistant to the major antibiotics used to treat tuberculosis poses a serious threat to public health. The highest priority in the fight against tuberculosis is the development of a vaccine that is more efficacious than the current vaccine - BCG. A vaccine more potent than BCG would have an impact on human health greater than virtually any other conceivable development in the fight against infectious diseases. Studies from this laboratory completed under the current grant established the importance of major extracellular proteins of M. tuberculosis in inducing both cell-mediated and protective immunity in the guinea pig model of pulmonary tuberculosis, a highly susceptible species that develops disease remarkably similar to human tuberculosis. Studies under the current grant also succeeded in developing technology for high level expression and secretion in native form of major M. tuberculosis extracellular proteins in a nonpathogenic rapidly growing heterologous host, allowing isolation and purification of 100 mg quantities of recombinant M. tuberculosis extracellular proteins for vaccine studies. In this grant application, we seek to build on the knowledge and experience gained in previous studies to develop a vaccine more potent than BCG in the highly relevant guinea pig model. We seek to develop and test live recombinant vaccines including recombinant BCG expressing major M. tuberculosis extracellular and cell-associated proteins and new non-live particulate vaccines formulated as liposomes and microspheres. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DEVELOPMENT OF A TUBERCULOSIS CURRICULUM Principal Investigator & Institution: Jones, Brenda E. Medicine; University of Southern California 2250 Alcazar Street, Csc-219 Los Angeles, CA 90033 Timing: Fiscal Year 2001; Project Start 01-JUL-1997; Project End 30-SEP-2003 Summary: The goal of this proposal is to develop and implement comprehensive, interdisciplinary curricula on tuberculosis for health care providers in academic and community settings. We will first establish interdisciplinary curricula at a Health Sciences campus, train faculty to disseminate these curricula, and adapt the curricula for use in the community. Our specific aims are: 1) Establishment of comprehensive, interdisciplinary tuberculosis curricula at a Health Sciences campus. The target audience will include physicians, physician assistants and nurses who care for most tuberculosis cases in urban areas, and primary care providers who evaluate most patients for tuberculosis infection. As part of this aim, we will use a Telemedicine system that allows transmission of high-quality images and interactive videoconferencing between faculty at a central location and trainees at geographically separate sites. The effectiveness of the tuberculosis curricula will be measured by: (1) objective tests of factual knowledge and clinical decision-making skills, using written tests and structured clinical examinations; (2) evaluation of changes in clinical practice patterns, using focused chart reviews. 2)Training Health Sciences faculty to provide instruction in tuberculosis. Faculty will be trained to disseminate the curricula developed in aim 1. Regularly scheduled forums will be established to provide ongoing education and discussion among trainees and faculty, and the quality of faculty teaching will be assessed and improved through evaluations by trainees, peers and experts. 3) Expansion of educational efforts to the community. Using the educational infrastructure of Tuberculosis Control of Los Angeles county, we will adapt the tuberculosis curricula developed in aim 1 for delivery to community health care providers, including those who work with populations in which special problems are encountered, including the homeless, immigrants, HIV-infected persons and inmates of correctional institutions. Computer-based interactive programs using CD-ROM technology and/or the Internet will be used for some target audiences. Supervisory physicians and nurses at Tuberculosis Control will be trained to disseminate these curricula. The efficacy of these curricula will be assessed by objective measures of clinical decision- making skills and clinical practice patterns. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DEVELOPMENT OF AN IMPROVED MYCOBACTERIA CULTURE MEDIUM Principal Investigator & Institution: Somoskovi, Akos L.; Life Biomed, Llc 2800 Millers Way Dr Ellicott City, MD 21043 Timing: Fiscal Year 2002; Project Start 15-AUG-2002; Project End 14-SEP-2004 Summary: (provided by applicant): The clinical signs and symptoms of tuberculosis are not specific. Therefore, laboratory methods play a crucial role in establishing the diagnosis, monitoring the therapy and preventing transmission of the disease. Laboratory results must be not only accurate, but also rapidly available. However, the culture techniques most commonly employed world-wide for the diagnosis of tuberculosis are time-consuming. Recently it has been shown that the spent culture supernatant of Mycobacterium tuberculosis and active components (phopholipids and peptides) derived from it can enhance the growth of tubercle bacilli and allow small inocula to initiate growth in liquid culture. The aims of the proposed study are: (1) To
Studies 35
evaluate the growth enhancing/resuscitating factors as additives to current liquid and solid medium culture systems using lab strains of M. tuberculosis. Experiments will be carried out in both liquid media (7H9, 7H12B Bactec and MGIT) and solid medium (7H1 1 agar), with different inoculum sizes on both fresh and 01 cultures. Serial dilutions of a fresh culture representing the varying bacterial load of smear positive and smear negative clinical specimens can provide information about the growth enhancing effect of the examined agents, while inocula from old cultures will enable investigation of the resuscitation effect of the agents. (2) To evaluate the effect of opitmal growth enhancing factors on drug susceptible and drug-resistant clinical isolates of M. tuberculosis. The effect of selected phospholipids and peptides (based on the results of the above-detailed experiments) will also be studied both on 50 drug susceptible and drug-resistant clinical isolates. (3) To evaluate the effect of growth enhancing factors on drug susceptibility testing. Drug susceptibility testing will be performed on media containing the growth enhancing factors to rule out any interference and to determine the appropriate MIC values. At the end of the Phase I study the optimal components will be selected for formulation of a new culturel medium with commercial potential for improved diagnosis of tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DEVELOPMENT OF IMMUNE EVASION IN TUBERCULOSIS Principal Investigator & Institution: Ernst, Joel D. Professor; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001 Summary: Acquired immunity to intracellular pathogens depends on T cell recognition of HLA-bound microbial peptides. Pathogens that occupy phagosomes are controlled by CD4+ cells that recognized microbial peptides bound to HLA class II. While these interactions occur in response to Mycobacterium tuberculosis, they are only partially efficacious: the human immune system is unable to completely resolve infection with M. tuberculosis. M. tuberculosis has been found to inhibit class II antigen presentation, but the mechanisms has not been determined. The goal of this project is to define the mechanism of inhibition of class II antigen present in human macrophages infected with M. tuberculosis. We have found that M. tuberculosis causes a decrease of cell surface class II (HLA-DR) in macrophages, without a decreased in the total cellular HLA-DR pool. This suggests that M. tuberculosis alters trafficking of class II. We will further characterize the decreased surface expression of class II by determining whether it is directly related to the number of intracellular M. tuberculosis and by comparing expression of HLA-DR on infected macrophages and dendritic cells. We will test the hypothesis that HLA-DR is sequestered in M. tuberculosis phagosomes, and will determine whether HLA-DR is sequestered due to failure of exchange of exogenous peptides for CLIP. We will also further characterize the functional effects of M. tuberculosis down-regulation of HLA-DR. We will determine whether down-regulation of class II inhibits CD4+ cell recognition of extracellularly loaded M. tuberculosis antigens and whether M. tuberculosis antigens and whether M. tuberculosis inhibits allogeneic responses. Finally, we will test the hypothesis that inhibition of class II antigen presentation is an inherent property of mycobacteria by comparing the response of CD4+ T cell lines that recognize a DR3-restricted epitope of M. tuberculosis hsp65 when it is expressed by mycobacteria or by an alternative vehicle. We anticipate that our results will provide new information on the pathogenesis of tuberculosis, and may identify alternative means of delivering M. tuberculosis antigens for vaccination. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DEVELOPMENT OF SYSTEM FOR TREATMENT OF TUBERCULOSIS Principal Investigator & Institution: Tsukamoto, Takuji; Chemica Technologies, Inc. 325 Sw Cyber Dr Bend, OR 97702 Timing: Fiscal Year 2001; Project Start 05-SEP-2001; Project End 28-FEB-2003 Summary: Tuberculosis causes a staggering burden of mortality world-wide, killing 2 million people annually. To cure tuberculosis, prolonged therapy of at least 6 months is required, because antituberculosis drugs do not penetrate well into macrophages, which are a major reservoir of M. tuberculosis. To devise antituberculosis medications that will shorten the duration of therapy, we will use microparticles to target the delivery of enzyme-cleavable lipophilic derivatives of isoniazid and rifampin to infected macrophages. Once inside the macrophage, the microparticles degrade and release the lipophilic drugs, which are then transported across the M. tuberculosis cell envelope. Our specific aims are: 1. To synthesize and characterize palmitic acid derivatives of isoniazid and rifampin. 2. To determine the rates of esterase or amidase hydrolysis of these derivatives. 3. To evaluate the capacity of these derivatives to kill M. tuberculosis. If this proposal is successful, a phase II proposal will be submitted to perform additional studies to evaluate these agents combined with microparticles in cell culture systems in vitro and in animals. This project will explore the feasibility of a strategy that can be applied to the treatment of infection caused by many intracellular organisms, including viruses, fungi and parasites. PROPOSED COMMERCIAL APPLICATIONS: The technology we will develop in this program has the potential to significantly improve the treatment of diseases due to pathogens that reside in macrophages, such as tuberculosis and HIV infection. In the treatment of tuberculosis, targeted-delivery systems of enzyme- cleavable lipophilic drugs are presently not available. This strategy has the potential to increase the efficacy of treatment, reduce the dosage of drugs required, reduce side effects of treatment, improve patient compliance, and minimize development of drug resistance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DIABETES AND TUBERCULOSIS PILOT Principal Investigator & Institution: Perez, Adriana; Assistant Professor; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2003; Project Start 03-MAR-2003; Project End 29-FEB-2008 Summary: Screening for Mycobacterium tuberculosis infection in those at high risk for developing clinical tuberculosis (TB) is a key strategy of the Centers for Disease Control and Prevention (CDC) to control the spread of TB in the US. This is particularly important at the US/Mexico border, where converging socioeconomic and demographic factors contribute to the higher burden of TB when compared to the US national average. This region also has high prevalence of type II diabetes (DB), which has long been known to predispose to active tuberculosis. Based on this information, we hypothesize that DB contributes to the burden of TB in the Hispanic population from four Texan counties along the Mexican-American border. Four counties in the Lower Rio Grande Valley (LRGV) will be analyzed in this pilot project: Hidalgo, Cameron, WilIacy and Starr. A secondary analysis of the Texas hospital discharges during 2001 will be implemented using ICD 9 codes for case and control selection. Unconditional multiple logistic regression analysis will be used to evaluate the relationship between tuberculosis and tentative associated factors. Health production functions will be estimated, using unconditional multiple logistic regression as well. The specific aims of the project are: Aim 1: Determine the degree of association of DB and TB in Hispanics in
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the LRGV Aim 2: Determine the strength of the association between TB and its associated risk factors (besides DB), with emphasis on underlying medical conditions. Aim 3: Estimate health production function that will identify relevant socio-economic indicators of TB and DB. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DIAGNOSIS & PATHOGENESIS OF TUBERCULOSIS: AIDS OPPORTUNISTIC INFECTION Principal Investigator & Institution: Didier, Peter J.; Tulane University of Louisiana New Orleans, LA 70118 Timing: Fiscal Year 2001 Summary: The objective of this pilot project is to continue the characterization of the tuberculosis model in rhesus monkeys. We previously found that high dose inoculation (six million bacteria) of Mycobacterium tuberculosis H37Rv and Erdman strains produced lethal lung disease within five to ten weeks. Monkeys developed clinical signs of dyspnea, coughing, anorexia, and weight loss 4-6 weeks after inoculation. Skin testing with Old tuberculin became mildly positive after 5 weeks while PPD skin tests remained negative. Animals developed extensive necrotizing granulomatous lesions in the inoculated half of the lung with spread to the contralateral lung and bronchial lymph node in animals with H37Rv, and to the liver and kidney in animals with the Erdman strain. Animals vaccinated with culture filtrate from H37Rv and challenged with two million bacteria developed the same clinical disease as unvaccinated controls. However, low dosage inoculation of a pair of animals with each strain (<300 Erdma n, <30 H37Rv) failed to produce clinical disease within 18 weeks. Lesions in three of four animals were confined to the bronchial lymph node. To confirm this finding, four more animals per strain have been inoculated with low dosages of bacteria. All animals have strongly positive skin tests but remain clinically normal four weeks after inoculation. Characterization of this model by culture, blastogenesis, flow cytometry, and cytokine detection continues. Results contradict old literature and indicate that tuberculosis in rhesus monkeys may present as a chronic disease based on the dose of the inoculum. This suggests that future studies using low dose inoculation will be feasible in SIVinfected monkeys to study tuberculosis as an opportunistic infection. FUNDING Venture Research PUBLICATIONS Scollard DM, Didier PJ, Dietrich MA, Bohm Jr RP. Selective increases in CD45RO(+) CD38(+) T cells in 1? and 2? responses to BCG in rhesus macaques. [Abstract]. Proceedings of the 9th International Immunology Congress, San Francisco, CA, July 23-29, 1995. Scollard DL, Didier PJ, Bohm Jr RP. A method to assess changes in rhesus lymphatic T cells in vivo during local immune responses. [Abstract]. Second Annual Molecular Biology and Biotechnology Conference, Baton Rouge, LA, February 9-10, 1996. Didier PJ, Blanchard JL, Gormus BJ. Pulmonary tuberculosis in normal rhesus monkeys infected with Mycobacterium tuberculosis H37Rv. [Abstract #159]. Amer Soc Trop Med Hygiene, 57(3):156, 1997. Didier PJ, Blanchard JL. Chronic tuberculosis produced by low dosage of M. tuberculosis (Erdman) in rhesus monkeys. [Abstract #734]. Amer Soc Trop Med Hygiene 59(3):361, 1998. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DIAGNOSTICS FOR AIDS-RELATED PEDIATRIC TB, PERU Principal Investigator & Institution: Oberhelman, Richard A. Professor; Tropical Med & Parasitology; Tulane University of Louisiana New Orleans, LA 70118
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Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 31-JUL-2005 Summary: (provided by applicant)Tuberculosis is the major infectious cause of mortality among AIDS patients in the developing world, and HIV infection has been shown to increase mortality from tuberculosis five-fold in parts of Subsaharan Africa. Increasingly, HI V-infected children in developing countries are becoming infected with Mycobacterium tuberculosis (Mtb) and dying at an early age, presenting new dilemmas that differ from those facing adults with HIV-Mtb coinfection. The diagnosis of pediatric TB is complicated by inefficient and expensive methods to recover Mtb and vague diagnostic criteria. This project will evaluate novel approaches to the diagnosis of AIDSrelated pediatric TB in a hyperendemic setting using 1) rapid, cost-effective Mtb culture and susceptibility methods based on direct microscopic observation techniques and 2) alternative non-invasive specimens such as nasopharyngeal aspirates (NPA) and stool to detect Mtb. An optional component will assess improved rapid detection of Mtb by a semi-nested polymerase chain reaction assay (N2 PCR), a technique appropriate for regional reference laboratories in developing countries. Our preliminary data show a high correlation between culture results and N2 PCR results in adults (sputum PCR) and children (stool and NPA PCR) with tuberculosis, and mean time to detection of Mtb by our microscopic observation method was 9 days (at a fraction of the cost of rapid methods used in the U.S.). This is a collaborative effort between PRISMA, a Peruvian private voluntary organization, two U.S. universities (Tulane and Johns Hopkins), and a Peruvian university (Cayetano Heredia). Two hundred-sixty children with pulmonary disease meeting clinical criteria for TB disease (including at least 100 HIV-infected) from the Hospital del Nino, Lima, Peru, and 260 age-matched controls from both high- and low-risk communities will be enrolled. Mtb will be detected in gastric aspirates (cases only), NPAs, and stool by new and traditional culture methods and by N2 PCR. Children with a positive N2 PCR but without clinical evidence of TB requiring antituberculous therapy will be followed longitudinally. These new diagnostic methods have tremendous potential to improve and simplify the diagnosis of pediatric tuberculosis in low-income countries with limited laboratory resources. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EFFICIENCY
DIETARY
DEFICIENCIES
AND
TUBERCULOSIS
VACCINE
Principal Investigator & Institution: Mcmurray, David N. Professor; Medical Microbiol & Immunology; Texas A&M University Health Science Ctr College Station, TX 77843 Timing: Fiscal Year 2001; Project Start 30-SEP-1982; Project End 31-JAN-2005 Summary: The ultimate aim of this project is to elucidate the mechanisms by which protein deficiency interferes with vaccine-induced resistance to pulmonary tuberculosis. New tuberculosis vaccines will be tested, ultimately, in malnourished humans. Thus, the focus of this project has never been a more urgent research priority. Previous work with a highly relevant guinea pig model, employing low-dose aerosol exposure to virulent Mycobacterium tuberculosis, has revealed diet-induced defects in trafficking, activation, and interactions of immune cells. The recent acquisition of cDNA clones for guinea pig chemokines (MCP-1, IL- 8, RANTES) and cytokines (IFNgamma, TNFalpha, TGFbeta, and IL- 1beta) provides a unique opportunity to apply molecular biological approaches to test four hypotheses: (a) protein deficiency affects trafficking of immune cells into inflammatory exudates by interfering with the production/function of chemokines; (b) abnormal granuloma formation in protein-deficient guinea pigs results from alterations in the production/function of TNFalpha; (c) failure of immune lymphocytes from protein- deficient guinea pigs to activate macrophages to suppress
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intracellular M. tuberculosis is due to decreased production/function of paracrine (IFNgamma) or autocrine (IL- 1beta, TNFalpha) cytokine signals; and (d) TGFbetamediated suppression of T lymphocytes and/or deactivation of macrophages leads to loss of control of intracellular mycobacteria in protein deficiency. Recombinant proteins and polyclonal antibodies will be produced for each of the chemokines and cytokines. Protein- deficient and well-nourished, BCG-vaccinated or nonvaccinated guinea pigs will be challenged by the pulmonary route and levels of these molecules assessed in freshly isolated or cultured cells by Northern blot and RT-PCR (for mRNA), or by bioassay (TNFalpha, TGFbeta) or ELISA. A tuberculous pleuritis model, previously established in the laboratory, and bronchoalveolar lavage will be used to assess the role of chemokines by instillation of recombinant chemokines or specific anti-chemokine antibodies. The effects of recombinant TNFalpha or anti-TNFalpha on granuloma formation will be determined. TGFbeta activity will be blocked in vivo by the injection of anti-TGFbeta antibodies or recombinant decorin. These experiments will provide important new insights into the contributions of these molecules to loss of tuberculosis vaccine efficacy observed in malnourished subjects. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DIRECT DETECTION OF DRUG RESISTANT MTB WITHOUT PCR Principal Investigator & Institution: Wu, Whei K.; Applied Gene Technologies, Inc. 6190 Cornerstone Ct E, #101 San Diego, CA 921214701 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JAN-2003 Summary: (provided by applicant): Drug resistance is a significant problem in the fight against the bacterial pathogen Mycobacterium tuberculosis. Resistance to rifampin, a first-line antituberculosis drug, is almost always the result of mutations in the rpoB gene of the pathogen. In order to speed the diagnosis of drug resistant tuberculosis, it is possible to detect these mutations by a variety of available methods. However, all such methods require the PCR reaction, which is too expensive for routine use in most countries. There is a critical need for new diagnostic methods that detect genetic markers of rifampin resistance at a fraction of the cost of current methods. Our Tessera Array Technology detects single-copy chromosomal genes of M. tuberculosis without the need for PCR amplification. The method involves high-affinity labeling of pathogen DNA in crude or partially purified lysates, hybridization of the labeled DNA to sequence-specific probes, and detection of the labeled, hybridized DNA. A test for detecting M. tuberculosis has been developed by AGTI and is currently in pre-clinical trials. We propose to expand the test for simultaneous taxonomic detection of M. tuberculosis and genotypic detection of mutations that cause rifampin resistance. The specific aims of Phase I of the project are to adapt the Tessera platform to accommodate probes for rpoB; to enhance test sensitivity by optimizing cell lysis methods; and to conduct a non-clinical trial on known rifampin-sensitive and rifampin-resistant clinical isolates of M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DISSEMINATED TUBERCULOSIS IN HIV INFECTION Principal Investigator & Institution: Von Reyn, C Fordham. Medicine; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, NH 03755 Timing: Fiscal Year 2001; Project Start 01-AUG-2000; Project End 31-JUL-2005 Summary: Disseminated infection (mycobacteremia) with Mycobacterium tuberculosis (dMTB) has been documented by our group in 10-25 percent of patients with HIV
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infection in Africa using lysis- centrifugation blood cultures. Unlike pulmonary tuberculosis (pMTB), most cases of dMTB are not recognized and death ensues rapidly. Thus, in developing countries dMTB may be a more important cause of HIV-associated mortality than pMTB. Risk factors for dMTB have not been identified and it is not known if most cases are due to primary infection, reactivation or re- infection. We hypothesize that most cases of dMTB are due to primary MTB infection in patients without prior infection with MTB or non-tuberculous mycobacteria (NTM). Mycobacterial immunization in early HIV infection is a potential strategy to prevent dMTB. Mycobacterium vaccae (MV) is an investigational vaccine prepared by heat inactivation of an NTM, and has been shown to be protective against MTB in several animal models. Studies conducted by our group indicate that a 5-dose series of MV is safe in patients with HIV infection and induces a durable cellular immune response to MTB antigens in persons with prior BCG immunization. Our hypothesis is that MV immunization will reduce the risk of HIV-associated dMTB by 50 percent. Our specific aims are: (1) to define risk factors for HIV-associated disseminated tuberculosis and to assess the relative contributions of primary infection, reactivation and re-infection in the pathogenesis of disseminated tuberculosis, and (2) to assess the safety and efficacy of a 5-dose schedule of inactivated MV vaccine for the prevention of HIV-associated pulmonary and disseminated tuberculosis in persons with prior BCG immunization. 2274 HIV-positive patients with prior BCG immunization and 100 HIV-negative controls will be entered in a 5-year study in Zambia. Baseline evaluation will include history, chest x-ray, dual skin tests with purified protein derivative (PPD) and Mycobacterium avium sensitin (MAS), and whole blood assay for interferon-gamma production in response to MV sonicate, PPD, ESAT-6 (a protein antigen unique to MTB) and MTB antigen 85. Subjects with PPD reactions greater than or equal to 5 mm will receive 6 months of prophylaxis with isoniazid. All subjects will be randomized 1:1 to receive a 5dose series of MV or placebo over 12 months with repeat skin test and in vitro studies at 14 months. Subjects will be followed every 3 months for 3-5 years to assess new pMTB (microbiologic or clinical diagnosis) or dMTB (microbiologic diagnosis). All isolates will have susceptibility tests and IS6110 DNA fingerprinting performed. Potential risk factors for dMTB, including baseline PPD test results, will be assessed in placebo and vaccine groups. Vaccine efficacy against dMTB and pMTB in HIV-positive subjects will be determined, and post immunization interferon gamma responses used to identify a surrogate marker of efficacy. The proposed study has important implications for the reduction in mortality from HIV-associated tuberculosis and for design of future trials of new vaccines against tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DNA GYRASE AND QUINOLONE RESISTANCE IN TUBERCULOSIS Principal Investigator & Institution: Drlica, Karl A. Associate Professor; Public Health Research Institute 225 Warren St Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 30-JUN-2003 Summary: (Adapted from the applicant's abstract): Drug resistance is a major problem for anti-tuberculous chemotherapy and is one of the main reasons why compounds such as fluoroquinolones are not first line agents. Two recent advances, using a variety of bacteria, encourage refinements of fluoroquinolones. First, resistance arises stepwise from mutations in two independent targets, DNA gyrase and DNA topoisomerase IV. Thus a first step resistant gyrase mutant can be used to screen new fluoroquinolones for derivatives that require two mutations to confer resistance. Since double mutations occur less frequently that single mutations by many orders of magnitude, resistance to
Studies 41
the new compounds should arise very rarely. Second, the double mutant concept was experimentally validated: a C8-methoxyl group reduced the acquisition of resistance by at least three orders of magnitude. Two approaches are proposed to define how these principles apply to fluoroquinolone resistance in Mycobacterium tuberculosis. In one, fluoroquinolone structural variants will be examined for their ability to prevent resistance. Prevention of resistance will be correlated with avid killing of moderately resistant strains 1) in pure culture under growing and non-growing conditions, and 2) inside cultured human macrophages. The generality of the conclusions will be tested using a wide variety of clinical isolates of M. tuberculosis. In the other approach, resistance will be studied by examining nucleotide sequences of genes encoding gyrase and topoisomerase IV. Mutants will be selected for resistance to various fluoroquinolone congeners, some of which are already known to elicit different alleles. Selected mutants will be tested for 1)cross-resistance, 2) adaptation to changing environment, and 3) invasion of human macrophages. Already available are many new compounds, two novel assays (killing resistant mutants and prevention of resistance), a human macrophage infection system, and a large collection of clinical isolates of M. tuberculosis. Significant progress is expected toward finding fluoroquinolones that will prevent resistance from arising in M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DRUG DEVELOPMENT FOR MDR-M TUBERCULOSIS Principal Investigator & Institution: Dick, James D. Pathology; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2003; Project Start 15-MAR-2003; Project End 28-FEB-2008 Summary: (provided by applicant): Tuberculosis is one of the oldest and most common infectious diseases of man. The disease is epidemic with approximately 1.8 billion people or one third of the world's population currently infected. The HIV epidemic and the emergence of multi-drug resistant M. tuberculosis (MDR-TB) has led to a critical need for the discovery and development of new and effective therapeutic agents for the treatment and control of tuberculosis. The b-sulfonylcarboxamides are a novel class of compounds which have potent in vitro activity against pathogenic mycobacteria with minimal inhibitory concentrations (MICs) ranging from 0.78 micrograms per ml to 12.5 micrograms per ml for M. tuberculosis, including MDR-TB. In addition, these compounds are highly specific for pathogenic mycobacteria, maintain activity in the presence of human serum, and are not cytotoxic for mammalian cells at 40 times their MIC for M. tuberculosis. Recent studies in our laboratories have demonstrated the antimycobacterial activity of the b-sulfonylcarboxamides to be the result of inhibition of a potentially unique pathway/target involved in central energy metabolism. The longterm objectives of this grant proposal are to determine the molecular target and mechanism of action of this novel class of compounds, with subsequent optimization of drug structure, synthesis, and preclinical drug development. This will be accomplished through: (1) characterization and identification of the target/pathway using available genomic and proteomic technologies, (2) confirmation and validation of candidate target(s) through microbiologic, biochemical, and genetic comparisons between resistant and susceptible mycobacteria, (3) determination of the three dimensional structure of the identified target, (4) design and synthesis of optimum small molecule inhibitors of the identified target/pathway (5) in vitro and in vivo toxicity and efficacy testing in a murine model of tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DRUG DISCOVERY FOR PERSISTENT TUBERCULOSIS Principal Investigator & Institution: Sacchettini, James C. Professor & Wolfe-Welch Chair; Biochemistry and Biophysics; Texas A&M University System College Station, TX 778433578 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-AUG-2004 Summary: Tuberculosis (TB) is the leading cause of death by an infectious disease in the world. The synergy between TB and AIDS is also very apparent. TB is the primary cause of death for individuals co-infected with the human immunodeficiency virus (HIV). HIV infection is the greatest single risk factor known for progression from TB infection to active TB disease in developing nations, where as many as 40-80% of individuals with the acquired immunodeficiency syndrome (AIDS) will also develop TB. Conversely, coinfected patients with active TB show enhanced viral replication and accelerated progression of AIDS. Although Tb is a treatable disease, current chemotherapy regimens require a complex strategy of 3 drugs for at least six months. Such prolonged therapy is necessary because conventional drugs are poorly effective against a sub-population of latent and drug-tolerant bacterial 'persisters'. Most patients will not adhere to this treatment regimen. Patients who default suffer increased rates of treatment failure, relapse, and drug resistance. New drugs against persisting mycobacteria are urgently needed to counter the problem of patient non-compliance. The development of new drugs would be greatly facilitated by the identification of bacterial 'persistence factors' as candidate drug targets. A significant step towards this goal was our recent demonstration that the glyxoylate shunt enzyme isocitrate lyase, ICL, is essential for persistence of Mycobacterium tuberculosis, in vivo. In addition, we have demonstrated that ICL is expressed at high levels in M. tuberculosis living in murine macrophages. Building on these observations, we will take a multi-discipline approach to define the role of the glyoxylate shunt and fatty acid catabolism in persistence. Enzymes found essential to persistent organisms will be characterized and used in high throughput drug screening and structure based drug-design. Promising lead compounds will be tested for safety and efficacy in a mouse model of chemotherapy, latency, and relapse. Indeed, the primary goal of this proposal is to develop new chemotherapeutics for the treatment of persistent M. tuberculosis infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DRUG RESISTANT TUBERCULOSIS Principal Investigator & Institution: Montellano, Paul; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2002; Project Start 01-SEP-1997; Project End 31-AUG-2007 Summary: Drug resistant tuberculosis is a major opportunistic infection in AIDS patients and resistance to the available drugs is growing. Tuberculosis infection both increases the infectivity of the HIV virus and is a major cause of death in AIDS patients. This project focuses on the role of selected oxidation-reduction processes in the development of Mycobacterium tuberculosis drug resistance and the use of this information for the design of new antitubercular agents. Isoniazid, a first line drug, is activated by the KatG catalase peroxidase to a reactive intermediate. This intermediate, or one of its further reaction products, is responsible for interaction with the cell wall biosynthetic enzymes InhA and KasA-AcpM, the ultimate targets of the drug. Resistance to isoniazid is largely caused by mutations that disable KatG and is associated with the enhanced expression of AhpC and AhpD, two unrelated non-heme alkylhydroperoxidases (peroxiredoxins) that compensate for the loss of KatG. Inhibition
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of AhpC/AhpD in conjunction with isoniazid treatment is a potentially useful mechanism for counteracting isoniazid resistance. In the expiring grant period, AhpC and AhpD were cloned, expressed, and partially characterized and the crystal structure of AhpD was determined. Building on these results, further structural and mechanistic studies are to be carried out with AhpC/AhpD, and this information is to be used to develop potent and selective inhibitors of these two proteins. Ethionamide is another antitubercular prodrug. It is activated by the enzyme EtaA rather than by KaG but the activated species also appears to act at the level of InhA andor KasA-AcpM. Ethionamide resistance is due to impairment of EtaA activity and is associated with elevations in AhpC/AhpD similar to those observed with isoniazid. We have cloned and expressed EtaA and shown it to be a flavoprotein that catalyzes two sequential step in the activation of ethionamide and other thioamide drugs. We propose to complete characterization of EtaA, including the nature of the reactive intermediate, the mechanisms of its interactions with InhA and KasA-AcpM, and its biological importance. The information is to be used to synthesize agents that inhibit one of these two target proteins and, if warranted, EtaA. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DRUG TRANSFERASES
TARGETING
M.
TUBERCULOSIS
ARABINOSYL
Principal Investigator & Institution: Lee, Richard Edward. Assistant Professor; Pharmaceutical Sciences; University of Tennessee Health Sci Ctr Health Science Center Memphis, TN 38163 Timing: Fiscal Year 2001; Project Start 30-SEP-2000; Project End 31-MAY-2003 Summary: (adapted from applicant's abstract): Tuberculosis is still a major cause of morbidity and mortality worldwide with the number of cases rising due to the HIV pandemic and the emergence of drug resistant strains. To combat these problems more effective antibiotics must be developed with better pharmacokinetics and lower toxicity than the drugs currently used. It is the aim of this proposal to study the chemistry, biochemistry and genomics of arabinose metabolism in M. tuberculosis and the effects of the front line drug ethambutol on the important mycoloylarabinogalactan (MAG) biosynthetic cascade. MAG is a unique lipidated polysaccharide surrounding and protecting the tubercular bacilli from the host immune system where it has found its evolutionary niche. The proteins involved in MAG biosynthesis are excellent drug targets because they are essential for growth of the organism and are unique to mycobacteria. However, a fundamental lack of knowledge of the genes involved prevents these targets from being developed. This proposal initiates the synthesis of advanced cell wall mimetic acceptors and the development of new arabinosyl transferase assays. Using these assays the relationship between the alpha (1-3) arabinosyl transferase activity and the frontline drug ethambutol will be defined. These assays will also allow detailed analysis of the mechanism of beta (1-2) arabinosyl transfer and aid in identification of the beta (1-2) transferase. The role of the protein encoded embCAB operon in arabinan biosynthesis will be explored, and its sensitivity to ethambutol and novel antimycobacterials examined. Most importantly the knowledge gained from these studies will initiate a thorough medicinal chemistry effort involving these targets for the development of new, more effective and less toxic treatments against tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: EARLY PULMONARY HOST DEFENSE IN M. TUBERCULOSIS INFECTIO Principal Investigator & Institution: Sherman, David R. Senior Scientist; Pediatrics; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2003; Project Start 30-SEP-1999; Project End 31-JUL-2004 Summary: Although a lung is a portal of entry for this M. tuberculosis and chronic pneumonia is the most common disease caused by M. tuberculosis infection, the specific nature of the pulmonary host response to this pathogen is relatively unknown. The central goal of the studies outlined in this proposal is to determine whether components of the early pulmonary immune response are critical determinants for resistance or susceptibility to disease caused by M. tuberculosis. The specific aims will address the role of TNF-alpha, beta chemokines and the B7/CD28/CTLA4 costimulation pathway in the initiation and amplification of the pulmonary host response to aerosol infection with M. tuberculosis. Preliminary studies using transgenic mice with a local inhibition of TNF-alpha in the lung (SPCTNFRIIFc mice) indicate that blockade of TNF-alpha selectively in the lung results in early deaths, severe lung pathology and an alteration of antigen specific immunity. These studies also indicate that transgenic mice with local inhibition of the immune response in the lung are a useful model to study pulmonary host defense. Further studies are proposed to compare the response that develops in the lung vs. regional lymph nodes in wildtype and SPCTNFRIIFc mice. To investigate the role of beta chemokines in the pulmonary host defense, transgenic mice that secrete a virally encoded inhibitor of all chemokines has been generated. The phenotype of these mice will be determined then used to investigate the role of beta chemokines in the initiation of the pulmonary host response to M. tuberculosis. To investigate the role of the B7/CD28/CTLA4 costimulation pathway in the initiation and amplification of the pulmonary host response to M. tuberculosis, transgenic mice that secrete an inhibitor of the B7/CD28/CTLA4 co stimulation pathway will be generated, characterized then used in experiments examining the immune response to M. tuberculosis. To determine the relative importance of the intrapulmonary component of the immune response vs the systemic immune response, in each of the project specific aims, mice will be included with both local inhibition of the immune response and systemic inhibition. In the studies examining the pulmonary host response to M. tuberculosis, infections will occur via the aerosol route because inhalation is the usual route of infection in humans. The primary endpoints will include bacterial burden, lung histology, survival and the phenotypic and functional characteristics of the immune response. The proposed studies should advance the existing knowledge of the specific nature of the pulmonary immune response to M. tuberculosis, will directly test the role of these mediators in host defense against M. tuberculosis, suggest important targets for novel therapies and elucidate role of specific mediators of the host immune response that are critical for the induction of an antigen specific immune response by candidate vaccines. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: EDUCATION IN TUBERCULOSIS--A NEED IN PUERTO RICO Principal Investigator & Institution: Rodriguez, William; U.S. Dept/Vets Affairs Medical Center Affairs Medical Center San Juan, PR 00933 Timing: Fiscal Year 2001; Project Start 01-JUL-1997; Project End 30-JUN-2002 Summary: During the last seven years we have seen dramatic changes in the epidemiology and incidence of tuberculosis in the United States. Despite improved
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methods of diagnosis and treatment, tuberculosis is increasing. Likewise, in Puerto Rico, tuberculosis cases have increased and occur disproportionately in certain high risk groups as HIV-infected persons. Over the last 3 decades as tuberculosis cases diminished, so did tuberculosis education. Medical students at the University of Puerto Rico School of Medicine, as well as housestaff, postgraduate fellows, and faculty receive an inadequate amount of training in tuberculosis and tuberculosis control. Moreover, the Puerto Rican community has little means of learning about this increasing public health threat. The primary goals of the curriculum development program in this application are the following: 1) to provide an integrated curriculum in tuberculosis that emphasizes principles and practices relevant to tuberculosis control at all levels of medical education; 2) to improve tuberculosis education in Puerto Rican community focusing on the providers caring for ("high risk" clients; 3) to develop a faculty capable of providing high quality tuberculosis instruction. The proposed curriculum development plan involves the following major components; a) creation of a multidisciplinary advisory committee; b) development of instructional materials that emphasize the importance of tuberculosis and tuberculosis control in case-based problem solving sessions; c) increased didactic and case-based teaching during the clinical rotation of medical students as well as housestaff postgraduate fellows; d) training of faculty responsible for tuberculosis education; e) improved continuing education in tuberculosis for community physicians and health care workers through a number of innovative courses and collaborations with the Department of Public Health and other community organizations. Research related to tuberculosis control will be fostered by support of student research projects as well as the continuation of the candidate's research program and the many collaborations that will result from this award. Evaluation of the curriculum development program will be conducted by an experienced group of physician and health educators through a stepwise and analytical manner. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EFFLUX INHIBITORS FOR MYCOBACTERIUM THERAPIES Principal Investigator & Institution: Markham, Penelope N. Director of Research; Influx, Inc. 2201 W Campbell Park Dr Chicago, IL 60612 Timing: Fiscal Year 2001; Project Start 01-MAR-2001; Project End 31-DEC-2001 Summary: (Verbatim from Applicant's Abstract): New drugs are desperately needed for tuberculosis both to reduce the long duration of treatment and to cure and stop transmission of multiple-drug-resistant Mycobacterium tuberculosis. Recent studies, including the completion of the TB genome sequence, suggest that efflux pump inhibitors may play a significant role in the intrinsic resistance of M. tuberculosis to most common antibiotics. Influx has identified several low molecular weight inhibitors of the NorA efflux pump in Staphylococcus aureus which demonstrated synergistic activity with ciprofloxacin and which dramatically reduced the frequency of emergence of ciprofloxacin-resistant mutants. In preliminary in vitro experiments with M. tuberculosis, two of these compounds demonstrated marked synergy when combined with two tetracyclines and a macrolide. We propose to a) test additional congeners of these inhibitors to determine structure-activity relationships with respect to M. tuberculosis, b) determine the substrate specificity of the effected efflux pump by testing for synergy with a wide variety of antimicrobials, c) determine the effect of the inhibitors on the frequency of emergence of drug-resistant mutants and d) with a variety of TB drug-susceptible and resistant clinical isolates, and e) assess synergistic activity of inhibitors with antimicrobial agents in a macrophage model of TB infection. It is
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anticipated that these studies will result in the identification of one or more lead compounds that would be assessed for activity in animal models of acute and chronic TB in a phase II project. In addition these compounds would serve as starting points for synthesis of additional congeners. PROPOSED COMMERCIAL APPLICATION: The envisioned commercial product is a combination of tetracycline, a macrolide, or possibly another antibiotic with an efflux pump inhibitor. This combination can form a powerful anti-TB agent which can be effective against resistant strains and prevent further emergence of resistance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EMERGING MDR TUBERCULOSIS ACROSS THE US/MEXICO BORDER Principal Investigator & Institution: Mccormick, Joseph B. None; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2003; Project Start 15-SEP-2003; Project End 31-AUG-2005 Summary: (provided by applicant) Our application will create dynamic databases to analyze and generate preliminary data on the precise patterns of transmission of tuberculosis (TB), especially multidrug resistant tuberculosis (MDR-TB), across South Texas and North Eastern Mexico. This will be achieved through a binational consortium that will share social, demographic, and geographic data on tuberculosis cases from each of their respective areas. The aims of the consortium are 1) aggregate existing sociodemographic, drug resistance and molecular fingerprinting databases currently held separately by consortium members 2) merge, geocode and analyze the aggregated database 3) establish common protocols and procedures for testing of drug resistance, extraction of M. tuberculosis DNA, and molecular characterization of isolates 4) evaluate social network analysis as a method for improving the tracking of MDR-TB transmission. These analyses link individual cases over 3-5 years with molecular fingerprints, drug susceptibility patterns, sociodemographic characteristics and geographic location, allowing regional understanding of the distribution and dynamics of DR and MDR-TB transmission across the border. These data will be used to improve control techniques and become the basis of hypotheses that will be tested in future, more elaborate research applications. These applications will include epidemiologic and translational research studies using common protocols for binational projects in emerging diseases, specifically MDR-TB. The geographic area of the consortium encompasses 5 counties of the Lower Rio Grande Valley (LRGV) from the Gulf of Mexico to Laredo, and the Mexican border states of Tamaulipas and Nuevo Leon. The consortium will be led by the Dean and faculty of the University of Texas School of Public Health Regional Campus at Brownsville, less than a mile from the border and contributing expertise in molecular microbiology, epidemiology, biostatistics, social network studies and spatial-temporal analyses. Leading tuberculosis researchers in Houston, San Antonio and Monterrey will play key roles in strengthening the capacity to conduct specialized public health, epidemiologic, and molecular based research on MDR-TB in the study area. Border consortium participants are from all the public health investigatorities responsible for TB elimination in both countries. An important defense of MDR introduction by bioterrorism is understanding how transmission of these organisms may differ from non resistant TB (if at all) and detailed knowledge of local strains of MDR that will allow quick distinction between known circulating strains of MDR TB and newly introduced strains. This application will be instrumental in addressing all of these issues. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ENHANCEMENT OF TUBERCULOSIS VACCINE BY THYMOSIN Principal Investigator & Institution: Tuthill, Cynthia W.; Sciclone Pharmaceuticals, Inc. 901 Mariner's Island Blvd San Mateo, CA 94404 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2004 Summary: (provided by applicant): Tuberculosis (TB) is a chronic infectious disease. Although most of the earths' 5.7 billion inhabitants have received the attenuated M. bovis BCG vaccine, many have still become infected and are at risk for developing clinical disease. The difficulty of compliance with lengthy and costly treatment regimens, coupled with the emergence of multi-drug resistant strains and the global AIDS crisis, have contributed to an emerging tuberculosis pandemic. An effective prophylactic vaccine is desperately needed. Most recent vaccines use non-viable bacterial proteins, lipids, or DNA; these require added adjuvants to generate a T helper 1 cellular response. We propose to evaluate the effects of thymosin, a synthetic 28 amino acid peptide with demonstrated Th1 immunomodulator properties, as an adjuvant. A well-characterized murine aerosol model of infection will be used with viable, virulent M. tuberculosis. We will evaluate the effect of thymosin on the level of antigen-specific IFNg production by circulating T cell populations; the reduction of bacterial load in lung and spleen; and conduct histopathological analyses of lung tissues to determine immune responsiveness and protective granuloma formation. Findings that indicate superior performance to BCG vaccinated controls will provide the basis for further evaluations in the more sensitive guinea pig model of infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: EXPRESSION OF TUBERCULOSIS IN THE LUNG Principal Investigator & Institution: Ellner, Jerrold J. Professor and Executive Vice President; Medicine; Univ of Med/Dent Nj Newark Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 31-JUL-2005 Summary: (Adapted from the Applicant's Abstract): Mycobacterium tuberculosis infects a third of the worlds' population and TB is the leading cause of morbidity and mortality due a single infectious agent. However, only 5-10% of M. tuberculosis-infected subjects without an underlying immunodeficiency develop disease during their lifetimes. Therefore protective immunity is induced in the majority of subjects. Understanding correlates of protection against M. tuberculosis in humans is needed to better direct efforts in the development of antituberculosis vaccines. The PI suggests that increased susceptibility to M. tuberculosis infection by patients with IFNgamma receptor I deficiency and successful therapeutic use of IFNgamma in refractory mycobacterial infections indicates the importance of IFNgamma in immunity against mycobacteria. However, during the previous funding period the PI found increased levels of IFNgamma in the lungs of patients with TB. Thus, the question is raised as to the effectiveness of IFNgamma and/or involvement of other factors in protective immunity. In the present competitive renewal application, the PI proposes to define pulmonary correlates of protective immunity by comparing several immunological parameters in TB patients, healthy household contacts of TB patients (tuberculin skin test positive), and community control subjects. Bronchoalveolar and blood mononuclear cells obtained from each study subject will be utilized to characterize antigen-specific cytokine induction, killing of M. tuberculosis, CTL activity against M. tuberculosisinfected targets and mediators involved in these effector functions (iNOS, granzyme, perforin, granulysin, FasL) (specific aim 1). According to the PI, this first part of the work should identify correlates of protective immunity as differences between
48 Tuberculosis
protective immune responses (in skin-test-positive, healthy household contacts) and failed immune responses (patients with TB). Correlates of protection identified in aim #1 should then be used to assess induction of protective immunity and chemokine expression by vaccination of humans with BCG. Two strains of BCG having different efficacy and reactogenicity, and two routes of vaccine administration (oral and intracutaneous) will be compared (specific aim 2). The PI states that this proposal attempts to define new parameters of immunological protection in humans and to rationally assess the impact of BCG strain variation and the route of BCG administration on the induction of protective immunity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: FUNCTION OF CD8 T CELLS IN TUBERCULOSIS Principal Investigator & Institution: Flynn, Joanne L. Associate Professor; Molecular Genetics & Biochem; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2002; Project Start 30-SEP-1997; Project End 31-MAR-2007 Summary: (provided by the applicant): The immune response against Mycobacterium tuberculosis results in control, but not elimination, of infection. T cells are a crucial component of this response. This proposal extends our current work on examining the CD8 T cell subset and the effect of these cells in tuberculosis. It is clear that the naturally induced immune response is insufficient to resolve a M. tuberculosis infection. Understanding how the CD8 T cell response develops and is modulated over the course of infection may provide clues to augmenting this response to provide better control of infection. In this proposal, we will focus on following CD8 T cell responses to specific antigens, focusing on the function of the CD8 T cells at various times post-infection. Specifically, cytokine production and cytotoxic ability will be tested during acute, chronic, memory and reactivation states. Our hypothesis is that the CD8 T cell response and function wane during a chronic infection, and boosting this response would result in improved control of the infection. In addition, preliminary data indicates a role for CD4 T cells in maintaining CD8 CTL function, and the mechanisms responsible for this will be investigated. Our long term goal is to have a clear picture of the CD8 T cell response in tuberculosis, including antigen specificity, function, evolution, and maintenance. This information will impact directly on vaccine development, since it appears that stimulation of both CD4 and CD8 T cells will be necessary to provide adequate protection against tuberculosis. To this end, our specific aims are: Aim 1. To examine evolution in the antigen specific CD8 T cell responses during M. tuberculosis infection. Aim 2: To investigate the function of CD8 T cells in M. tuberculosis infection. Aim 3: To assess the development, maintenance and function of memory and recall CD8 T cell responses in tuberculosis. Aim 4: To determine the effects of CD4 T cells on CD8 T cell maintenance and function in tuberculosis. Animal models will be used in these studies, and we have adapted a variety of functional assays for CD8 T cells for use with lung cells. These complementary aims will provide a definitive picture of the CD8 T cell response in tuberculosis, and contribute to a greater understanding of the challenges facing vaccine development and design against this disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: FUNTIONAL GENOMICS OF INTERFERON FOR TUBERCULOSIS Principal Investigator & Institution: Pine, Richard I. Assistant Member; Public Health Research Institute 225 Warren St Newark, NJ 07103
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Timing: Fiscal Year 2001; Project Start 10-SEP-2001; Project End 31-JUL-2006 Summary: Interferon-gamma (IFNgamma) is a cytokine released by TH1 lymphocytes in tuberculosis and is an essential mediator of the cellular immune response, producing macrophage activation. Signaling is transduced through activation of STAT-1, a latent transcription factor, and leads to induced expression of other transcription factors. STAT-1 and the other transcription factors like IRF-1 together increase expression of effector genes such as iNOS. We have been conducting a clinical trial of aerosolized IFNgamma as treatment for pulmonary tuberculosis and have reported that patients with multi-drug resistant tuberculosis improved on this regimen. We now report that aerosolized IFNgamma induces STAT-1 and IRF transcription factors in lung cells. Treatment also markedly reduces HIV-1 levels in the lungs of AIDS patients with tuberculosis, providing further evidence of enhanced lung immunity produced by pharmacological doses of IFNgamma. THP-1 macrophages and alveolar macrophages are similar with regard to induction of STAT-1, IRF-1 or IFR-9 (also called p48 and ISGF3-gamma) after infection with M. tuberculosis (M.tb) or IFNgamma treatment. THP-1 macrophages respond to IFNgamma with increased IRF-9 mRNA but do not change its transcription, demonstrating that IRF-9 is up-regulated by posttranscriptional mechanisms such as splicing or message stability. We used functional genomics to identify other IFNgamma responsive genes. Of 4375 genes present on a high density filter array, 48 were induced and 13 were repressed after 4 hours of IFNgamma stimulation. Only 15 of these 61 genes have been reported to be responsive to IFNgamma Finally, with the goal of assaying genome wide transcription rates, we have developed a novel assay for transcription using Br-UTP labeled nascent RNA. Our goal is to identify genes unique to the cellular immune response during tuberculosis in vivo and genes induced or repressed by IFNgamma and/or M.tb. ex vivo and in vivo. We propose to use functional genomics to identify the genes induced and repressed in: 1) THP-1 macrophages infected with M.tb and/or treated with IFN-gamma in vitro 2) Alveolar macrophages infected with M.tb and/or treated with IFNgamma ex vivo and 3) Bronchoalveolar lavage cells from tuberculosis patients before and after aerosolized IFNgamma. We will then assay transcription rates of the genes that are IFN responsive to define the contribution of transcriptional and post transcriptional regulation to mRNA abundance. We hypothesize that like IRF-9, other genes important to the IFNgamma response are post-transcriptionally regulated in macrophages. This analysis will further the understanding of cellular immunity in tuberculosis and gene expression regulated by IFNgamma. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENE EXPRESSION IN MYCOBACTERIUM TUBERCULOSIS Principal Investigator & Institution: Clark-Curtiss, Josephine E. Research Associate Professor; Biology; Washington University Lindell and Skinker Blvd St. Louis, MO 63130 Timing: Fiscal Year 2001; Project Start 01-FEB-2000; Project End 31-JAN-2004 Summary: Tuberculosis, one of the great scourges of humankind, is the leading cause of death worldwide from a single infectious disease. Although the incidence of TB has again begun to decline in the U.S., TB remains a significant health problem in this country, most frequently affecting the elderly, the homeless, individuals with AIDS and immigrants from nations where TB is endemic. Although the causative agent of TB, Mycobacterium tuberculosis, was identified a century ago, knowledge about fundamental physiological capabilities, the genetics and the mechanisms of pathogenicity of M. tuberculosis is only now beginning to emerge. We have identified
50 Tuberculosis
several genes which are expressed by M. tuberculosis H37Rv when the bacilli are growing in human macrophages in culture, but which are not expressed by the bacilli when they are growing in laboratory broth culture. We hypothesize that these genes and their products may be important in the survival and growth of M. tuberculosis in macrophages and may contribute to the pathogenicity of the tubercle bacilli. We propose to (1) determine the contribution of specific macrophage-expressed gene products to survival and growth of M. tuberculosis in macrophages by molecular and genetic characterization of genes on a cosmid that appear to be coordinately expressed, a putative response regulator gene, and mceD, a gene that has been shown to enhance survival of E. coli in cultured macrophages; and (2) to identify and characterize genes and gene products of M. tuberculosis that are expressed at early times after phagocytosis, at late times, and throughout growth in human macrophages in culture. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENE MACROPHAGES
EXPRESSION
OF
M.TUBERCULOSIS
WITHIN
Principal Investigator & Institution: Mcdonough, Kathleen A. Research Scientist; Wadsworth Center Empire State Plaza Albany, NY 12237 Timing: Fiscal Year 2001; Project Start 01-JUL-2000; Project End 30-JUN-2005 Summary: (Adapted from the Applicant's Abstract): The long term objective of this proposal is to gain a better understanding of how Mycobacterium tuberculosis establishes infection so that effective strategies can be developed to prevent it. A multidisciplinary approach will focus on the interaction of tubercle bacilli with macrophages at the molecular, genetic and cellular levels, with an emphasis on M. tuberculosis gene expression within macrophages. The specific aims are: 1) Identifying M. tuberculosis genes that are induced when bacteria are within macrophages by 2D gel electrophoresis coupled with mass spectrometry. 2) Identifying class-specific regulatory motifs among intracellular induced promoters using a combination of molecular and computational techniques. 3) Characterizing the roles in the M. tuberculosismacrophage interaction of selected intracellular induced M. tuberculosis genes. Assays will include bacterial survival, replication and trafficking in macrophages. 4) Assessing the role of cAMP signaling in M. tuberculosis within macrophages by defining the distribution and expression among mycobacteria of genes encoding novel cyclase and cyclic NMP binding proteins; estimating the minimum number of cAMP-responsive proteins using 2D gels; and determining the effects of a novel adenylate cyclase gene knock-out on M. tuberculosis interaction with macrophages using tissue culture, microscopy, and 2D gel analyses. This work will contribute to our understanding of the factors needed for the establishment of tuberculosis infection and disease and will identify potential targets for tuberculosis vaccines, therapeutics, and diagnostic purposes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENES TUBERCULOSIS
FOR
METHY1-BRANCHED
WALL
LIPIDS
AND
Principal Investigator & Institution: Kolattukudy, Pappachan E. Professor and Directpr; Neurobiotechnology Center; Ohio State University 1800 Cannon Dr, Rm 1210 Columbus, OH 43210 Timing: Fiscal Year 2001; Project Start 15-JAN-2000; Project End 31-DEC-2003
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Summary: Resurgence of tuberculosis (TB) and other mycobacterial infections associated with AIDS threaten human health world-wide. Emergence of drug resistant TB makes it critical to discover new drug targets. Cell walls of Mycobacterium tuberculosis contain a variety of unusual lipids with many types of multiple methylbranched fatty acids that are unique to pathogenic mycobacteria. These lipids are thought to play important roles in the ability of the pathogens to resist antimicrobial agents and evade the defense reactions of the host. Biosynthesis of these unique lipids containing multiple methyl-branched fatty acids could offer ideal targets for new anti mycobacterial drugs. Genomic sequencing revealed that a remarkably unique feature of the mycobacterial genome is that it contains an unusually large number of genes involved in lipid metabolism. Based on the homology to mycocerosic acid synthase (mas) we have identified two classes of polyketide synthase (pks)-like genes which contain a full complement of active site domains that should be involved in the catalysis of all of the steps required for the synthesis of multimethyl- branched fatty acids: Class 1 mas-like genes mas-like genes (msl1, msl2 and msl3) that are highly homologous to mas and Class 2 (msl4m msl5, msl6 and msl7) which show a lesser degree of homology to mas. These open reading frames (ORFs) most probably encode the more than eight classes of methyl-branched fatty acids found in M. tuberculosis. To elucidate the function of these genes and to examine their possible role in the host-pathogen interaction, we propose to pursue the following specific aims: 1) Determine the functions of Class 1 mas-like genes, ms1, msl2 and msl3 of M. tuberculosis by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 2) Determine the functions of Class 2 (msl4, msl5, msl6 and msl7) mas-like genes in M. tuberculosis genome, by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 3) Determine whether lack of specific lipids caused by the above gene-disruptions affects host-pathogen interaction and virulence. The results will help identify cell wall lipids critical for pathogenesis that might be suitable targets for new anti-mycobacterial drugs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC DISSECTION OF MYCOBACTERIAL INFECTION Principal Investigator & Institution: Schurr, Erwin; Associate Professor; Mc Gill University James Admin. Bldg., Room 429 Montreal, PQ H3A 2T5 Timing: Fiscal Year 2001; Project Start 14-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) The objective of the studies proposed in this application is to identify genotypic combinations of mice and mycobacteria that result in significant alterations of host responses to experimental mycobacterial infection. Susceptibility or resistance to experimental infection will be defined by determination of median survival time, weight loss, mycobacterial load in the lung and spleen, and in addition various immunological parameters in lung and other tissues. In these experiments, the host genome will be varied by use of 37 recombinant congenic strains (RCS), which are derived from inbred progenitors that are either susceptible to tuberculosis (A/J, abbreviated A) or resistant to tuberculosis (C57BL/6J, abbreviated B). The AcB/BcA RCS are now fully inbred and genotyped with a dense set of genomewide microsatellite markers. These strains will be infected with a panel of Mycobacterium tuberculosis and M. bovis strains that have been deleted for genome regions associated with attenuation of BCG vaccines. The infection protocol in the RCS will reveal informative, significant deviations from the "expected" or "parental" disease phenotypes which signify the presence of quantitative trait loci (QTL) with strong effect
52 Tuberculosis
on phenotype expression and possibly specific gene(s) interaction between the host and pathogen. In RCS carrying QTL with strong effect on phenotype expression, changes in gene expression level in both lung macrophages and intracellular mycobacteria will be revealed by microarray analysis. The knowledge to what extent host responses in mycobacterial infections are a reflection of specific host pathogen combinations will be crucial for our understanding of the epidemiological flow of M. tuberculosis through an exposed population. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC FACTORS AND SUSCEPTIBILITY TO TUBERCULOSIS Principal Investigator & Institution: Goldfeld, Anne E.; Cbr Institute for Biomedical Research 800 Huntington Ave Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 30-SEP-1997; Project End 30-JUN-2004 Summary: The host factors and mechanisms involved in the pathogenesis of pulmonary tuberculosis in the setting of AIDS as well as in the normal host are poorly understood. The long term objective of this project is to further identify and to characterize host factors that predispose individuals to develop pulmonary tuberculosis. Preliminary data indicate an association of an HLA allele, the HLA-DQB1*0503 class II allele, with susceptibility to pulmonary tuberculosis in Cambodia. Specific Aim 1 will determine whether the same or different HLA alleles are associated with TB progression in a genetically distinct population from South Africa. The TNF-alpha gene occurs in the class III region of the major histocompatibility complex (MHC) and its protein product plays a critical role in the containment of tuberculosis infection. Studies will include the identification of polymorphisms and microsatellites in the tumor necrosis factor-alpha (TNF-alpha) gene, and their association to tuberculosis susceptibility. Specific Aim 2 will characterize the mechanisms of activation of the TNF-alpha gene by mycobacteria. By using the TNF-alpha gene promoter as a target, these studies could define the molecular mechanisms of the activation of this important gene by mycobacteria. Specific Aim 3 proposes to investigate the genetic and molecular determinants of anergy in the setting of pulmonary tuberculosis. This aim will investigate whether there is an HLA association with anergy or lack of DTH, and will characterize the cytokine profiles and T cell proliferative responses in anergic pulmonary tuberculosis patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: GENETIC TUBERCULOSIS
STUDIES
OF
HUMAN
SUSCEPTIBILITY
TO
Principal Investigator & Institution: Scott, William K. Associate Research Professor in Medicine; Medicine; Duke University Durham, NC 27706 Timing: Fiscal Year 2001; Project Start 10-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) Tuberculosis (TB) is currently and historically an enormous public health problem. Approximately one-third of the world's population are currently infected with Mycobacterium tuberculosis (M. tuberculosis) and TB accounts for over 25% of preventable adult deaths world-wide. Despite the high infection rate, only about 10% of people infected with M.tb ever become sick with active TB. Evidence suggests that progression to active TB is influenced by host genetic factors. For example, the epidemiology of TB suggests that genetic selection takes place after introduction of M. tuberculosis to the population; genetically susceptible individuals succumb to the infection and relatively resistant individuals survive to reproduce. As well, twin studies demonstrate higher concordance rates for TB among identical twins,
Studies 53
compared to fraternal twins. Mouse models of mycobacterial infection have identified several potential susceptibility loci, such as the gene named Nramp1, as well as several cytokine and cytokine receptor genes. Family-based linkage studies and case-control studies of candidate genes in humans suggest roles for these and other genes associated with development of TB in humans. In light of these observations, we propose a familybased association study of candidate genes for TB susceptibility. To accomplish the goal of identifying genes influencing susceptibility to TB we specifically propose to: 1) Ascertain 1,000 parent- child triads (500 Caucasian, 500 African-American) from North and South Carolina for genetic studies of TB susceptibility genes. 2) Test candidate genes in the first 500 parent-child triads. Multiple single nucleotide polymorphisms (SNPs) will be genotyped in each gene and analyzed using family- based tests of association; significant results will be followed-up in the remaining 500 triads. 3) Examine the relationship between candidate genes and other clinical variables such as PPD skin test results, disease severity, treatment relapse and failure, and presence of extrapulmonary disease. 4) Evaluate gene-gene and gene-environment interactions using multivariable models and data reduction techniques such as the multifactor dimensionality reduction (MDR) method. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC SUSCEPTIBILITY TO MYCOBACTERIUM TUBERCULOSIS Principal Investigator & Institution: Kramnik, Igor; Assistant Professor of Immunology & Infe; Immunology/Infections Diseases; Harvard University (Sch of Public Hlth) Public Health Campus Boston, MA 02460 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 30-JUN-2006 Summary: (provided by the applicant): It is projected that a total of 225 million new cases of tuberculosis will occur between 1998 and 2030. The outcome of primary infection with Mycobacterium tuberculosis (MTB) varies and, in immunocompetent hosts, imparts only a 10 percent lifetime risk of developing clinical disease. The significant variation in tuberculosis susceptibility among immunocompetent individuals remains unexplained. Differences in the outcome of tuberculosis infection in the setting of similar risk factors support a significant role of the host genetic background in predisposition to progression towards clinical disease. Therefore, identification of genetic factors associated with susceptibility to tuberculosis will have important implications for controlling the disease. We use a mouse experimental model of infection with virulent strain of MTB to characterize genes that are responsible for control of tuberculosis infection in immunocompetent hosts. We have identified and mapped to a 2 cM interval on mouse chromosome 1 a novel locus (sst1) that significantly contributes to control of growth of virulent MTB primarily in the lungs. Observations on its phenotypic expression demonstrate that genetic mechanisms controlling infection with virulent MTB are distinct from those that control an avirulent vaccine strain of M bovis BCG. Having generated a set of sst1-congenic inbred strains of mice, we propose to use them (1) to isolate the sst1-candidate genes by positional cloning; (2) to identify genetic polymorphism responsible for tuberculosis susceptibility; (3) to identify cells and functional pathways affected by the sst1 polymorphism. The proposed project will identify the nature and mechanism of action of the sst1 in mice, which in the future should permit isolation of its human homologue and the analysis of its role in tuberculosis susceptibility in humans. The understanding of genetically determined mechanisms that operate during the course of tuberculosis infection in the lung will provide new insights into the pathogenesis of tuberculosis and suggest improved strategies for treatment and prevention of the disease.
54 Tuberculosis
Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC/IMMUNOLOGICAL BASIS OF TUBERCULOSIS AND ANERGY Principal Investigator & Institution: Delgado, Julio C.; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant): The purpose of this Clinical Investigator Development Award is to prepare the candidate, Dr. Julio C. Delgado, MD, for a career as an independent investigator in infectious diseases with a special emphasis on the genetic and immunological mechanisms associated with susceptibility to tuberculosis infection. The applicant proposes a 5-year training and research program that will prospectively identify novel host factors that predispose individuals to develop pulmonary tuberculosis in a population from Cambodia and characterize the events associated with the anergic state induced by tuberculosis infection in the intact human host. In Specific Aim 1, he will test the hypothesis that HLA molecules are functionally associated with tuberculosis progression and differential immune response to PPD and whether polymorphisms of the promoter region of certain cytokines are associated with impaired immune response against tuberculosis infection. In Specific Aim 2 and 3, he will characterize immunological and chromatin structural mechanisms associated with anergy to PPD in a cohort of tuberculosis patients from Cambodia with persistent and specific lack of response to PPD. Tuberculosis infection and pathogenesis are the result of a balance between the virulence of a particular invading organism and the host immune response. Susceptibility to tuberculosis in human populations is likely to be due to a complex interaction between several genetic, immunological and environmental factors. The experiments proposed in this application would attempt to identify host genetic factors and unveil abnormal immunological mechanisms leading to increase risk of clinical tuberculosis disease. It is anticipated that these results will be useful in the development of genetic and immune therapeutic strategies to combat tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: GENETICS OF M TUBERCULOSIS PERSISTENCE & DRUG TOLERANCE Principal Investigator & Institution: Gomez, James E. Lab/Infection Biology; Rockefeller University New York, NY 100216399 Timing: Fiscal Year 2001; Project Start 01-APR-2001 Summary: (provided by the applicant): One of the primary reasons that our arsenal of drugs has failed to lead to the successful control of tuberculosis is the ability of the etiologic agent, Mycobacterium tuberculosis (Mtb), to persist within the host tissues, where the drugs that readily kill this bacterium in vitro require prolonged periods of time to achieve similar effects. The extended duration of chemotherapy required to cure a human (6-12 months) is one of the greatest barriers to effective TB control. Poor patient compliance may lead to chronic infection, either active or latent, and the emergence of drug resistant Mtb. The underlying biology of persistent Mtb infection is poorly understood, and therefore, the objectives of this study are as follows: I) to better characterize the "persistent state" by examining drug efficacy and bacterial division at a variety of stages of infection in the mouse model; 2) to identify Mtb genes required for persistence in a mouse model using signature-tagged mutagenesis (STM); and 3) to identify Mtb genes that are required for the increased tolerance of Mtb to
Studies 55
chemotherapeutic agents in vivo, again using STM in the mouse model. Achievement of these aims will provide a basis for the rational development of novel therapeutics that target persistent bacilli, thereby shortening the time required to achieve a cure. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GLUTAMINE SYNTHETASE INHIBITORS FOR TUBERCULOSIS THERAPY Principal Investigator & Institution: Atkins, William M. Medicinal Chemistry; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2002; Project Start 01-JUL-2002; Project End 30-JUN-2005 Summary: (Provided by the applicant:) Infection by Mycobacterium tuberculosis (MBT) is a significant complication experienced by many AIDS patients. Recently, the enzyme glutamine synthetase (GS) has become a new therapeutic target for MTB, due to the demonstration that secretion of enzymatically active MBT GS is required for survival and pathogenecity of the organism. The long-term goal of the proposed research is to develop inhibitors of MBT GS with potential therapeutic use. Many in vitro inhibitors of bacterial GS's have been documented, but none are useful clinically. In order to initiate MBT GS inhibitor design, a portion of this proposal is aimed at understanding its molecular properties, in comparison to the well studied E. coli GS. If comparable to the E. coli GS, then a new strategy will be pursued to obtain inhibitors that are more potent and selective than any previously described compounds. Specifically, the highly symmetrical ring structure of GS will be exploited to design a library of multivalent inhibitors which bind to the flexible loop on several subunits, in contrast to the monovalent inhibitors previously targeted individually to the active sites. The specific aims are: 1) To determine whether structural modification of the central loop on each subunit results in loss of enzyme activity. Because the modification of the central loop of the E. coli GS does lead to loss of activity, it is anticipated that this will be the case for MBT GS; 2) To design, synthesize and screen libraries of multivalent inhibitors targeted to the central loops of MBT GS, and to E. coli GS, for 'proof-of-principle.' Demonstration of the utility of multivalent inhibitors targeted to the central loops of MBT GS would provide a new rationale for GS inhibition and possibly for tuberculosis therapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: GLYCOPROTEINS OF MYCOBACTERIUM TUBERCULOSIS Principal Investigator & Institution: Belisle, John T. Associate Professor; Microbiology, Immunology & Pathology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2001; Project Start 01-FEB-1999; Project End 31-JAN-2004 Summary: (Adapted from the Applicant's Abstract): In response to the NIH Program Announcement calling for more basic information regarding the biology, immunology and pathogenesis of this disease, the investigators present the hypothesis that past failures in vaccine development against tuberculosis have been due to the inability to emulate the immunogenicity of the whole bacillus due to failure to recognize the importance of appendages that covalently modify key protective protein antigens. The differential immunological reactivity of native and E. coli recombinant forms of mycobacterial proteins as well as our own preliminary data support this principle. Recently, the investigators provided definitive chemical proof that some M tuberculosis proteins are glycosylated. They have now extended these studies to demonstrate that the M tuberculosis proteome possesses a number of such structures and that glycosylation plays a direct role in immunological recognition of M tuberculosis
56 Tuberculosis
proteins, facts which now portend new approaches to vaccine and serodiagnostic antigen development. To address the issue of the contribution of aprotein components to immunological recognition of protein antigens, they will (i) exploit their proven strengths in protein chemistry to demonstrate the relative abundance of glycoproteins in the M tuberculosis proteome and provide a precise chemical characterization for a large number of these molecules; (ii) apply this structural information, some of which is already in hand, to immunological assays in both the human and murine systems, to provide a detailed understanding of how the host's acquired immune system responds to M tuberculosis glycoproteins; and (iii) initiate studies of the biosynthesis of the posttranslationally modified, and specifically mannosylated, proteins, which will yield information and new tools for full evaluation of the general biological, particularly immunological, significance of protein glycosylation and lipidation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GROWTH ACTIVATING FACTOR FOR MYCOBACTERIUM TUBERCULOSIS Principal Investigator & Institution: Griffin, John P.; Mycos Research, Llc 217 Racquette Dr, #6 Ft. Collins, CO 80524 Timing: Fiscal Year 2001; Project Start 01-JUL-2001; Project End 30-JUN-2002 Summary: (provided by applicant): In this proposal we are requesting funding for proof of principle research into Mycobacterial Growth Activating Factors (GAFs). The longterm objective of these studies is the production of a commercial product that would significantly reduce the time required for diagnosis and drug susceptibility screening of tuberculosis. The aims of this proposal are to: (1) establish the growth promoting characteristics of GAFs using both clinical and laboratory isolates of M tuberculosis. (2) Evaluate the potential use of GAFs in drug susceptibility testing. (3) Determine the effects of GAFs in a clinical setting. And (4) Evaluate derivatives of our defined peptides for potential activity and test for synergistic combinations. If successful, these products could lead to multiple innovative uses that range from the improvement of diagnoses in developing nations to significantly faster identification of efficacious therapeutic drug regimens. PROPOSED COMMERCIAL APPLICATION: MycoQuick Growth Media: A culture medium supplement that reduces the time required for the culture of slow growing mycobacteria species. This in turn will significantly reduce the lengthy time required for laboratory diagnosis of infection with M. tuberculosis. Rapid diagnosis will also permit the timely initiation of drug therapy in these patients and permit rapid drug resistance profiling. MycoQuice Diagnostic Kit: A diagnostic kit for M- tuberculosis, specifically targeted for use under laboratory conditions commonly encountered in developing nations. This product will incorporate the MycoQuick growth medium in pre-prepared assemblies with or without drugs commonly prescribed and antituberculosis therapy. MycoQuick Kit will facilitate both rapid diagnosis and drug susceptibility profiling in a format conducive for use under the less stringent conditions found in distant third world sites. This product may have a significant impact in these regions where tuberculosis is endemic. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: HEAT SHOCK PROTEIN BASED VACCINE FOR TUBERCULOSIS Principal Investigator & Institution: Mo, Annie Xy.; Antigenics, Inc. 34 Commerce Way Woburn, MA 01801 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 30-NOV-2003
Studies 57
Summary: (provided by applicant): Mycobacterium tuberculosis is a continuing health threat both in the United States and the rest of the world with an estimated two billion people currently infected worldwide. Antibiotic regimens requiring lengthy direct observation of patients and the emergence of drug resistant strains create a pressing need for novel therapies. One such novel therapy is the use of heat shock protein to adjuvant a tuberculosis peptide vaccine for optimal generation of cell-mediated immune response. Heat shock protein-peptide complexes are recognized by HSP receptors on antigen presenting cells. Peptides chaperoned by HSP are then represented by MHC Class I and II molecules leading to cell mediated immunity including stimulation of antigen-specific CD4+ and CD8+ T cells. Cell mediated immunity is an important major protective mechanism in tuberculosis. The ability of heat shock protein-peptide complexes to elicit T cell responses may address a shortcoming of traditional tuberculosis vaccines that primarily elicit antibody responses. The proposed product will consist of Mycobacterium tuberculosis antigens complexed in vitro to mammalian heat shock protein 70. Total cytosolic and culture filtrate fractions from M tuberculosis will be obtained and subjected to proteolysis with highly selective proteases to generate a large array of peptides containing T cell epitopes. Murine heat shock protein 70 will be complexed to the TB peptide extract and utilized as a vaccine. The product will be evaluated in mice for induction of cellular immune responses, assessed by antigen-specific cytotoxicity, cytokine response, and proliferation. The ultimate purpose of the research funded by this grant is to ascertain whether it will be possible to utilize the heat shock protein technology to generate a novel and efficacious therapeutic vaccine. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HETEROGENEITY OF T CELLS IN M TUBERCULOSIS INFECTION Principal Investigator & Institution: Boom, W Henry. Professor of Medicine; Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 01-FEB-1989; Project End 31-MAR-2002 Summary: (Adapted from the applicant's abstract): T cells have a critical role in regulating the protective immune response to Mycobacterium tuberculosis. As a result of studies supported by AI-27243, our understanding of the interaction between human T cells and macrophages in response to M. Tuberculosis has increased substantially during the last five years. First, M. Tuberculosis antigens activate three major T cell subsets from healthy tuberculin positive donors: V delta2 + gamma delta TCR bearing T cells (gamma delta T cells and CD8+ alpha beta (ab) TCR+ T cells (CD 8 T cells) in addition to CD4+ ab T cell receptor (TCR) bearing T cells (CD 4 T cells. Second, all three T cell subsets produce IFN-gamma and can serve as cytotoxic effector cells (CTL) against M. Tuberculosis infected macrophages, fitting into a Th-1 like pattern. These findings allow refinement of the model in which control of mycobacterial growth and maintenance of immunity requires the activation not only of CD4+ T cells and gamma delta T cells, but also of CD8+ T cells. This model postulates that there is a defined sequence of T cell subset recruitment and that the antigen processing mechanisms used by M. Tuberculosis infected mononuclear phagocytes are critical in the recruitment of T cell subsets and in determining the antigen repertoire recognized by CD4+, CD8+ and gamma delta T cells. In addition, the model postulates that both secretion of macrophage activating cytokines and CTL effector function are necessary for protective immune responses to M. Tuberculosis. There are three specific aims to test the postulates of this model: 1. To further characterize a 10-14KD M. tuberculosis antigen for V delta2+ gamma delta T cells, to determine if the antigen can be detected on the
58 Tuberculosis
surface of mononuclear phagocytes for presentation to V delta2+ gamma delta T cells and to determine if gamma delta T cells activated by this antigen enhance the production by mononuclear phagocytes of chemokines (MIP-1alpha/beta, RANTES) and cytokines (IL-12, IL-15) that promote T cell recruitment and differentiation into CTL. 2. To characterize the antigen processing pathways of M. tuberculosis antigens for class II MHC presentation to CD4+ T cells, using the 30kD 85B antigen as model antigen. 3. To characterize the repertoire of antigens, the antigen-processing pathway and CTL function of CD8+ T cells reactive to M. tuberculosis in comparison to CD4+ and gamma delta T cells. These studies will provide insight into the regulation of human T cell subsets involved in protective immunity to M. tuberculosis, which is necessary for the design of improved vaccines, and immunotherapies for tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HIV PREVENTION/ALCOHOL TREATMENT IN A RUSSIAN TB SAMPLE Principal Investigator & Institution: Fleming, Michael F. Professor; Family Medicine; University of Wisconsin Madison 750 University Ave Madison, WI 53706 Timing: Fiscal Year 2001; Project Start 28-SEP-2001; Project End 31-AUG-2003 Summary: (provided by applicant): HIV infection, tuberculosis, and hazardous drinking are of increasing concern in Russia. While the number of officially registered cases of HIV infection as of October 31, 2000 was 69,120, experts expect an exponential increase in the number of Russian people infected with the human immunodeficiency virus in the next 10 years. Incident cases of TB and multi drug resistant tuberculosis in Russia are among the highest in the world. Mortality rates exceed 10% with over 125,000 persons receiving medical treatment for active TB in 1999. Hazardous drinking is common and a major source of morbidity and mortality in Russia. Alcohol use also complicates prevention and treatment programs for HIV infection and TB. Russia shares borders with 13 countries. The global health implications of a large reservoir of HIV infection and tuberculosis in a country that crosses 8 time zones is enormous. Now is the time to prevent the spread of HIV infection and tuberculosis in Russia and neighboring countries. Potential strategies include testing of HIV prevention programs (i.e., Project RESPECT) and treatment programs to reduce rates of hazardous drinking. This proposal is designed to increase our understanding of HIV risk factors, and to increase the role and treatment of alcohol use disorders in the prevention of HIV infection in the context of a TB treatment population. This proposal will conduct the following 3 studies in St. Petersburg and Ivanovo, Russia: a) a prevalence study designed to assess the frequency of HIV risk factors and alcohol use in a patient population being treated for tuberculosis (n=600); b) a naturalistic study designed to assess the effects of alcohol use on HIV risk behaviors and TB treatment outcomes (n=200); c) a combined day-treatment program for HIV risk reduction and alcohol treatment (n=80). The data from these studies will provide new information. The findings are expected to facilitate the development, testing and implementation of HIV prevention and alcohol treatment programs. It is hoped that these programs can reduce the spread of HIV infection and TB in Russia and neighboring countries. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: HIV/TB INTERACTION IN THE LUNG Principal Investigator & Institution: Weiden, Michael D. Medicine; New York University School of Medicine 550 1St Ave New York, NY 10016
Studies 59
Timing: Fiscal Year 2001; Project Start 01-APR-1999; Project End 30-MAR-2004 Summary: We have shown that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro, we recently showed that monocytes increase HIV-1 replication after infection with M. tb. but, surprisingly, we now show M. tb infection inhibits HIV-1 replication in macrophages. Suppression of HIV-1 replication is associated with repression of the HIV-1 LTR and induction of ISGF-3, an interferonalpha/beta (IFN)-specific transcription-factor complex. Monocytes do not induce ISGF-3 after IFN treatment or infection with M. tb. Intact C/EBP sites in the HIV-1 LTR negative regulatory element (NRE) are required for promoter repression. Macrophages but not monocytes induce an inhibitory 16-kDa C/EBP-Beta isoform coincident with the HIV-1 LTR repression. Low-dose IFN-Beta represses the HIV-1 LTR and induces the 16kDa inhibitory C/EBP-Beta. During LTR repression, C/EBP-Beta is present in over 95 percent of the NRE/protein complexes. Mouse bone- marrow macrophages deficient in an IFN receptor do not produce the C/EBP-Beta repressor after an inflammatory stimulus. Taken together, these data suggest that, in vitro, proinflammatory stimulation of macrophages produces an antiviral IFN response that induces a C/EBP-Beta transcriptional repressor and inhibits LTR-mediated transcription. In vivo, alveolar macrophages from uninflamed lung are like macrophages treated with IFN-Beta in vitro; both strongly express inhibitory 16-kDa C/EBP-Beta and inhibit HIV-1 replication. Pulmonary tuberculosis abolishes C/EBP-Beta expression and induces a novel C/EBP DNA binding protein in involved lung segments of both HIV-1- infected and immunocompetent patients. Stat-1 homodimer (an IFN-gamma-specific transcription factor) is also induced, but only in immunocompetent tuberculosis patients. A plausible hypothesis for these observations is as follows: In the absence of inflammation, alveolar macrophages are primed by low doses of IFN-Beta. The cellular immune response in pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression in both HIV-1-infected and immunocompetent patients. The failure to produce IFNgamma in AIDS patients allows high-level viral replication. This proposal will investigate the regulation of IFN-mediated immunity in uninflamed lung and in tuberculosis. It will also model stimulation of viral replication in tuberculosis and assess how HIV-1 infection alters IFN response. It will investigate the mechanism and effects of inhibitory C/EBP-Beta induction. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HOST & PATHOGEN DETERMINANTS OF M TUBERCULOSIS LATENCY Principal Investigator & Institution: Kaplan, Gilla; Full Member; Public Health Research Institute 225 Warren St Newark, NJ 07103 Timing: Fiscal Year 2002; Project Start 15-SEP-2002; Project End 30-JUN-2007 Summary: (provided by applicant): Our aim is to define the host and bacterial factors that characterize the clinical and microbiologic latency which distinguish human tuberculosis. We hypothesize that in human tuberculosis, persistent/latent infection is associated with a different immune response than active chronic disease. For Mycobacterium tuberculosis to survive in the face of these varying host immune responses, it must alter its physiology through differential changes in gene expression. Thus a particular clinical state associated with a specific state of immunity will elicit a corresponding profile of genes expressed by the infecting organism ("molecular mirror"). We will utilize M. tuberculosis infected lung tissue obtained from surgical resection of TB patients with active disease, or asymptomatic persistent infection, or recurrent disease. These tuberculous lung specimens will be used to characterize the
60 Tuberculosis
host immune response by histology and immunohistology combined with real time RTPCR with molecular beacons to quantitate leukocyte gene expression in the lung. Our collaborator, Dr. John McKinney, will use the same tissues to identify the patterns of M. tuberculosis gene expression in each type of disease. In addition, we will use the rabbit model of M. tuberculosis infection to generate animals with either active disease or persistent asymptomatic infection (latency). Rabbits will be infected with clinical strains of M. tuberculosis of differing degrees of virulence to recreate the range of human disease. Tissues from rabbits with each form of the disease will be used to characterize the cellular response to infection in the lung and to elucidate the patterns of M. tuberculosis gene expression associated with either active disease or persistent asymptomatic infection (latency). We will also infect rabbits with M. tuberculosis mutants selected to be defective for persistence in murine models and establish whether these mutants retain their persistence defect in the rabbit model. By combining the results obtained from the rabbit studies and the human studies, we will validate our experimental animal model as a mirror of human disease and use the model to identify the molecular correlates of M. tuberculosis latency. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HOST DEFENSE FUNCTIONS OF M. TUBERCULOSIS LIPOGLYCAN Principal Investigator & Institution: Chan, Edward D. Associate Professor; National Jewish Medical & Res Ctr and Research Center Denver, CO 80206 Timing: Fiscal Year 2001; Project Start 15-JUL-2001; Project End 31-MAY-2006 Summary: (Adapted from the Applicant's Abstract): Tuberculosis (TB) is the leading cause of death by an infectious agent. Macrophages play a pivotal role in the control of Mycobacterium tuberculosis through the expression of nitric oxide (NO.) and TNFalpha. NO plays an important mycobactericidal role in murine TB and is increasingly recognized to be important in humans. The overall hypothesis of this proposal is that macrophages, by specific surface receptor(s), recognize mycobacterial cell wall products to initiate iNOS- and TNFa-induction. Hence, the focus of this proposal is to: i) determine the receptor and signaling mechanisms which regulate iNOS and TNFa following exposure to the mycobacterial cell wall component lipoarabinomannan (ManLAM) and ii) to determine the significance of each of these components in an in vitro model of infection. Based on experiments showing that macrophages from the Toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice produced significantly lower levels of NO than TLR4-intact C3H/HeN macrophages in response to IFNg + ManLAM, we hypothesize that ManLAM engages a TLR to induce iNOSNO*/ TNFa expression. Since non-mannose capped LAM (AraLAM) from M. smegmatis induced greater NO about expression than ManLAM, we hypothesize that ii) the exposed arabinose residues on ManLAM or AraLAM are the components of ManLAM that bind to its putative TLR. Since initial studies show that the mitogenactivated protein kinases (MAPKs) and NFkB signaling pathways regulate iNOS and TNFa expression, we hypothesize that the proximal kinase MAP/ERK kinase kinase (MEKK) is a pivotal regulator for ManLAM-induction of iNOS and TNFa. Lastly, because TLRs recognize pathogen-derived molecules and enhance host-defenses, we hypothesize that blocking one or more of the TLRs will enhance the growth of M. tuberculosis and inhibit NO* and TNFa expression. These hypotheses will be addressed by three specific aims: 1. To determine the ManLAM structures that mediate the induction of iNOS-NO*/TNFa and the receptor that mediates these ManLAM effects. 2. To investigate the role of the MAPK and NFkB signaling pathways in ManLAM- and other lipoglycan-induced iNOS-NO* and TNFa. 3. To elucidate the role of the TLRs,
Studies 61
MAPK and NFkB signaling pathways, and ManLAM in controlling the growth of M. tuberculosis in mouse and human macrophages and to correlate effects on growth with NO* and TNFa expression. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HOST DETERMINANTS OF INFECTIOUSNESS IN TUBERCULOSIS Principal Investigator & Institution: Fennelly, Kevin P. Assistant Professor; Medicine; Univ of Med/Dent Nj Newark Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-AUG-2004 Summary: Both experimental and epidemiological data suggest that there is a wide range of infectiousness among patients with tuberculosis. Although much research has focused on the virulence of the organism, little is known of the host determinants of infectiousness. A novel Cough Aerosol Sampling System (CASS) can isolate and quantify (viable airborne Mycobacterium tuberculosis from individual patients with pulmonary tuberculosis. We hypothesize that the production of viable aerosols of M. tuberculosis determined by this method is correlated with infectiousness. Our central hypothesis is that most of the variability of infectiousness in tuberculosis is explained by non-immunological host factors including the duration of antimycobacterial therapy with drugs to which the organism is susceptible, the strength and frequency of coughing, and the physicochemical properties of the sputum. The first specific aim is to correlate viability assessed by the air sampling method used in the CASS and infectiousness using the mouse and guinea pig models. The second aim is to determine if the infectiousness of tuberculosis decreases rapidly with appropriate antimycobacterial therapy. The third aim is to assess whether cough strength and frequency are associated with the quantity of viable aerosol. The fourth aim is to determine if infectiousness is associated with physicochemical properties of sputum. These data may alter clinical practice and public health control measures. Changes in tuberculous aerosol viability associated with drug therapy may provide insight into the basic biology of the mycobacterial cell wall. Similarly, the rapid changes in cough strength and frequency may suggest mechanisms in the pathophysiology of cough. Verifying the correlation between quantitative aerosol cultures and infectiousness using animal models may validate the use of the cough aerosol sampling system at the bedside. Data obtained from patients with tuberculosis may in turn validate the use of animal models of infection. This approach may open a new field of investigation of the host determinants of infectiousness, which could be extended to other respiratory infectious agents in the future. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: HOST RESPONSE TO TB AND AIDS Principal Investigator & Institution: Rom, William N. Chief, Pulmonary and Critical Care Divis; Medicine; New York University School of Medicine 550 1St Ave New York, NY 10016 Timing: Fiscal Year 2001; Project Start 30-SEP-1997; Project End 31-AUG-2002 Summary: The recent tuberculosis (TB) epidemic in the United States has been fueled by several factors, including underfunded public health programs, overcrowding in urban homeless shelters and prisons, continuing immigration to the United States from countries with a high incidence of TB, and the HIV epidemic. The increased susceptibility to tuberculosis infection and disease among HIV-infected patients is directly related to impaired host immunity. In recent years, key components of the host
62 Tuberculosis
response to tuberculosis have been elucidated, and it now seems increasingly clear that a Th1-type T-lymphocyte response is associated with a good outcome in TB patients. In addition, there is increasing evidence that HIV-infected patients have impaired TH1 number and function, with a resultant deficiency of interferon-gamma (IFN-g), a key effector cytokine in host immunity in tuberculosis. This project proposes to study the role of T-helper cells in human tuberculosis, with a specific focus on the role of IFN-g in the pathogenesis of tuberculosis in patients with and without HIV infection. This project will study the role of Th1 cells in general and IFN-g in particular both at the clinical level as well as at the basic level so that we might understand both the regulation of gene expression of this cytokine as well as its interaction with other cytokines and inflammatory effector cells in the lung itself, both in HIV positive and negative patients. The specific aims are to: 1) modulate the Th phenotype in BAL cells and HIV-1 replication in active pulmonary tuberculosis by aerosol treatment with interferon-gamma; 2) investigate mechanisms by which IFN-gamma contributes to host defense against tuberculosis in HIV-positive and HIV-negative persons; and 3) analyze mechanisms of gene regulation effected by IFN-gamma treatment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HOST RESPONSES TO MYCOBACTERIUM INFECTION IN ZEBRAFISH Principal Investigator & Institution: Ramakrishnan, Lalita; Assistant Professor; Microbiology; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2007 Summary: (provided by applicant): Tuberculosis is the leading cause of death from infectious diseases worldwide, with increasing consequences due to the HIV epidemic. The existing vaccine, BCG, has marginal efficacy. Infection and disease are caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. In the face of a complex immune response, the bacteria can persist indefinitely in granulomas, tight aggregates of highly differentiated macrophages and other immune cells. As with most infectious diseases, the impact of the different aspects of host immunity on tuberculous infection and disease are not well understood. To better understand the host responses to tuberculous infections, we will exploit the fact that Mycobacterium marinum, a close relative of M. tuberculosis is a natural pathogen of zebrafish, causing a tuberculosis-like disease. Zebrafish are genetically tractable vertebrates that are used as models of disease and development. We have exploited the optical transparency of the zebrafish embryo to visualize M. marinum infection of embryonic macrophages in real time. The infection parallels adult tuberculosis in many ways. We will use the zebrafish-M, marinum infection model to study the role of host immune genes in infection. We will use whole mount in situ hybridizations to determine which host immune markers thought to be important in tuberculosis, are expressed in the developing embryo. We will determine if their expression is changed during infection. We will inactivate these genes using morpholino technology and determine by real time visualization when and how they act in infection. We will determine their role for the different facets of infection, from early macrophage migration in response to M. marinum to granuloma formation. The ability to visualize infection in real time in zebrafish embryos with functional inactivation of individual host genes will allow us to study the role of the host immune system in unprecedented detail. The proposed experiments will inherently yield new information about the role of various immune genes in zebrafish development. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 63
•
Project Title: HTS FOR PROTEIN SPLICING INHIBITORS AS TB DRUGS Principal Investigator & Institution: Paulus, Henry; Director; Boston Biotechnology Corporation 64 Grove St Watertown, MA 02472 Timing: Fiscal Year 2003; Project Start 01-MAY-2003; Project End 30-APR-2004 Summary: (provided by applicant): One-third of the world' s population (2 billion people) are infected with Mycobacterium tuberculosis and 5-10% of these suffer active tuberculosis, leading to nearly 3 million deaths annually. An alarmingly growing number of patients in developed country are suffering from multidrug-resistant TB, which is essentially refractory to therapy by existing drugs. The mainline antituberculosis drugs need thus be supplemented by additional drugs against new targets, until those, too, succumb to resistance mechanisms. The aim of the proposed research is to discover drugs that inhibit the post-translational processing of the M. tuberculosis DnaB and RecA proteins by protein splicing. The DnaB and the RecA proteins play essential roles in DNA replication and DNA repair, respectively. Because in virulent strains of M. tuberculosis these proteins are synthesized as inactive precursors that need to be activated by protein splicing, the inteins that interrupt these proteins and promote the protein splicing reaction constitute anti-tuberculosis targets. This application focuses on developing in vitro fluorescent assays for DnaB and RecA protein splicing based on the activation of Green Fluorescent Protein (GFP) from inactive fusions with the DnaB or RecA intein, which can be adapted as a high-throughput screening system for protein splicing inhibitors. Inhibitors found with these screens will be for specificity and antibacterial activity using both in vitro and in vivo assay systems. Preference will be given to inhibitors which inhibit splicing of the DnaB as well as the RecA intein so as to interfere both with DNA replication and DNA repair. Because protein splicing occurs only in unicellular organism and in mycobacteria as the only pathogens, such inhibitors should be highly specific antimycobacterial agents with no effect on other bacteria associated with humans nor on the human host. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: HUMAN AND TUBERCULIN COA BIOSYNTHETIC ENZYME STRUCTURES Principal Investigator & Institution: Izard, Tina; St. Jude Children's Research Hospital Memphis, TN 381052794 Timing: Fiscal Year 2003; Project Start 01-SEP-2003; Project End 29-FEB-2008 Summary: (provided by applicant): Coenzyme A (CoA) is the major acyl group carrier for all organisms and is essential for cell survival. In bacteria, the last two reactions of CoA biosynthesis are mediated by the enzymes phosphopantetheine adenylyltransferase (PPAT), which generates dephospho-CoA (dPCoA), and dephospho-CoA kinase (DPCK), which yields CoA. In contrast, in higher eukaryotes a bifunctional enzyme, CoA synthase, catalyzes both of these final reactions. The substrate of PPAT is rate-limiting and the enzyme is feedback regulated by CoA, indicating that PPAT is an excellent target for developing novel antibiotics. We have solved the crystal structures of PPAT from Escherichia coli, which exists as a hexamer, and of PPAT bound to its substrates and to its product dPCoA. These studies identified the residues involved in substrate/product binding and revealed an in-line displacement catalytic mechanism. Finally, our Preliminary Studies of the PPAT:CoA crystal structure revealed an unusual mechanism of feedback inhibition of PPAT by CoA. The structure of the monomeric DPCK enzyme (from Haemophilus influenzae) has recently been solved and suggests an induced-fit reaction mechanism typical of kinases. Collectively, these
64 Tuberculosis
structures form the foundation for the design of PPAT- and DPCK-specific inhibitors, an important issue in the context of treating drug-resistant bacteria, in particular tuberculosis. Like other bacteria, Mycobacterium tuberculosis requires PPAT and DPCK, and although the catalytic mechanisms of tuberculin PPAT and DPCK are likely identical to their homologs in other bacteria, there are significant differences predicted for the structures of these enzymes, especially DPCK. Recombinant M. tuberculosis PPAT and DPCK proteins have been produced and we have crystallized tuberculin PPAT. Experiments in Specific Aims #1 and #2 will determine the native and substratebound crystal structures of M. tuberculosis PPAT and DPCK. In addition, the structures of tuberculin PPAT bound to its product dPCoA, and to its inhibitor CoA will be solved. The design of PPAT- and DPCK-specific inhibitors requires that they not inhibit human CoA synthase. The monomeric nature, predicted structure, and coupled catalytic functions of CoA synthase indicate that regulation of this enzyme is unique, and a novel N-terminal domain may play a regulatory role. Experiments in Aim #3 will determine the native and substrate-bound crystal structures of human CoA synthase. The proposed crystallographic studies are essential for the development of novel inhibitors that selectively target M. tuberculosis PPAT and DPCK. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HUMAN TUBERCULOSIS
T
CELL
RESPONES
TO
MYCOBACTERIUM
Principal Investigator & Institution: Lewinsohn, David M. Assistant Professor; Medicine; Oregon Health & Science University Portland, OR 972393098 Timing: Fiscal Year 2001; Project Start 15-SEP-1998; Project End 31-AUG-2003 Summary: Tuberculosis is the leading single cause of infectious disease mortality worldwide. Immunity to tuberculosis depends upon the cellular immune system. Hence, developing an improved vaccine will require characterization of T cell antigens recognized early during the course of infection, and an understanding of the mechanisms by which T cells recognize and respond to Mtb infected cells. Two novel proteins (TbH9 and 38.1) from Mtb that are potent T cells antigens have recently been cloned and expressed in our laboratory. We have found that these proteins elicit vigorous T cell proliferation and interferon-gamma production from healthy PPD positive individuals, and that one of these proteins is preferentially expressed in cells infected with live Mtb. However, little is known about the response to these proteins in persons who have been recently infected or in those with active tuberculosis. Additionally, several lines of evidence, including the susceptibility of mice rendered deficient in beta2 microglobulin to infection with Mtb, have suggested an important role for CD8+ cytotoxic T lymphocytes (CTL) in immunity to tuberculosis. Using peripheral blood derived dendritic cells infected with Mtb, we have isolated human CD8+ CTL clones to Mtb. This is the first demonstration of human Mtb specific CD8+ T lymphocytes, and further characterization of these cells will be an important part of this proposal. This proposal rests upon two major hypotheses: 1) Protective T cell responses to Mtb arise early in the course of infection and are associated with the production of interferon-gamma (IFN-gamma), and 2) MHC class Ia restricted and non restricted CD8+ CTL that recognize Mtb infected cells are generated during the course of infection. The specific aims are: 1) To characterize human Mtb specific T cell responses in diseased and healthy infected individuals. 2) To characterize human CD8+ T cell responses to Mtb. The research methods for achieving these goals include a collaborative project with Seattle-King County Tuberculosis Clinic to study human T cell responses to recombinant Mtb antigens during the evolution of disease. In addition, the relative
Studies 65
contribution of MHC class Ia restricted and non-restricted CD8+ CTL will be evaluated in healthy PPD positive individuals. The mechanisms by which Mtb gain entry into the MHC class Ia will be explored using selective inhibition of antigen processing. The mechanisms of antigen processing and nature of the antigen or MHC class Ia nonrestricted clones will also be evaluated. Finally, mammalian expression vectors for the proteins TbH9 and Ag 85b will be constructed, and used to elicit antigen specific CD8+ CTL. The proposed studies will allow for greater understanding of T cell responses to tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: HUMAN TUBERCULOSIS
T
CELLS
ANTIGENS
OF
MYCOBACTERIUM
Principal Investigator & Institution: Reed, Steven G. Chief Scientific Officer; Infectious Disease Research Institute 1124 Columbia St, Ste 600 Seattle, WA 98104 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-AUG-2004 Summary: (Adapted from the Applicant's Abstract): This proposal is aimed at identifying and characterizing T cell antigens of M. tuberculosis which may be important vaccine candidates. To identify novel Mtb antigens which may be relevant in protective immunity against tuberculosis we have used a variety of antigen discovery methods which emphasize expression cloning with anti-M. tuberculosis antisera and human T cells. Eight vaccine candidates (Mtb 8.4, Mtb 9.8, Mtb 9.9, Mtb 11, Mtb 19, Mtb 32, Mtb 39, Mtb 40) have so far been selected based on their ability to stimulate PBMC from donors infected with M. tuberculosis. These eight antigen genes will be evaluated as recombinant antigens or as naked DNA, alone or in combination, for immunogenicity and protection against tuberculosis in three animal models including cynomolgus monkeys, mice, and guinea pigs. These models will be used to assess T cell responses as well as protection. In addition, we will emphasize the use of human T cell expression cloning to identify other vaccine candidate antigens. These will be characterized in the in vitro and animal models as above. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNOLOGICAL TUBERCULOSIS
BASIS
OF
SUSCEPTIBILITY
TO
Principal Investigator & Institution: Behar, Samuel M. Assistant Professor; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-AUG-2004 Summary: The long term objectives of this project are to understand the immunological and genetic basis for susceptibility to tuberculosis. As the effectiveness of cellular immunity to Mycobacterium tuberculosis determines whether infection evolves into clinical disease, a better understanding of the immune response and the genes that govern it will facilitate a more rational approach to development and use of vaccines, drugs, and biological therapy. An aerosol route of inoculation will be used to infect intact mice that are either inherently resistant or susceptible to tuberculosis. The local immune response in the lung will be compared to the systemic response in the spleen using a variety of techniques including the quantitation of T cell subsets, intracellular cytokines staining, and RNAse protection assays in order to define immunological parameters that indicate a protective immune response. Other investigations will ascertain which genetic loci are critical for disease resistance. With this insight, these techniques will be applied to interventional models to determine how different
66 Tuberculosis
treatment strategies modify the immune response during primary infection (in the case of vaccines and biological treatments) or after subsequent rechallenge (in the case of chemotherapy). Finally, two different models that approximate the cell mediated defects observed in HIV/AIDS will be examined to determine how the immune response is altered to M. tuberculosis infection in the absence of CD4 plus T cells, and whether therapeutic interventions can beneficially augment the innate and adaptive immune responses under these conditions. The specific aims are as follows: Aim 1. Characterize the immunological differences between mouse strains that are either resistant or susceptible to infection with Mycobacterium tuberculosis after aerosol inoculation. Aim 2. Determine the genetic derangements that correlate with susceptibility and resistance. Aim 3. Identify how the immune response to tuberculosis changes as a consequence of therapeutic interventions in an animal model. Aim 4. Characterize the immune response to tuberculosis in mouse models that simulate the abnormalities of cell mediated immunity typical of HIV/AIDS patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNOLOGICAL DIAGNOSIS OF TUBERCULOSIS Principal Investigator & Institution: Gennaro, Maria L. Associate Member; Public Health Research Institute 225 Warren St Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 30-SEP-1994; Project End 31-MAR-2003 Summary: (Adapted from the applicant's abstract): The increasing worldwide incidence of tuberculosis, which is the leading cause of death from a single infectious disease, and its frequent association with HIV-1 infection have rendered current laboratory diagnostic methods inadequate. Microscopic examination of sputum has low sensitivity, and culture techniques are too slow. DNA sequence amplification by PCR is sufficiently sensitive only in patients with multibacillary pulmonary TB. New diagnostic methods are urgently needed. The PI believes that serology presents an attractive approach. Serodiagnostic tests are simple, rapid, inexpensive and do not require the presence of mycobacteria in the clinical specimens. The present lack of a highly accurate immunoassay for TV is primary due to insufficient knowledge of the humoral immune response to TV and to a limited availability of purified mycobacterial antigens. During the current funding period, the PI developed a panel of eleven recombinant, highly purified antigens of M. tuberculosis and found a remarkable patient-to-patient variation in antigen recognition. The PI has also developed a strategy for multi-antigen-based serodiagnosis of TB. Antigens will be selected for a multi-antigen cocktail for diagnosis of active TB in the general population by identifying antigens that are highly seroactive and that are recognized by sera from patients with paucibacillary and multibacillary pulmonary TB. To minimize false positive results due to immune responses that are not associated with active TB, antigens for the cocktail will be chosen from those that are preferentially recognized by active TB patients rather than asymptomatic tuberculin reactors, BCG vaccines, and individuals with inactive TB. Cocktail development will be pursued by assessing the behavior of selected antigens when combined in solid phase. Peptides displaying immunodominant, M. tuberculosis complex-specific B cell epitopes on highly seroreactive proteins will be utilized to minimize antigen interference or loss of assay specificity that may be observed when antigens are combined. Special emphasis will be placed on development of diagnostic assays for TV in children and in HIV-1infected individuals. Because of the rapid progression from infection to active TB in children, antigens recognized by serum antibodies in pediatric TB may not be the same as those recognized by the adult populations. The PI will select antigens recognized by sera from children with TB to develop serodiagnostic assays specific for pediatric TB.
Studies 67
Another high risk group for rapid disease progression and high TB-associated mortality is the HIV-1-infected population. The PI's studies indicate that, although the overall antibody response is decreased in HIV+ TB, recognition os certain antigens of M. tuberculosis is unaltered in the presence of concomitant HIV-infection. Based on these findings, the PI proposed to develop serodiagnostic assays that meet the special needs of this population. The PI also anticipates that they will be able to identify early serological markers of TB infection in household contacts of patients with active TB. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNOSUPPRESSION IN MYCOBACTERIAL DISEASES Principal Investigator & Institution: Toossi, Zahra J. Associate Professor; Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 01-JAN-1982; Project End 31-JUL-2002 Summary: The investigators' observations to date indicate a central immunomodulatory role for transforming growth factor beta (TGF-beta), a potent immunosuppressive molecule, in human tuberculosis. The sustained support of research at CWRU has delineated mechanisms that may explain the failure of expression of protective immunity during active tuberculosis, and the paradox of skin test anergy in a disease caused by an organism with adjuvant-like properties. M. tuberculosis, its PPD, and its cell wall antigens induce monocytes to production of TGF-beta. TGF-beta is present in tuberculous granulomas and is produced by mononuclear phagocytes of patients with tuberculosis. TGF-beta affects several events in T-cell activation, inhibits the cytokines important in development of a TH 1-like response (IFN-gamma, IL-12), and directly suppresses the TH 1 cytokines (IL-2, IFN-gamma). Further, TGF-beta may promote the expression, or synergize with other suppressive mediators (IL-10), thus amplifying immunosuppressive circuits in situ. A natural inhibitor of TGF-beta, decorin, binds and inactivates TGF-beta and may be clinically useful in diseases such as tuberculosis that are associated with excess TGF-beta activity. Previous observations and the preliminary data support the following hypothesis; the polysaccharide and protein antigens of virulent M. tuberculosis bias the cytokine profile of mononuclear phagocytes towards excess TGF-beta activity. The immunopathogenesis of active tuberculosis involves altered regulation of T-cell activation by TGF-beta which may lead to growth arrest and/or apoptosis of T-cells and an attenuated TH1 response. Down modulation of TGFbeta activity, for example, using the natural inhibitor decorin is expected to enhance such a TH1 response, and, therefore, may be useful as an adjunct to chemotherapy. Specific Aims are: 1. To examine the role of interaction of intact M. tuberculosis through its 30 kD antigen and ManLAM, with fibronectin and mannose receptors on monocytes in expression and activation of TGF-beta. 2. To examine the modulation by TGF-beta of early events in T-cell activation, the growth arrest and/or apoptosis of T-cells stimulated by M. tuberculosis and its products; and the role of TGF-beta in initiating immunosuppressive circuits involving IL-10. 3. To characterize the T-cell dysfunction of patients with active pulmonary tuberculosis, its relationship to TGF-beta and other suppressive cytokines (IL-10) and its reversal by inhibitors of TGF- beta. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: IMPACT OF TUBERCULOSIS OF HIV INFECTION IN UGANDA Principal Investigator & Institution: Whalen, Christopher C. Associate Professor; Epidemiology and Biostatistics; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106
68 Tuberculosis
Timing: Fiscal Year 2001; Project Start 01-JUL-1992; Project End 31-AUG-2004 Summary: (Adapted from Investigator=s Abstract) The World Health Organization estimates that 10 million HIV-infected individuals will die of tuberculosis (TB) during the decade of the 1990's. Although HIV-infected patients with TB respond to effective antituberculous therapy, their prognosis remains poor. Early deaths are often attributable to TB, but later deaths result from complications of HIV infection other than tuberculosis. Recent epidemiologic observations indicate that TB reduces survival and increases the rate of opportunistic infections in HIV-infected patients. Mounting evidence from immunologic and virologic studies supports the concept of copathogenesis in which immune activation triggered by tuberculosis, and mediated by cytokines such as tumor necrosis factor-alpha (TNFa), stimulates viral replication, increases viral load, and accelerates HIV infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INDUCTION OF TH1 IMMUNITY IN TUBERCULOSIS Principal Investigator & Institution: Salgame, Padmini; Associate Professor; Microbiology and Immunology; Temple University 406 Usb, 083-45 Philadelphia, PA 19122 Timing: Fiscal Year 2003; Project Start 01-FEB-2003; Project End 31-JAN-2008 Summary: (provided by applicant): Prevention of infectious diseases through vaccination remains a priority goal for scientists and public health officials. The high risk of developing multidrug-resistant Mycobacterium tuberculosis is a serious limitation to drug therapy, and consequently development of efficacious vaccines is critical for the successful eradication of tuberculosis. Vaccination against tuberculosis would rely on the immune system's ability to generate an appropriate protective response that can then be successfully garnered when the host is challenged with M. tuberculosis. Although we know that Th1 immunity is critical for protection against tuberculosis, yet the cells and mechanisms that are involved in generating a protective Th1 immunity are far from completely understood. In this proposal we combine two complementary approaches, a hypothesis-driven and low-through put approach and a global microarray analysis to ask mechanistic questions regarding how protective Th1 immunity to M. tuberculosis is generated. Therefore the central goal of this proposal is to examine the mechanism for the distinct cytokine response from dendritic cells and macrophages following M. tuberculois infection, and study its impact on the development of M. tuberculosis-reactive CD4 + Th1 cells in vivo: from naive to effector. Overall we expect to obtain from these studies an integrated appreciation of the dynamics of induction of Th1 immunity in tuberculosis. Successful completion of these studies will provide innovative strategies for vaccine development and also provide new modalities of modulating the immune response to shorten chemotherapy and/or overcome drug resistance. The specific aims that will address the goals of the proposal are: Aim 1. Corroborate the theory that signaling from TLRs is sufficient for IL-12 production from dendritic cells, and that macrophages require an additional signal to synergize with TLR signaling for IL-12 production. Aim 2. Examine the molecular basis for differential IL-12 regulation in M.tuberculosis stimulated dendritic cells and macrophages. Aim 3. Test the hypothesis that dendritic cells are critical for Th1 cell priming, and that macrophages are elemental for initiating the granulomatous response. Aim 4. Compare the reprogramming of the dendritic cell and macrophage transcriptome in response to M.tuberculosis alone and in response to M.tuberculosis and interferongamma. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 69
•
Project Title: INHIBITION TUBERCULOSIS
OF
MACROPHAGE
INNATE
IMMUNITY
IN
Principal Investigator & Institution: Kusner, David J. Associate Professor; Internal Medicine; University of Iowa Iowa City, IA 52242 Timing: Fiscal Year 2003; Project Start 15-AUG-2003; Project End 31-JAN-2008 Summary: (provided by applicant): Tuberculosis (TB) is one of the world's greatest health problems, causing approximately 3 million deaths per year. Despite continuing increases in global morbidity and mortality, therapeutic and preventative options for TB remain severely limited. The central feature of TB pathogenesis is infection and intracellular survival of Mycobacterium tuberculosis (Mtb) within human macrophages. Following phagocytosis, Mtb evades the normally potent antimicrobial defenses of innate immunity by inhibiting the maturation of its phagosome to a microbicidal phagolysosome. The molecular mechanisms by which Mtb blocks phagosomal maturation and survives intracellularly are incompletely understood. The long-term goal of this project is to define the molecular mechanisms of tuberculous pathogenesis, to provide a foundation for improved therapies and vaccines. Recently, we demonstrated that live, virulent Mtb, but not killed Mtb, inhibit macrophage Ca2+signaling, and that this defect in host activation directly contributes to inhibition of phagosomal maturation and promotion of the bacilli's intracellular survival. Important gaps in our knowledge include: (1) the mycobacterial determinants responsible for inhibition of macrophage Ca2+-signaling, and (2) the macrophage targets of Mtbinduced inhibition during this critical phase of the host-pathogen interaction. The hypotheses are: (a) sphingosine kinase (SK) is a critical target of macrophage deactivation by live Mtb, and (b) inhibition of SK is causally related to defective Ca2+signaling, inhibition of phagosome maturation, and the survival of Mtb within human macrophages. We will investigate these hypotheses by pursuing the following Specific Aims: (1) Characterize the activation of macrophage SK during phagocytosis of killed Mtb and its role in Ca2+-signal transduction and phagosome maturation. (2) Determine whether inhibition of SK-mediatedCa2+-signaling by live Mtb is causally related to defective phagosome maturation and intracellular viability. In Aims 1 and 2, pharmacological, biochemical, and genetic approaches will be used to modulate specific signaling pathways. (3) Determine the component(s) of Mtb responsible for inhibition of macrophage SK- and Ca2+-mediated activation. A genetic approach of screening a transposon mutant library of Mtb and a biochemical approach of direct assessment of subcellular fractions of Mtb for effects on macrophage SK-andCa2+-mediated signal transduction will be undertaken. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INHIBITION M.TUBERCULOSIS
OF
MYOCOLIC
ACID
BIOSYNTHESIS--
Principal Investigator & Institution: Jacobs, William R. Investigator; Microbiology and Immunology; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2001; Project Start 01-JUL-1998; Project End 30-APR-2003 Summary: Tuberculosis and disseminated Mycobacterium avium cause significant morbidity and death in individuals with Human Immunodeficiency Virus (HIV) infection. Globally, tuberculosis is a leading cause of death for patients infected with HIV. The emergence of M. tuberculosis strains resistant to two or more anti-tuberculosis drugs have compromised control strategies. M. avium Complex (MAC) causes disseminated infection and increased mortality in up to 40 percent of patients with
70 Tuberculosis
AIDS. There is an urgent need to develop new, potent anti-mycobacterial drugs. Mycolic acids are integral and unique parts of the mycobacterial cell wall and the biosynthetic enzymes represent attractive targets for the development of novel drugs. Towards that goal, we are using a multi-disciplinary approach employing mycobacterial genetics, biochemistry, x-ray crystallography, molecular biology, organic chemistry and animal models to develop novel inhibitors. We have discovered a gene, named inhA, which was found to be a primary target to isoniazid (INH) and ethionamide (ETH). Point mutations within the structural gene, or overexpression of inhA confers INH- and ETHresistance in mycobacteria. Biochemical studies reveled that inhA encodes an NADHspecific enoyl-acyl carrier protein (ACP) reductase which prefers long chain fatty acids as substrates, consistent with its role in mycolic acid biosynthesis. The three dimensional structure of resistant and sensitive forms of InhA were determined. We demonstrated that INH is a pro-drug which, upon activation by a catalase-peroxidase forms a covalent adduct to NADH bound on the InhA enzyme. Armed with the knowledge of InhA structure and function, we have generated a series of long-chain fatty acid analog inhibitors that contain a triple bond between C2 and C3: the 2-alkynoic acids (KOAs). The KOAs inhibit the InhA enzyme and have activity against MAC, and against both INH- susceptible and INH-resistant M. tuberculosis. Furthermore, we have screened a combinatorial library of compounds against the InhA enzyme and identified two new classes of compounds with activity against InhA that inhibit M. tuberculosis and MAC growth. In this proposal, we intend to expand upon these successes and continue to elucidate the key targets of mycolic acid biosynthesis with the aim of developing novel drugs against M. tuberculosis and M. avium. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INHIBITION OF TUBERCULAR MYCOTHIOL PATHWAYS Principal Investigator & Institution: Knapp, Spencer A. Chemistry and Chemical Biology; Rutgers the St Univ of Nj New Brunswick Asb Iii New Brunswick, NJ 08901 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): Drug-resistant tuberculosis now threatens a large portion of the earth's population, and the development of new treatments for tuberculosis infection has become a national and international priority. Mycobacterium tuberculosis depends on a low molecular weight thiol, "mycothiol," to maintain a reducing intracellular environment and to remove exogenous electrophilic agents. Disruption of the enzymatic pathways of mycothiol biosynthesis and/or mycothiolbased detoxification could leave M. tuberculosis vulnerable to drugs, oxygen, and other stress factors, and constitutes a new tactic for the control of tuberculosis. The objective of this project is to develop inhibitors of the mycothiol-related enzymes of M. tuberculosis, and eventually to design new and successful treatments for tuberculosis. Three enzymes will be targeted initially: mycothione reductase, mycothiol S-conjugate amidase, and inosityl GIcNAc deacetylase, although others, including a cysteine ligase and a cysteine transacetylase, could be added. This work will be guided by enzymatic assays conducted by collaborators using existing screens, and by preliminary results that already indicate that substantial structural simplification in designing mycothiol analogues is possible. First, the minimum substrate requirements for the M. tuberculosis enzymes will be defined. Then, inhibitors based on these minimum structures will be synthesized and evaluated. New methods for the synthesis of mycothiol-analogous compounds will be developed, and new ideas for enzyme inhibitor design will be explored. The most active compounds will be taken as leads for
Studies 71
further analogue development and for increasing the bioavailability, and metabolic stability in M. tuberculosis itself.
potency,
specificity,
Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INHIBITORS OF SULFATE ASSIMILATION AS NEW THERAPY FOR TB Principal Investigator & Institution: Hemmerich, Stefan; Thios Biotechnology, Inc. 747 52Nd St Oakland, CA 94609 Timing: Fiscal Year 2003; Project Start 15-MAY-2003; Project End 14-NOV-2003 Summary: (provided by applicant): Mycobacterium tuberculosis (M. tuberculosis) is a major pathogen of global importance. Despite the availability of chemotherapy and the Bacille Calmette-Guerin vaccine, M. tuberculosis continues to claim more lives than any other single infectious agent. Recent years have seen increased incidence of tuberculosis in both developing and industrialized countries, the widespread emergence of drugresistant strains and a deadly synergy with the human immunodeficiency virus. The limitation of current therapy originates from the widespread occurrence of antibiotic resistant strains and also the requirement for prolonged and uninterrupted administration of antibiotics. Thus, new classes of antibiotics are desperately needed. Sulfur-containing metabolites, including the essential amino acids cysteine and methionine, and the virulence factors mycothiol and sulfolipid-1, are crucial for the infectivity of M. tuberculosis. Their biosynthesis involves the sulfate assimilation pathway, which has no counterpart in humans. Since the enzymes in this pathway comprise a central hub of metabolism and are unique to bacteria, we propose that these enzymes are prime targets for anti-mycobacterial therapy. This proposal focuses on 5'adenosinephosphosulfate (APS) reductase (CysH) as a target for small molecule drug development. In preliminary work, we have 1) confirmed the APS reductase activity of M. tuberculosis CysH using genetic complementation in E. coli, and 2) demonstrated that genetic deletion of CysH from a pathogenic strain of M. tuberculosis renders the mutant unable to establish infection in a mouse model of tuberculosis. In specific aim 1 of this proposal we shall screen a compound library for inhibitors of M. tuberculosis sulfate assimilation using an E. coli complementation assay. In specific aim 2 we shall set up cell-free homogenous assays for recombinant M. tuberculosis CysH, examine our leads from the primary screen for their ability to directly inhibit this enzyme, and optimize these leads through medicinal chemistry. In specific aim 3 the best compounds will be examined using in vitro susceptibility studies of clinical isolates of M. tuberculosis. In phase II of this study, the inhibitor leads from our feasibility study will be examined for bacteriostatic and bacteriocidic efficacy in mouse models of tuberculosis. The long-term objective of this study is to develop novel antibiotics for more effective treatment of human tuberculosis and other mycobacterial diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: INNOVATIVE APPROACHES FOR TUBERCULOSIS CONTROL IN BRAZIL Principal Investigator & Institution: Lapa E Silva, Jose R.; Federal University of Rio De Janeiro Cidade Universitaria Rio De Janeiro, Timing: Fiscal Year 2002; Project Start 22-SEP-2002; Project End 21-SEP-2003 Summary: (provided by applicant): Tuberculosis is still an important cause of human suffering and economic loss in Brazil and beyond. A concerted effort to address all the questions related to TB control is needed, involving Universities and Health
72 Tuberculosis
Departments. The Millenium TB Network was organized in 2001 with the goal to generate new knowledge about the disease; to train highly qualified professionals with a critical view of the problem and to transfer the new information to the health system. For many years, leading American academic institutions working in Brazil recognized the importance of this initiative. The major goal of this proposal is to develop a competitive application for a Comprehensive ICOHRTA-AIDS/TB Cooperative Agreement linking Federal University of Rio de Janeiro and other collaborating sites in Brazil and Johns Hopkins University, Cornell University and UC Berkeley as collaborating sites in the U.S. The specific aims are 1) To establish a multilateral collaborative training program in tuberculosis control with investigators and physicians from Brazil and Johns Hopkins, Cornell, and Berkeley Universities.2) To establish a network system between the different institutions and individuals participating in the program in Brazil and USA in order to promote frequent consultation between the partners to define the type of didactic and laboratory training in health sciences pertinent to tuberculosis control. 3) To identify the needs in infra-structure, human rights protection, administration necessary to the establishment of a long-term term collaborative program on TB and AIDS/TB research. The major task of this new consortium will be to identify possible interactions between these partners, in order to guarantee efficiency in the actions. The goal will be to link separate projects, funded by several sources, into one multicenter and organized research and training program to address the TB problem at the national and, in the future, continental levels. During the planning year, projects that share similar objectives will be linked, needs at the collaborating sites, identified, and a common training and research agenda in TB will be developed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INTERNET CONNECTION FOR 5 TUBERCULOSIS INSTITUTIONS Principal Investigator & Institution: Miller, Paula F. Assistant Regional Director; Project Hope 255 Carter Hall Ln Millwood, VA 22646 Timing: Fiscal Year 2002; Project Start 15-JAN-2002; Project End 31-AUG-2003 Summary: (provided by applicant): The goal of the Five Country Internet Access Project is to provide TB specialists, medical faculty, and medical students in the Central Asia Republics (CAR) - Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan with access to up-to-date medical information and resources, particularly those relating to Tuberculosis diagnosis and treatment that are readily available on the Internet. The specific objects of the projects are to: 1) Purchase equipment and service for connectivity for 6 library/learning centers; 2) Train 25 project personnel and 4500 users for the Internet; 3) Provide extended Internet access to over 10,000 medical professionals; 4) Establish a Tuberculosis materials database in each country through downloading, cataloging and initiating distribution of lists of materials available from library/learning centers in each country via e-mail; (5) Provide an Internet search center for materials on Tuberculosis for TB specialists, primary health care professionals, nurses and others located in 30 remote e-mail- connected TB pilot sites of countries of the CAR through submission of requests to the central library/learning center. The World Health Organization has identified the lack of training and updated knowledge of TB among Primary Health Care staff as one of several constraints hampering TB control in the Central Asia Republics. This Internet Access Project will build upon current TB diagnosis and treatment efforts currently being implemented by Project HOPE, the requesting organization, and strengthen capacity by improving exchange of information. The project will be initiated at the request and collaboration of the host
Studies 73
countries and is consistent with Project HOPE's vision and programming in the region. The projected plan for getting Internet access for the five TB institutes will begin with a consultant Internet specialist working with local Internet specialists and providing training of trainers specific to e-mail, the tuberculosis websites and provide the teaching methodologies to be Used in training at the TB institute sites. The consultant will assist with the preparation of an Internet Procedure Manual that outlines the use of Internet in a step-by-step format. Following the Training of Trainers (TOT), a two-page pamphlet in the local language will be written and printed by the local Internet specialist and may be given to each professional coming to the library/learning center to orient them how to use the Internet. The libraries/learning centers will be assisted by the Project HOPE office staff in each country to organize the connection of the phone line and modems and provide the training schedule for the staff. The local Internet specialist will go to each country to train the staff in the library learning center using the local language. Additionally, each site will be provided 750 pamphlets outlining procedure to use with medical staff requesting access to the Internet. Each library/learning center will keep a database of medical personnel accessing the Internet and report these statistics to the local HOPE office quarterly. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INTRACELLULAR MACROPHAGES
INFECTION
OF
HUMAN
ALVEOLAR
Principal Investigator & Institution: Park, David R. Medicine; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2001; Project Start 20-JUL-1997; Project End 30-JUN-2003 Summary: (Adapted from applicant's abstract) Legionella pneumophila and Mycobacterium tuberculosis, the etiologic agents of Legionnaires' disease and tuberculosis respectively, are facultative intra-cellular pathogens which parasitize human alveolar macrophages and blood monocytes recruited to the site of lung infection. An effective host response to these infections depends on cell-mediated immunity, and cytokine networks modulate this response. The overall hypothesis driving this proposal is that L. pneumophia and M. tuberculosis subvert the human alveolar macrophage cytokine response to facilitate their own survival by inducing permissive host cytokines which disarm the protective antimicrobial mechanisms of the phagocyte. The specific aims are: 1) to define the role of cytokine activation of human alveolar macrophages in determining the tolerance or elimination of intracellular infection by L. pneumophila and M. tuberculosis; 2) to determine whether apoptosis of infected alveolar macrophages diminishes the viability of intra-cellular L. pneumophila and M. tuberculosis; 3) to develop and use a virulent L. pneumophila strain transduced with the A victoria green fluorescent protein gene to co-localize intracellular bacteria and specific components of the human alveolar macrophage host response in infected cells. The experimental approach to the specific aims will use an established in vitro model in which human alveolar macrophages are infected with L. pneumophila or M. tuberculosis. Effects of cytokines on intracellular growth will be measured using selected combinations of recombinant cytokines and blocking antibodies. Specific interactions signaling cytokine induction by live bacteria and cell wall glycolipids will be assessed by blocking complement receptors, CD14, and LBP. Macrophage apoptosis will be assessed by cellular morphology, Annexin V binding, and by DNA fragmentation assays, and associated changes in bacterial viability will be quantitated. Individual cell response by infected cells will be assessed by flow cytometry and fluorescent microscopy co-localization of green fluorescent protein expressing bacterial
74 Tuberculosis
strains with selected markers of host defense. These studies will provide important insights into the human host responses to L. pneumophila and M. tuberculosis infections. A thorough understanding of these responses will be necessary for the development of rational immunotherapeutic approaches to the treatment of Legionnaires' disease and tuberculosis, which will be increasingly important for the treatment of immunocompromised patients and infections with drug-resistant strains. (End of Abstract) Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IRON TUBERCULOSIS
REGULATION
&
VIRULENCE--MYCOBACTERIUM
Principal Investigator & Institution: Manabe, Yukari C. Medicine; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2004 Summary: The candidate is completing a three year infectious disease fellowship and has devoted the last 2 years to basic science research in tuberculosis. In particular, the candidate is interested in studying gene regulation in tuberculosis to elucidate virulence genes and protein antigens which may make important drug and vaccine targets. Through the research conducted under this grant, the candidate would like to establish herself as a junior faculty member at an academic medical institution and continue to pursue tuberculosis disease pathogenesis using molecular biologic techniques. In collaboration with Dr. John Murphy, a leader in iron-dependent regulons in diphtheria, and Dr. Richard Silver, who has developed a reproducible macrophage model of tuberculosis infection, the candidate will have the collaborative resources necessary for the successful completion of this project. The sponsor, Dr. William Bishai, has wellestablished expertise in mycobacterial gene regulation and tuberculosis disease pathogenesis. To complement her research, the candidate will attend approximately 3 hours per week of infectious disease and TB-related conferences. She also plans to take courses in molecular biology, tuberculosis, vaccine development, and immunology in the School of Medicine and the School of Public Health and Hygiene. Fueled by antibiotic resistance and HIV infection, the global burden of tuberculosis infection is staggering. The elucidation of virulence mechanisms and protein antigens for new antimicrobials and vaccines has moved to the forefront. Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of multiple important infectious disease pathogens. Using the corynebacterial, iron- dependent DtxR regulon as a model, the candidate will study the mycobacterial homologue, ideR and its regulon. Preliminary results have shown that an iron-independent, corynebacterial DtxR mutant hyperrepressor attenuates Mycobacterium tuberculosis virulence in a murine model. A similar mutant of the mycobacterial ideR has been constructed and will be tested in vitro using a gel-shift assay, and in vivo in a murine and macrophage models. Taking advantage of the DtxR/IdeR "iron box", a palindromic DNA binding sequence present in all DtxR-regulated genes, other genes of interest have been identified in silico and will be examined. Elucidation of this iron-dependent IdeR regulon may lead to the identification of virulence determinants, and novel antigens for vaccines and therapeutics. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 75
•
Project Title: RESISTANCE
KATG
PEROXIDASE
IN
ISONIAZID
ACTIVATION
AND
Principal Investigator & Institution: Owen, Whyte G.; Mayo Clinic Rochester 200 1St St Sw Rochester, MN 55905 Timing: Fiscal Year 2002; Project Start 01-MAY-2002; Project End 30-APR-2006 Summary: (Adapted from the Applicant's Abstract): Tuberculosis is one of the leading causes of death worldwide from an infectious disease. Isoniazid is a front line antibiotic used in the treatment of tuberculosis. The Mycobacterium tuberculosis heme catalase/peroxidase KatG is responsible for activating isoniazid to a reactive biocidal species. Antibiotic resistance to isoniazid is a growing concern and can occur by deletions or point mutations in the katG gene. One of these, a serine-to-threonine substitution at position 315, KatG(S315T) is found in about 50 percent of clinical isolates resistant to isoniazid. Our research seeks to understand the mechanism of isoniazid activation and molecular basis for drug resistance caused by the S315T and other point mutations in KatG using biochemical, genetic, and spectroscopic techniques. KatG will be purified from recombinant and native sources and the activities and spectroscopic properties compared. Besides a catalase/peroxidase activity, KatG can also catalyze several other reactions including Mn2+ peroxidase, P450-like oxygenase, and peroxynitritase activities. Which of these or possibly other redox reactions are responsible for isoniazid oxidation and activation will be tested using wild-type and mutant KatG proteins to investigate the mechanism of activation. Genetic methods investigating the role of superoxide in isoniazid activation are proposed. Optical, EPR, NMR, and resonance Raman spectroscopies are revealing subtle differences in the heme active site of wild-type KatG and KatG(S315T). These spectroscopic techniques will be applied to various forms of wild-type and mutant enzymes in order to elucidate the molecular basis for reactivity toward isoniazid and the reduced rate of isoniazid oxidation by KatG(S315T). NMR relaxation measurements and x-ray crystallography will be used to map the isoniazid binding site on both enzymes to determine whether subtle differences in distance and/or orientation are responsible for the reduced turnover of drug by the mutant enzyme. Steady-state and rapid kinetic techniques will follow ligand-binding rates to the heme iron and the formation and decay of reactive intermediates in the catalytic cycle for both wild-type KatG and KatG(S3 1ST) to determine whether the S3 1ST mutation affects one of the steps in the catalytic cycle. EPR spectroscopy and spin-traps are being used to trap reactive intermediates. The stable products formed in this reaction are being characterized by mass spectrometry to reveal chemical information about the nature of intermediates in the reaction of isoniazid oxidation by KatG. Site directed mutagenesis of residues also implicated in isoniazid resistance other than S3 15 will be generated and the effects on enzyme activities and spectroscopic properties examined to determine structural information about the mutant enzymes. Analogs of isoniazid will be used as additional biochemical and spectroscopic probes of the reaction mechanism and molecular basis for drug resistance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: LOSS OF OXYR IN M TUBERCULOSIS Principal Investigator & Institution: Deretic, Vojo P. Professor; Molecular Genetics & Microbiol; University of New Mexico Albuquerque Controller's Office Albuquerque, NM 87131 Timing: Fiscal Year 2001; Project Start 01-APR-1998; Project End 31-MAR-2003
76 Tuberculosis
Summary: (Adapted from the applicant's abstract): M. tuberculosis is a natural mutant in the putative central regulator of oxidative stress response, oxyR. All M. tuberculosis strains investigated to date have nearly identical lesions in oxyR, and the elimination of oxyR function may have coincided with or directly participated in the evolution of M. tuberculosis into the potent contemporary human pathogen. The loss of oxyR has a dual significance for M. tuberculosis pathogenesis and treatment of tuberculosis. First, this surprising phenomenon reflects the complexity of host-parasite interactions and its continuing analyses are expected to reveal strategies used by M. tuberculosis for survival in the host. Second, the loss of oxyR and the associated dysfunction of oxidative stress responses most likely contribute to the exceptional sensitivity of M. tuberculosis to isonicotinic acid hydrazide (INH). The goals of this proposal are: (i) to delineate the effects of the loss of oxyR on M. tuberculosis response to reactive oxygen and nitrogen intermediates. This will be accomplished by identifying genes and functions affected by the loss of oxyR, analyzing alternative modes of their regulation, and characterizing M. tuberculosis response to reactive oxygen and nitrogen; (ii) to determine whether and how oxyR inactivation and the resulting dysfunction of oxidative stress responses contribute to the exceptional sensitivity of M. tuberculosis to INH. This will be examined by complementation of the defect in M. tuberculosis using a functional mycobacterial oxyR, by inactivation of oxyR in model mycobacteria, and by determination of the effects that such modifications have on INH sensitivity in vitro and in vivo; (iii) to investigate the role of the loss of oxyR in M. tuberculosis virulence and pathogenesis using mouse bone marrow derived macrophages and human monocytes, and models of tuberculosis in mice and guinea pigs. In addition to conventional analyses of M. tuberculosis survival and histopathology, GFP-based technologies developed in the PI's laboratory will be employed to monitor mycobacterial localization, and potential tissue- and infection stage-specific expression of oxidative stress response genes (e.g. ahpC). These experiments will delineate the effects that the loss of oxyR has on host-pathogen interactions in tuberculosis. The proposed studies are expected to improve understanding of the elusive virulence attributes of M. tuberculosis, and may help explain, at least in part, its high sensitivity to the front-line antituberculosis agent INH. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M TB SURVIVAL REGULATORY GENES Principal Investigator & Institution: Bishai, William R. Associate Professor; Molecular Microbiol and Immun; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-APR-1997; Project End 31-MAR-2003 Summary: (Adapted from the applicant's abstract): This is a revised application in which the PI proposes to analyze an alternate sigma factor of Mycobacterium tuberculosis which may be involved in the intracellular survival of the tubercle bacilli. The PI points out that the ability of M. tuberculosis to adapt to a variety of environmental conditions is an important feature of its pathogenicity. Many bacteria use alternate sigma factors for RNA polymerase to upregulate response genes specific for a needed adaptation. Using degenerate PCR, the PI has identified an alternate sigma factor from M. tuberculosis which he has called SigF. M. tuberculosis SigF is homologous to the SigF and SigB proteins of Bacillus subtilis, which are involved in regulation of sporulation and general stress response gene expression, respectively. Not only does M. tuberculosis SigF have homology with B. subtilis SigF and SigB, but it is preceded by an antisigma and a possible anti-antisigma homologue, just as are the B. subtilis sigma
Studies 77
factors. In vitro (i.e., broth-grown cultures), the expression of the M. tuberculosis SigF is upregulated by stress conditions and entry into stationary phase. In vivo, its expression increases 10-fold upon entry into murine macrophages. Hence, the PI hypothesizes that the M. tuberculosis SigF may govern an intracellular survival regulon. In this application, the PI proposes to determine (i) the in vitro and in vivo phenotypes of an M. tuberculosis sigF mutant, (ii) how SigF is regulated (i.e., is an antisigma partner switching mechanism involved?), and (iii) the identities of SigF-dependent genes. The PI anticipates that understanding how M. tuberculosis adapts for intracellular survival may lead to novel drug and vaccine targets for tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M DESIGN/EVALUATION
TUBERCULOSIS
CTL
EPITOPES:
VACCINE
Principal Investigator & Institution: Clayberger, Carol A. Associate Professor; Cardiovascular Surgery; Stanford University Stanford, CA 94305 Timing: Fiscal Year 2003; Project Start 15-FEB-2003; Project End 31-JAN-2008 Summary: (provided by applicant): Tuberculosis (TB) is the leading cause of death from a single infectious agent (Mycobacterium tuberculosis (Mtb)), causing approximately 3,000,000 deaths each year. Although TB can be effectively treated with a combination of antibiotics, drug resistant Mtb strains have recently emerged which are classified as Category C biological agents. Thus, it is widely felt that the long term control of TB will require the development of a more effective vaccine. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the current anti-TB vaccine, is quite variable in its ability to protect against TB but is effective against tuberculosis meningitis, suggesting that for the foreseeable future, new TB vaccines will be given as an adjuvant or boost to BCG. Thus, understanding the immune response to both Mtb and BCG is critical for the development of an improved vaccine for TB. An increasing body of evidence indicates that both CD4+ and CD8+ T lymphocytes are critical to a protective immune response against Mtb. However, little is known about the antigens targeted by protective immune responses against Mtb in humans. Such information is required for the rational development and clinical evaluation of new, more effective TB vaccines. We propose here to characterize the human CD4+ and CD8+ T cell response to a panel of Mtb antigens in order to identify correlates with protective immunity. Antigens to be tested include proteins as well as peptide epitopes restricted by HLA-A2, an allele expressed by approximately 50% of the population. Some of these proteins and epitopes were selected from a subset of Mtb genes that are highly expressed under specified conditions and whose products are predicted to localize to the extracellular milieu, while the remainder represent previously identified HLA-A2 restricted epitopes. The T cell response to these antigens will be evaluated in peripheral blood leukocytes from three different groups of BCG immune and/or Mtb infected individuals: i. Neonates immunized a birth with one of 4 strains of BCG; ii. Individuals infected with Mtb but who do not progress to disease (latent TB infected individuals); and iii. PPD+ TB patients and PPD- "anergic" TB patients. Some of these peptide epitopes will be used to develop epitope oligomers which will be used to analyze anti-Mtb responses In vitro and in vivo. Lastly, the localization and function of Mtb peptide specific memory T cells will be studied in vivo. Correlates of protective immunity can be used to identify or prioritize protective antigens and vaccine candidates, to optimize vaccine dosing, schedules, adjuvants, etc., and to provide early evidence of efficacy. For TB, which takes years to decades to develop after infection with Mtb, immune correlates with protection are an attractive, and perhaps essential, supplement to efficacy trials.
78 Tuberculosis
Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M TUBERCULOSIS EVASION OF THE INNATE IMMUNE RESPONSE Principal Investigator & Institution: Fortune, Sarah M. Immunology/Infections Diseases; Harvard University (Sch of Public Hlth) Public Health Campus Boston, MA 02460 Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-MAY-2006 Summary: (provided by applicant): This proposal describes a five year, twopart career development program in the study of Mycobacterium tuberculosis. This program will be carried out in the laboratory of Dr. Barry Bloom with the additional guidance of Dr. Eric Rubin, both in the Division of Immunology and Infectious Diseases at the Harvard School of Public Health. The candidate is trained in Infectious Diseases and has the long term goal of establishing an independent laboratory studying M. tuberculosis pathogenesis with a particular focus on mycobacterial interaction with the host immune response and vaccine development. The didactic component of this program involves courses in molecular biology and immunology through Harvard University. The research component of this program involves exploration of two putative mechanisms of immune evasion by M. tuberculosis, inhibition of IFN-gamma signaling and inhibition of inducible IL-12 production in infected macrophages. The specific aims of the research proposed will be: 1) to determine whether Toll-like receptor 2 (TLR-2) activation inhibits IFN-gamma signaling or inducible IL-12 production; 2) to identify the M. tuberculosis genes required for inhibition of IFN-gamma and IL-12 signaling by screening a transposon mutagenized library of M. tuberculosis; and 3) to characterize the genes identified in these screens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: M TUBERCULOSIS GENES INDUCED DURING MOUSE INFECTION Principal Investigator & Institution: Woolwine, Samuel C. International Health; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-JUL-2001 Summary: Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is responsible for the deaths of millions of individuals each year. One obstacle in the crusade against this pathogen is the ability of the organism to persist in an asymptomatic state, referred to as latency. Another rapidly increasing trend in tuberculosis is the emergence of multiply drug resistant strains of M. tuberculosis. In order to better understand the biology of M. tuberculosis during active or latent disease, and to identify new potential drug targets and candidate vaccine strains, a powerful technique known as differential fluorescence induction will be used to identify M. tuberculosis genes induced specifically during host infection. In this approach, a plasmid library of M. tuberculosis genomic DNA (gDNA) fragments fused to a promoterless gfp, encoding the green fluorescent protein, will be transferred into M. tuberculosis. The resulting clones will be used to infect Balb/c mice. At specific time points mice will be euthanized, and specific organs will be harvested and homogenized. The resulting homogenates will be analyzed in a fluorescence-activated cell sorter (FACS). In this way, fluorescent bacilli will be identified and collected.These bacilli harbor plasmids in which the M. tuberculosis gDNA fragments upstream of gfp contain promoters that are expressed in vivo. The collected bacilli will be cultured in vitro and then re-analyzed by FACS. During this round of analysis, those bacilli that exhibit low
Studies 79
levels of fluorescence will be collected and used to infect a second set of mice. By successive rounds of in vivo and in vitro propagation, a set of M. tuberculosis promoters will be identified that are specifically induced in the animal host. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M TUBERCULOSIS PHOP LIKE GENE IN MACROPHAGES Principal Investigator & Institution: Buchmeier, Nancy A. Associate Research Microbiologist; Pathology; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, CA 92093 Timing: Fiscal Year 2001; Project Start 01-APR-1998; Project End 31-MAR-2003 Summary: (Adapted from the applicant's abstract): The regulated expression of specific Mtb genes is likely required for the establishment of tuberculosis infections. One family of gene regulators present in M. tuberculosis is the two component regulators. Two component regulatory systems sense and respond to environmental stimuli. tcrA (for two component regulator) is a two component regulatory gene found in M.tb. tcrA is present in M. tuberculosis isolates and in M. bovis isolates, but is absent in mycobacterial strains which belong to the four nontuberculosis groups of mycobacteria. tcrA has a regulatory effect on Mtb protein expression as evidenced by multiple changes in the synthesis of Mtb proteins in a tcrA mutant compared to Mtb Erdman. The objective of this proposal is to evaluate the role of the tcrA regulatory system in the production of tuberculosis infections. A tcrA mutant of Mtb Erdman will be used to assess the importance of the tcrA gene in Mtb growth in cultured macrophages. The sensitivity of the tcrA mutant to toxic macrophage compounds will be evaluated and the morphologic appearance of macrophages infected with the tcrA mutant analyzed. Mice will be infected with the tcrA mutant using aerosol inoculation and the resulting infection compared with Mtb Erdman infections. tcrA regulated genes will be cloned, mutants in each gene will be constructed and each mutant evaluated for growth in cultured macrophages. The environmental conditions which activate the tcrA regulon will also be identified. These studies will be valuable in identifying MTb genes which contribute to Mtb pathogenicity and which may serve as targets for anti-tuberculosis treatment either in vaccines or for anti-mycobacterial drugs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: M TUBERCULOSIS SURVIVAL AND GROWTH IN VIVO Principal Investigator & Institution: Smith, Issar; Member; Public Health Research Institute 225 Warren St Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 01-MAR-1999; Project End 29-FEB-2004 Summary: (Adapted from the Applicant's Abstract): Despite the use of a generally safe vaccine for 70 years and effective antibiotics for 40 years, tuberculosis still kills approximately 3 million people annually. To develop new methods of diagnosis and therapy, it is necessary to find new targets, i.e., genes and proteins, in the causative organism, Mycobacterium tuberculosis. An understanding of mycobacterial gene expression is expected to provide much assistance in achieving this goal. In recent years, the investigator's laboratory has studied general and specific mechanisms of gene regulation, i.e., the structure and function of the basic mycobacterial transcriptional apparatus and the control of iron acquisition. Much has been learned regarding these regulatory systems in pathogenic and non-pathogenic mycobacteria, both biochemically and in vitro. Information gained in these initial experiments is now being used to study the in vivo regulation of M. tuberculosis genes and their functions during the infectious
80 Tuberculosis
process. The aims of this new grant proposal are: 1) identification of M. tuberculosis genes that are specifically induced, in vivo. An IVET promoter trap system has been developed. Several genes have been isolated thus far, and the search for new candidate genes will be continued; 2) studying the expression of M. tuberculosis genes in vivo. A sensitive and accurate real time RT-PCR method has been developed to quantitate the in vivo expression of M. tuberculosis genes that have been and will be identified in the experiments described in first aim. In addition, they can also measure the expression of other M. tuberculosis genes that they believe may be induced (or repressed) upon infection of macrophages. Several genes showing differential regulation in macrophages have already been identified and they will study the mechanisms by which they are induced or repressed after infection. Since some of these genes are iron regulated, they will also manipulate iron levels in macrophages to measure the affects on their expression; 3) elucidating the role of in vivo-induced M. tuberculosis genes in virulence. In the course of these studies, they expect to identify many genes that are induced in vivo. To determine their role in virulence, they will inactivate these genes in M. tuberculosis and will measure the survival and growth of the mutants in various macrophages and in mice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M. TUBERCULOSIS CELL WALL BIOGENESIS; NEW DRUGS; TBHIV Principal Investigator & Institution: Brennan, Patrick J. Professor; Microbiology, Immunology & Pathology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2003; Project Start 15-JUL-2003; Project End 30-JUN-2006 Summary: (provided by applicant): The incidence of HIV-associated tuberculosis has been increasing worldwide since the beginning of the AIDS epidemic, and is expected to rise even further in the future, especially in developing countries. The accelerating and amplifying influence of HIV infection is contributing to the increasing incidence of disease caused by multidrug-resistant strains of Mycobacterium tuberculosis. Development of new drugs against tuberculosis is thus important for control of both of the infections. Mycobacterial cell wall is an attractive target for rational drug design against tuberculosis, due to the fact that it forms a protective, almost impermeable barrier, on the surface of mycobacteria. Some of the most effective drugs currently used for the treatment of TB affect components of its backbone - mycolylarabinogalactanpeptidoglycan (mAGP) complex. Our long-term goal is to identify processes and enzymes involved in the mAGP assembly. Possible AG biosynthetic gene cluster has been recently identified in the genome of M. tuberculosis and thus the specific aims of this grant proposal are: 1. Identify the genes involved in mycobacterial galactan biosynthesis within AG biosynthetic cluster and determine their biological functions via cloning, overexpression and subsequent biochemical characterization. 2. Establish the function of the putative ABC transporter within the AG biosynthetic cluster by way of preparation and phenotypic characterization of the mutants/conditional mutants. The approach will help define one of the more complex pathways in microbial biochemistry and reveal reactions that should be exploitable for drug development. The research will be primarily carried out at Comenius University, Faculty of Natural Sciences in Bratislava, Slovakia in collaboration with Katarina Mikusova, as an extension of the NIH grant A1-18357. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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•
Project Title: M. TUBERCULOSIS SURVIVAL IN MACROPHAGES Principal Investigator & Institution: Friedman, Richard L. Professor; Microbiology and Immunology; University of Arizona P O Box 3308 Tucson, AZ 857223308 Timing: Fiscal Year 2001; Project Start 01-APR-2001; Project End 31-MAR-2004 Summary: (provided by the applicant): Central to the disease process in tuberculosis are the interactions of the bacilli with host macrophages. The infection of macrophages by Mycobacterium tuberculosis can be divided into four steps: adherence, entry, intracellular survival, and multiplication. This proposal will concentrate on one of these steps, survival. The role virulence factors of M. tuberculosis play in these complex interactions is virtually unknown. The aim of the proposed research is to identify, clone, and characterize genes and their protein products of M. tuberculosis which are required for intracellular survival within macrophages. Potential virulence factor genes of M. tuberculosis will be cloned by first constructing a recombinant library and incorporating it into the non-pathogenic Mycobacterium smegmatis. This rapidly growing mycobacterium is internalized and killed by macrophages. Clones with enhanced survival in macrophages will be identified and examined for the presence of M tuberculosis genes involved in survival. Using this system we have already isolated a M. tuberculosis gene, named eis (enhanced intracellular survival gene), which does enhance intracellular survival of M. smegmatis within macrophages. The primary focus of this grant application is the further characterization of the eis gene and its protein product Eis. The specific aims are: 1.Effect of eis Gene Inactivation on Survival and Multiplication of M tuberculosis in Macrophages and Mice. Mutations will be constructed in eis and introduced into the chromosome of both avirulent and virulent M tuberculosis (H37Ra and H37Rv) by allelic exchange. The ability of the eis knockout mutants to survive and replicate in the U-937 macrophage survival assay and in human mononuclear phagocytes will be tested and compared to the parental strain. Additionally, the ability of the eis mutants to persist and replicate in vivo in a mouse intravenous infection model will also be evaluated. 2.Mechanism(s) Whereby eis Enhances Survival and Multiplication of M tuberculosis in Unactivated and Interferongamma-Activated Macrophages. To learn how eis may enhance intracellular survival of mycobacteria in macrophages, survival in interferon-gamma-activated U-937 cells and human monocytes will be evaluated. The ability of M. smegmatis with and without eis, as well as wild-type M. tuberculosis and eis knock-out mutants, constructed in Specific Aim No.1, to survive/multiply in both unactivated and interferon-gamma-activated U937 cells and human monocytes will be determined. Studies will also be done to determine what role Eis may play in the ability of M. tuberculosis to resist known killing mechanisms operating in macrophages. 3.Properties of the Eis Protein and their Relationship to its Survival-Increasing Action. These studies will include: (I) intracellular localization of Eis in M. tuberculosis and M. smegmatis, (2) purification of the Eis protein, (3) screening of sera from tuberculosis patients for presence of antibody to Eis, and (4) measurement of eis gene expression in vitro and within macrophages using integrative reporter gene vectors. 4.Identification of Non-eis Survival Genes in a New M. tuberculosis DNA Library. In initial studies, eis-containing clones were the predominate clones isolated after the sixth passage in the U-937 macrophage survival assay. Such eis-containing clones are preferentially selected and appear to outcompete/dominate other M. tuberculosis genes which may also play a role in intracellular survival. Thus, in order to identify these other potential genes, a new M. tuberculosis plasmid library will be constructed with larger (10-12 kb) DNA inserts of genomic DNA from an H37Rv eis knockout mutant. This eis knockout library will then be screened for survival in the U-937 macrophage survival assay. Clones with enhanced survival will be isolated and further characterized.
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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: M.TUBERCULOSIS GENES REGULATING PERSISTENT INFECTION Principal Investigator & Institution: Zahrt, Thomas C,. Assistant Professor; Microbiol & Molecular Genetics; Medical College of Wisconsin Po Box26509 Milwaukee, WI 532264801 Timing: Fiscal Year 2001; Project Start 15-AUG-2001; Project End 31-MAY-2006 Summary: (provided by applicant) Tuberculosis is the leading cause of death in the world from a single infectious agent, and is responsible for more than 3 million deaths annually. The high mortality rate in individuals infected with Mycobacterium tuberculosis is due in part to its ability to parasitize macrophages and establish longterm, persistent infection in the host despite cell-mediated immunity. Although the current anti-tubercular drug arsenal is effective in treating individuals suffering from active disease, these drugs are ineffective in treating the 2 billion people that currently suffer from latent tuberculosis, or that are infected with multi-drug resistant strains of M. tuberculosis. One group of transcriptional regulatory determinants that may play a critical role in processes associated with M. tuberculosis latency is the two-component signal transduction systems. These systems mediate adaptation processes and have been shown to contribute to virulence and disease elicitation in other organisms. The goals of this study are to characterize further a two-component system of M. tuberculosis (MprA-MprB) that is required for the establishment and maintenance of persistent infection. In this proposal, we plan to: (i) Identify and characterize the genes regulated by the MprA transcription factor. The genes regulated by MprA will be identified from the M. tuberculosis chromosome using biochemical enrichment and genetic selection techniques, and will be characterized by gene inactivation, promoter expression analysis, and evaluation in model systems for infection. (ii) Analyze the in vivo expression profile of the MprA response regulator, and the genes regulated by MprA. This will be accomplished by expression analysis of these genes using GFP reporter technology, primer extension analysis, molecular beacon technology, and DNA microarray based analysis under physiologically relevant conditions. (iii) Delineate the effects of MprA de-regulation on host-pathogen interactions. This will be accomplished by examining effects of MprA loss or overexpression on M. tuberculosis virulence. Virulence studies will include bacterial survival and cytokine expression analysis as assayed in in vitro tissue culture systems and animal model systems of infection. These studies will also address the effects of MprA de-regulation on Mycobacterium bovis BCG attenuation. The proposal outlined here is expected to improve our understanding of genes required by M. tuberculosis for pathogenesis, and help better define the conditions encountered and responses utilized by M. tuberculosis during the latent stage of infection. We hope that the analysis of two-component systems will aid in the identification of genetic determinants for which novel anti-tubercular drugs can be developed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MACAQUE MODEL OF AIDS VIRUS-RELATED PULMONARY TUBERCULOS Principal Investigator & Institution: Chen, Zheng W. Associate Professor; Beth Israel Deaconess Medical Center St 1005 Boston, MA 02215 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-AUG-2004
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Summary: Mycobacterium tuberculosis (M.tb) disease remains an important lifethreatening problem. While HIV infection clearly predisposes the infected patients to developing primary and reactivation tuberculosis, protective human immunity to M.tb is poorly understood. We have recently shown that the intravenous infection of macaques with Mycobacterium bovis Bacille Calmette-Guerin (BCG) induces potent CD4+ and CD8+ T cell responses, which are associated with the resolution of BCG infections. In contrast, the systemic BCG infection in SIV-infected monkeys results in the chronic co-infection and the development of disseminated BCG disease resembling tuberculosis. Based on the results showing the BCG-driven T cell responses, we hypothesize that MHC-restricted T cells exert anti-mycobacterial immunity against pulmonary mycobacterial infections in the infected individuals. The depletion or dysfunction of these T cell responses will, therefore, result in the increased susceptibility to primary mycobacterial infection or reactivation tuberculosis. To test this hypothesis, we have developed the macaque model of pulmonary BCG infection to characterize anti- mycobacterium T cells as well as to study BCG latency in the lungs. In addition, we have made use of systemic SIV/BCG co-infection to develop an in vivo system in which to test the role of antiretroviral therapy in restoring anti-mycobacterial immunity. Employing these model systems, we will: I. Identify anti-BCG T-cell responses in the lungs and determine the correlation between these responses and the resolution of active pulmonary BCG infection. II. Determine the impact of SIV-induced damage of anti-BCG T cell immunity on the reactivation of the latent pulmonary BCG infection. III. Determine the utility of antiretrovirals for restoring anti-BCG pulmonary T-cell immunity in SIV-infected monkeys. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MACROPHAGE TUBERCULOSIS
GENE
EXPRESSION:
IMPACT
OF
M.
Principal Investigator & Institution: Ehrt, Sabine; Microbiology and Immunology; Weill Medical College of Cornell Univ New York, NY 10021 Timing: Fiscal Year 2001; Project Start 10-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) One third of the world's population is currently infected with Mycobacterium tuberculosis (Mtb) and every second, another person is newly infected with Mtb around the world. Each year an estimated 8 million people develop clinical disease and 1.87 million people die of TB. The primary host cell of Mtb is the macrophage and the Mtb-macrophage interaction is critical to every phase of Mtb's infectious cycle. Gene expression analysis (GEA) is the most effective technology currently available to record a quantitative picture of a cell's functional state, and therefore to compare cell states and types. The critical need for such a comparison in TB research is heightened when one takes into account the heterogeneity of macrophages by organ, species of origin and donor history. The goals of this study are to extend our understanding of macrophage biology relevant to tuberculosis (TB), and in so doing, to define the extent to which mouse macrophages can serve as surrogates for human macrophages. The latter question is key if functional genomics is used as a tool in TB research to test the course of infection by wild type and genetically modified Mtb in wild type and genetically modified mice. Specifically, we will extend our current use of high-density oligonucleotide microarrays, quantitative PCR and in situ hybridization (ISH) to analyze and compare the gene expression profiles of three distinct populations of primary cells: mouse bone marrow macrophages (BMM), mouse pulmonary alveolar macrophages (PAM), and human PAM. These relatively pure macrophage populations will be compared with the heterogeneous mixture of all cells in mouse lung. Both
84 Tuberculosis
human and mouse cells will come both from normal donors and individuals with active TB. Each cell population will be studied without further treatment, after infection in vitro with virulent Mtb, and/or after exposure to interferon gamma (IFNgamma), a cytokine critical for control of mycobacterial infection in both humans and mice. These comparisons will allow us to validate or qualify the use of mouse PAM, mouse BMM and human PAM as model systems for studying macrophage-Mtb interactions. The identification of genes regulated by Mtb in macrophages will generate hypotheses with respect to the role of these genes in pathogenesis. We will test at least one such hypothesis, that secretory leukocyte protease inhibitor (SLPI) plays a role in the pathogenesis of TB. Finally, we will share our GEA with others via the internet so that as many of the resulting hypotheses as possible can be rapidly and independently explored. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MCP-1 AND SUSCEPTIBILITY TO ACTIVE TUBERCULOSIS Principal Investigator & Institution: Flores-Villanueva, Pedro O.; Dana-Farber Cancer Institute 44 Binney St Boston, MA 02115 Timing: Fiscal Year 2002; Project Start 10-AUG-2002; Project End 31-MAY-2004 Summary: (provided by applicant): Our long-term objective is to develop new approaches for the treatment of tuberculosis (TB) by studying the host genetic factors controlling the development of effective cellular immunity against M. tuberculosis (Mtb). We have preliminary data showing that the genotype GG in the promoter region of MCP-1 is present in 50% of individuals susceptible to TB. This genotype produces this trait by a mechanism that may involve increased production of MCP-l and MCP-l inhibition of IL-12 production through the down-modulation of Osteopontin (Opn) receptor alphaVbeta33 in macrophages. In specific aim 1, we seek to confirm the association of the genotype GG with susceptibility to TB in a two-step case/control and a family study with parental samples of affected individuals and a TDT (TransmissionDisequilibrium Test). The sample for the case/control studies will comprise a large and rigorously characterized group of unrelated individuals with active TB and controls. Plasma samples from these individuals will be used to determine the levels of MCP-l, IL-12, IL-10, IFN-gamma and Opn to uncover the effect of MCP-l on the levels of these factors. With the same purpose, PBMC from a limited number (20 of each genotype) of these fully typed TB cases and controls will be stimulated in vitro with Mtb antigens and the levels of factors mentioned above measured. In specific aim 2, we will extend our preliminary studies to characterize in vitro the effect of MCP-l in alphaVbeta33 and CD44 Opn receptors, CD14 and TLR-2, and CD40 and CD154 expression and its relevance in the modulation of IL-12, MCP-l, IL-10, and Opn production in response to Mtb antigens. We also seek to determine if alphaVbeta3/CD44, CD14/TLR-2, and CD40/CD154 have additive effects in the induction of IL-12, and how they interact to modulate each other, and how MCP-l affects these pathways and these pathways interaction. In specific aim 3, we will explore in vitro how mycobacterial infection affects the production of MCP-l, IL-12, IL-10, and Opn and the expression of alphaVbeta3, CD44, CD14, TLR-2, CD40 and CD154. To achieve our goals in specific aim 1, Dr. Julio Granados, Chairman Investigator, Dept. Immunology, Mexican Institute of Nutrition, will be our consultant and collaborator. He and Dr. Moises Selman, from the Mexican Institute of Respiratory Diseases will recruit the TB cases and controls, process blood samples to isolate DNA, plasma, and PBMC. The PBMC will be stimulated in vitro in Dr. Granados' laboratory. The DNA samples will be sent to Boston where they will be typed. The plasma samples and culture supernatants will be kept frozen until we pick
Studies 85
them up (twice a year) and bring them to Boston in vaporize-liquid nitrogen tanks for further analysis. For specific aims 2 and 3, we will use leukophoresis products from fully type- selected individuals of a panel of 200 healthy donors residents in Boston. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MECHANISM BASED INHIBITION OF M TUBERCULOSIS ENZYMES Principal Investigator & Institution: Blanchard, John S. Professor; Biochemistry; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 30-SEP-1992; Project End 31-AUG-2007 Summary: (provided by applicant): Bacterial resistance to antibiotics is a clinically significant problem that threatens current paradigms of antibacterial chemotherapy. This is particularly true for the treatment of tuberculosis, where singly- and multiplydrug resistant clinical strains of Mycobacterium tuberculosis have been identified with increasing frequency. Very few compounds are selective antimycobacterial agents, and mycobacteria are intrinsically resistant to many antibiotics either because of the constitutive expression of degrading enzymes (e.g., Beta-lactamases) or the inability of antibiotics to penetrate the uniquely hydrophobic outer cell wall. The principal investigator and his group have previously focused their attention on the DAP/L-lysine biosynthetic pathway, and will use similar approaches to explore the three-dimensional structures and chemical mechanisms of M. tuberculosis enzymes involved in the biosynthesis of pantothenate, a vitamin in mammals, and an essential pathway in M. tuberculosis. Specifically, they will clone, express and purify the M. tuberculosis ilvGM, ilvC and ilvD gene products involved in branched chain and pantothenate biosynthesis, and the panB gene product involved in pantothenate biosynthesis. They will clone, express and purify the M. tuberculosis dxs- and dxr-encoded deoxyxylulose5-phosphate synthase and isomeroreductase for mechanistic comparison to the ilvCand ilvGM-encoded enzymes. They will also determine the structures and chemical mechanisms of aminoglycoside N-acetyltransferases, enzymes that are primarily responsible for clinical resistance to aminoglycosides in both Gram-negative and Grampositive bacterial pathogens. These enzymes catalyze a rich variety of chemistries, and plausible chemical mechanisms suggest that mechanism-based, and bi- and tri-substrate analogue inhibitors might be found. They will continue to use an integrated approach involving kinetic and chemical mechanism studies, three-dimensional structural studies, and site-directed mutagenesis to identify active site residues important for substrate and inhibitor recognition, and fulfill their long term goal of characterizing unique bacterial multi-step biosynthetic pathways that are absent in humans. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: METABOLOME OF NON-REPLICATING M. TUBERCULOSIS Principal Investigator & Institution: Pauli, Guido F. Inst for Tuberculosis Research; University of Illinois at Chicago 1737 West Polk Street Chicago, IL 60612 Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2004 Summary: (provided by applicant): It is now generally accepted that new drugs are needed to make global tuberculosis control a reality. The completion of the sequence of the M. tuberculosis genome has laid the foundation for greatly accelerating new drug discovery. While a limited number of studies of the TB transcriptome and proteome have been reported, these global macromolecular analyses are still not able to contribute directly to drug discovery except where gene/protein function are known or protein
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crystal structures are available. Elucidating the full complement of low molecular weight compounds in an organism -the metabolome - is becoming recognized as a complementary and perhaps the most tractable approach to a comprehensive understanding of any pathogen including the insight necessary for rational drug discovery. This R-21 proposal seeks to define the metabolome of the tubercle bacillus in the stage of non-replicating persistence (NRP), the physiological state considered to be responsible for the required long treatment duration for TB. The metabolome will be elucidated by high-resolution chromatography (CCC, LC) and -spectroscopy (MS, NMR). Initial studies will utilize a high biomass of BCG to evaluate several separation schemes in optimizing the resolution of (secondary) metabolites. This protocol will then be used to define the metabolome of M. tuberculosis in NRP. Subsequent experiments will focus on understanding the growth-phase specificity of unique metabolites produced under NRP. The information obtained will not only complement the extensive knowledge of the chemistry of the cell wall but is also expected to I) identify novel secondary metabolites possibly related to those recently described for other mycobacteria, 2) help to clarify events and identify low MW markers during the metabolic shift to a low oxygen environment especially considering that >70% of genes up-regulated during this adaptation are of unknown function, 3) provide leads for drug development through analoging around TB-specific compounds not known to occur in mammalian cells and 4) assist in the understanding of the mechanism of action of newly discovered anti-TB agents. The operating hypothesis for this study is that elucidating the metabolome of Mycobacterium tuberculosis will overcome limitations inherent in global macromolecular analyses with respect to gaining key insights into dormancy and drug discovery. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MHC TUBERCULOSIS
CLASS
1
MOLECULES
IN
IMMUNITY
AGAINST
Principal Investigator & Institution: Urdahl, Kevin B. Pediatrics; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 30-APR-2006 Summary: (provided by applicant): This proposal describes a 5 year training program for the development of an academic career in Pediatric Infectious Diseases. The principal investigator has completed residency training in Pediatrics at the University of Washington and is currently in his final year of fellowship training in Infectious Diseases at the same institution. This program will expand upon his current scientific skills, and promote new skills that will enhance his career development. Specifically, this program promotes the development of a career studying the immune response to tuberculosis. Dr. Michael Bevan will mentor the principal investigator's scientific development. Dr. Bevan is a Professor of Immunology, a Howard Hughes Medical Institute Investigator, and a world-renowned leader in the field of immunology. He has trained numerous postdoctoral fellows and graduate students who themselves have become recognized leaders in their fields. In recognition of this outstanding record, Dr. Bevan received the Excellence in Mentoring Award from The American Association of Immunologists in 2000. To enhance the training, the program will enlist the expertise of Dr. David Sherman, Assistant Professor of Pathobiology. Dr. Sherman is a molecular microbiologist with considerable experience with genetic manipulation of Mycobacterium tuberculosis (Mtb). In addition, an advisory committee of highly regarded scientists will provide scientific and career advice. Research will focus on understanding the roles of CD8+ T cells and MHC class I molecules in immune
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protection against tuberculosis. To approach these issues, a new system to monitor the activity of T cells specific for Mtb-derived antigens after aerosol infection of mice with M. tuberculosis will be established. This system will enable some of the most fundamental questions in the immunology of tuberculosis to be addressed with greater precision than is possible using currently available tools. The specific aims include: 1) Determining the locations, phenotypes, and functions of CD4+ and CD8+ T cells specific for an Mtb-derived antigen after aerosol infection of mice, 2) Determining the roles of CD4+ vs. CD8+ T cells specific for an Mtb-derived antigen in immunity against tuberculosis, and 3) determining the role of MHC class I molecules in regulating immune processes involved in protection against tuberculosis. In addition to providing important information about tuberculosis, these experiments will shed light on basic immune mechanisms that regulate host defense. The Division of Infectious Disease in the Department of Pediatrics at the University of Washington provides an ideal setting for training physician-scientists by advocating scientific exploration within any laboratory of the university. The training program described in this proposal, which draws on several interdepartmental collaborations, will establish a scientific niche for the principal investigator that will position him uniquely for a successful academic career. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PATHOGENS
MICROMACHINED
BIOSENSOR
FOR
MYCOBACTERIAL
Principal Investigator & Institution: Wavering, Thomas A.; Luna Innovations, Inc. 2851 Commerce St Blacksburg, VA 24060 Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-JUL-2003 Summary: (Provided by applicant): Mycobacteria are slender, nonmotile, gram-positive rods and do not produce spores or capsules. Nineteen Mycobacterium species are associated with disease in humans. The most dangerous of these diseases is tuberculosis, caused by infection with M. tuberculosis. Tuberculosis kills about 2 million people annually and infects another 8 million people each year. Treatment and diagnosis of the disease is complicated by current laboratory methods that can take from four to six weeks to establish a definitive identification of infection. New diagnostic systems are needed for earlier detection of tuberculosis, to better monitor the treatment of tuberculosis, and to study the epidemiology of the disease. Luna Innovations has assembled a team of researchers to develop a micromachined biosensor array that combines technology from silicon micromachining, optical fiber sensors, and biochemistry for measurement of mycobacterial pathogens. The sensing element of the biosensor array will be a microcantilever beam coated on one side with receptors. As pathogens bind to the coated beam, a biochemical induced surface stress causes the beam to deflect proportionally to concentration. The Phase I program will focus on developing the microcantilever biosensor array, demonstrating detection of mycobacterial pathogens in neat solutions, and comparing performance to known standards. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MODE OF ACTION OF PYRAZINAMIDE IN TUBERCLE BACILLUS Principal Investigator & Institution: Zhang, Ying E. Associate Professor; Molecular Microbiol and Immun; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218
88 Tuberculosis
Timing: Fiscal Year 2001; Project Start 01-DEC-1998; Project End 30-NOV-2003 Summary: (Adapted from the applicant's abstract): The long-term goal of this project is to improve our understanding of mechanisms of drug action and drug resistance in Mycobacterium tuberculosis and to provide knowledge important for designing new tuberculosis (TB) drugs. Two significant problems seriously compromise our ability to effectively control TB: the emergence of drug-resistant strains, and the lengthy 6 month TB chemotherapy. There is an urgent need to develop new TB drugs that are effective in both aspects. Pyrazinamide (PZA) is a paradoxical front-line TB drug, because while PZA has powerful in vivo sterilizing activity, through killing a population of semidormant organisms, and is thus involved in shortening the TB therapy to 6 month; PZA has no activity in vitro against M. tuberculosis under normal culture conditions except under acid pH (5.5). The mode of action of PZA in M. tuberculosis is unknown, and this project proposes to address this issue. The specific aims of the project are: (1) To determine the effect of pyrazinoic acid (POA), the active form of PZA, on membrane integrity and nutrient transport of M. tuberculosis. (2) To test the potential inhibition of POA on the NAD metabolism in M. tuberculosis. (3) To identify and characterize the efflux genes involved in pumping POA out of the mycobacteria. The health relatedness of this project lies in the possible design of new antituberculous drugs that may further shorten the current 6 month TB chemotherapy based on understanding the mode of action of PZA. The investigators hypothesize that POA as a lipophilic weak acid will partition into the lipid-rich membrane of M. tuberculosis and cause structural and functional damage to the membrane. This hypothesis will be tested in Aim I by EM analysis, by measuring the nutrient transport using radiolabeled amino acids, uracil and thymidine, and by measuring the possible effect of POA on the membrane potential. Because of structural similarity of POA to niacin, they hypothesize that POA may interfere with NAD metabolism in M. tuberculosis by feedback inhibition of NAD biosynthesis and by incorporation as a pseudo-NAD molecule without NAD function. This hypothesis will be tested in Aim 2 by using C 14-labeled precursors of NAD and by tracing C 14-POA in NAD molecule by mass spec and NMR analyses. Because they found the activity of efflux pumps extruding POA in various mycobacteria varies widely and correlates with their susceptibility to PZA, they propose to identify the POA efflux genes by testing putative efflux genes in the Sanger Centre M. tuberculosis genome database and by testing mycobacterial genomic DNA libraries for their ability to confer PZA resistance in M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MOLECULAR BEACONS FOR TUBERCULOSIS TESTING Principal Investigator & Institution: Meserol, Peter; Synectiq Corporation 66 Ford Rd, Ste 123 Denville, NJ 07834 Timing: Fiscal Year 2001; Project Start 01-SEP-2001; Project End 31-AUG-2003 Summary: Genome sequencing technologies have begun a revolution in the diagnosis of human diseases, and infections, and in the characterization of microbes. Specific mutations linked to cancer, metabolic disorders, as well as bacterial and viral drug resistance are often as small as a single nucleotide polymorphism (SNP). Unfortunately, DNA microarrays and the current generation of nucleic acid-amplification based techniques under development for the detection of SNPs and other genetic polymorphisms are technically complex and expensive. These factors are likely to limit the widespread availability of genetic testing. We have pioneered a simple, rapid, and robust method for DNA sequence identification and SNP analysis that uses new types of fluorescent probes called molecular beacons. Molecular beacons can be used to develop
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highly accurate and inexpensive polymerase chain reaction (PCR) assays, but these assays have been limited by the requirement for costly and complex detection instruments. Molecular beacons are ideally suited for use in assays that take maximum advantage of advances in polymer microfabrication technology, cooled CCD imaging systems, ink jet directed microprinting, and advanced image processing software modules. This proposal will develop inexpensive desktop systems and assays for genetic diagnosis and SNP detection. It will concentrate on developing urgently needed assays to simultaneously diagnose Mycobacterium tuberculosis, and detect drug resistance directly from clinical samples. The ultimate goal of this proposal is to design systems and applications that are sufficiently robust and inexpensive to be used virtually anywhere, to address a wide variety of diseases. PROPOSED COMMERCIAL APPLICATIONS: 2.1 Billion people harbor a TB infection. UNICEF reports that tuberculosis poses a serious risk to Asia's sustained socioeconomic development: In a recent National Intelligence Estimate, the Central Intelligence Agency singled out drugresistant TB - and especially its incidence among immigrants - as a potential threat to national security. Salomon and Murray report that world expenditure on TB therapy and diagnosis was $4.1 Billion (USD) in 1998. Diagnosis of TB worldwide is by sputum smear, with 50% accuracy. This new diagnostic is desperately needed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MOLECULAR BIOLOGY OF INFECTION WITH M TUBERCULOSIS Principal Investigator & Institution: Riley, Lee W. Professor of Infectious Diseases and Epi; Public Hlth Bio & Epidemiology; University of California Berkeley Berkeley, CA 94720 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 30-JUN-2003 Summary: (Adapted from the Applicant's Abstract): Little is known about the bacterial Factors that contribute to pathogenesis of TB. However, during year 01-02 of AI-35266, the PI identified and characterized a recombinant protein encoded by M. tuberculosis that promoted uptake of latex microspheres coated with the protein into nonphagocytic cells. Subsequently (Year 03), this 27 kDa protein was discovered to be a truncated version of a native 45 kDa surface protein of M. tuberculosis encoded by a 1239-bp gene designated as mceA. The 45 kDa protein is referred to as mycobacterium cell entry protein or Mcep (the truncated protein is designated Mcep1). Reagents that affect cytoskeletal rearrangement (cytochalasin D, nocodazole, and onodansylcadaverine) inhibited the association with HeLa cells of microspheres coated with Mcep1. In addition, Mcep 1 inhibited Fc-receptor mediated oxidative burst in murine macrophage J774. A highly basic 22-amino region near the N-terminus of Mcep1 was found to be the cell uptake active domain. From the recently completed genome sequence of M. tuberculosis, the mceA locus was found to be part of a cluster of at least 15 open reading frames (ORF), 4 of which encoded a possible membrane-spanning lipoprotein, all located downstream of an ORF resembling the E. coli nta operon transcriptional regulator. The new proposal will have three specific aims. The first aim will continue the analysis of the structure and function of Mcep and its physiologic relevance to M. tuberculosis pathogenesis, focusing on characterizing the role of Mcep on inhibition of oxidative burst in macrophages and cytoskeletal rearrangement in nonphagocytic cells. The physiologic relevance of Mcep will be studied by the analysis of M. tuberculosis disrupted in mceA. The second aim will analyze the genes that cluster around the mceA locus. This aim will determine whether any of these genes constitutes part of a secretion system for the surface-expressed Mcep, and whether any of them regulates the expression of Mcep during and after entry into cells. The third aim will seek to
90 Tuberculosis
determine whether there is a specific receptor on mammalian cells for Mcep, and, if so, its function in relation to Mcep- mediated pathogenesis. It is hoped that these studies will determine whether Mcep constitutes a virulence factor of M. tuberculosis. At the same time, they may reveal new insight into how a possible effector molecule in mycobacteria is delivered to the surface through plasma membrane and the lipid-rich cell wall layer. In addition, these studies may identify several potential targets for new chemotherapeutic agents or vaccine candidates. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MOLECULAR BIOLOGY OF MYCOBACTERIA AND THEIR PHAGES Principal Investigator & Institution: Hatfull, Graham F. Professor; Biological Sciences; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2001; Project Start 01-DEC-1989; Project End 30-JUN-2005 Summary: (provided by the applicant): Mycobacterium tuberculosis, the causative agent of human tuberculosis, kills more people than any other single infectious agent. The prevalence of TB is greatest in the developing world but its control in the United States has become severely complicated by the appearance of multidrug resistant strains of M. tuberculosis. There has recently been a sharp increase in the incidence of these MDRTB strains in developing countries. Effective control of tuberculosis requires improved and more rapid diagnostic methods, more efficacious vaccines, and better antimycobacterial drugs, particularly for treatment of multidrug resistant infections. With the recent advances in mycobacterial genetics and the determination of the complete genome sequence of M. tuberculosis, there is now renewed hope that a more sophisticated understanding of the physiology, genetics, and metabolism of M. tuberculosis will lead to novel strategies for controlling mycobacterial infections. Unfortunately, in spite of these genetic tools and genomic information, we know little about the molecular basis of the fundamental aspects of mycobacterial physiology-such as slow growth, their unique cell wall, and DNA replication-let alone the molecular basis of mycobacterial pathogenesis. Viruses are powerful tools for genetic analysis of a broad range of organisms, and the viruses of mycobacteria (mycobacteriophages) are no exception. The use of mycobacteriophages was instrumental in the establishment of mycobacterial genetics and the creation of cloning vectors for the introduction of DNA into mycobacteria. More recently, recombinant reporter mycobacteriophages have been proposed as clinical tools for rapid determination of drug susceptibilities of clinical isolates of M. tuberculosis. This project aims at understanding the intimate interface between mycobacteriophages and their hosts. This interaction begins with the association of free phage particles with bacterial cells followed by injection of phage DNA into the cell. Phage DNA may then either integrate into the host genome and be genetically silenced, or reprogram the cell to direct it towards phage gene expression and subsequent cell lysis. By exploring these events we will gain insights into the regulation of gene expression, the structure of the mycobacterial envelope, and the process of phage-mediated cell lysis. We will also use proteomic approaches to understand the influence of phage gene expression on that of its host. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: MOLECULAR TRANSMISSION
VS.
CONVENTIONAL
ASSESMENT
OF
TB
Principal Investigator & Institution: De Riemer, Kathy; Medicine; Stanford University Stanford, CA 94305
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Timing: Fiscal Year 2002; Project Start 30-SEP-1999; Project End 31-AUG-2003 Summary: An estimated one third of the world's population is infected with Mycobacterium tuberculosis, and it is the leading cause of death among adults by an infectious disease worldwide. TB control using DOTS (Directly Observed Short Therapy, short course) to successfully diagnose, treat and follow sputum-smear positive TB cases is a priority of the World Health Organization (WHO), but there is growing recognition that DOTS alone will not be enough to control or eradicate TB given the extended duration of treatment and the emergence of drug-resistant forms of M. tuberculosis. New methods are needed to quantify the impact of ongoing TB transmission and the importance of preventive strategies. The focus of this research will be an evaluation of community- based TB transmission dynamics at a field site in Southern Mexico. Molecular epidemiology provides important insights into the transmission dynamics of tuberculosis, but because it relies upon mycobacterial cultures, it can only track active cases. The proposed research will integrate conventional tuberculin skin test survey methodology into an ongoing population-based research project so that we may track infection as well as disease and test hypotheses about alternative strategies for controlling TB infection. The specific aims are: 1) to describe the epidemiology of TB infection caused by culture-confirmed cases of M. tuberculosis; 2) to identify risk factors for TB infection, stratified by the biologic characteristics of the strains of M. tuberculosis and the gender, age group, and social setting of the index cases; 3) to determine the specificity and predictive value of contact tracing by comparing the RFLP patterns of cases and those arising among their contacts; and 4) to develop cost- effectiveness models of appropriate screening strategies and interventions to control TB infection. The research will also generate important baseline data for the future testing of new diagnostics and vaccines. This project will provide an ideal vehicle for additional, mentored training and research to develop the PI's long standing career interest in international infectious disease research. The grant award will build on previous work and provide training in areas new to the candidate, such as biomedical techniques and cost-effectiveness modeling. Her technical training in epidemiology and biostatistics, prior epidemiologic research studies, and extensive familiarity with Latin America fit perfectly with the strengths and needs of a well established field site in Veracruz, Southern Mexico. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MOLECULAR-GENETIC ANALYSIS OF TB PERSISTENCE AND LATENCY Principal Investigator & Institution: Mckinney, John D. Assistant Professor; Lab/Infection Biology; Rockefeller University New York, NY 100216399 Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 30-JUN-2007 Summary: (provided by applicant): Tuberculosis (TB) is rivaled only by AIDS as a communicable cause of death. Infection is lifelong and may reactivate following an asymptomatic (latent) interval of variable duration. It is estimated that nearly two billion individuals worldwide have been exposed to Mycobacterium tuberculosis. Exposed (tuberculin-positive) individuals have a 5-10% lifetime risk of developing active TB. The risk rises to nearly 10% per year for individuals with HIV/AIDS, indicating a key role for the host immune response in maintaining TB latency. Chemoprophylaxis of latent TB to prevent reactivation requires months of drug therapy, a regimen that many individuals are unwilling or unable to complete. The development of better tools for TB control will hinge on the elucidation of the adaptive mechanisms that allow latent M. tuberculosis to persist in the face of host immunity and
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chemotherapy. Towards that goal, this application is focused on the identification of mycobacterial "persistence factors" and "defense factors" via signature-tagged mutagenesis (STM), a method for simultaneous screening of multiple mutants in mice. Two genetic screens are described. The persistence (per) screen will identify M. tuberculosis mutants that are specifically impaired in their ability to persist at later stages of infection in mice. The defense (def) screen will identify mutants whose attenuated phenotype in immune-competent mice is reversed in mice with specific immune deficiencies. These screens will identify mycobacterial genes involved in longterm persistence and defense against host immune mechanisms, respectively. The role of these genes in latent persistence will be assessed in the "Cornell model" of chemotherapy-induced latency and reactivation in mice. Our studies in the mouse will serve as a springboard for linked studies in humans. Relevance to human infection will be explored by analyzing the expression of per/def genes in the lungs of mice and humans via real-time RT-PCR with molecular beacons. Bacterial correlates of protection and pathogenesis will be identified by comparison of gene expression profiles in latent v. active human lesions, respectively. These studies will elucidate the host/pathogen interactions that determine the state of infection-latency or active disease-and may point the way to novel interventions against TB. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MONOCYTE RECRUITMENT IN PLEUROPULMONARY TB IN AIDS Principal Investigator & Institution: Antony, Veena B. Professor; Medicine; Indiana Univ-Purdue Univ at Indianapolis 620 Union Drive, Room 618 Indianapolis, IN 462025167 Timing: Fiscal Year 2001; Project Start 01-FEB-1996; Project End 31-JAN-2006 Summary: Tuberculosis is endemic among patients with AIDS and follows an aggressive course with poor localization of mycobacteria into granuloma and widespread infection. Granulomas are monocyte rich collections of cells which derive from the circulating peripheral blood monocyte (PBMC). Monocytes are recruited to the site of tuberculous infection by the interaction of C-C chemokines, (mainly monocyte chemoattractant protein-1, MCP- 1) and monocyte expression of the CCR2 receptor. It is the hypothesis of this proposal that recruitment and retention of the monocyte at the site of tuberculous infection is predicated not only on local release of MCP-1 but on the expression of CCR2 receptor on PBMC. CD4 depletion alters monocyte recruitment and retention by altering the in vivo expression of CCR2 on PBMC and macrophages. This inhibitory effect of CD4 depletion is due to the relative imbalance between Thi and Th2 cytokines. We have developed a model of pleural tuberculosis in CD4 -1- and CD4 +/+ mice to evaluate the Th1/Th2 regulation of the CCR2 receptor on PBMC. We will evaluate our hypothesis in our model of pleural tuberculosis in vivo as well as in vitro in PBMC and elicited pleural macrophages (PM). Our specific aims are 1) To determine the in vivo moncyte influx, granulonia formation, mycobacterial clearance and mortality utilizing our model of pleural tuberculosis in a CD4 knockout mouse model, CD4 -/mice and control, wild type CD4 +1+/- mice. 2) To determine the in vivo effect of CD4 depletion on the compartmentalized and peripheral expression of CCR2 receptor on PBMC and PM and the regulatory role of Th1 (IL2, IL-12) and Th2 (11-10, JL-4) cytokines inCD4 -/- and CD 4 +/+ mice with pleural tuberculosis. 3) To determine the in vitro molecular and cellular regulation of CCR2 expression in PBMC and PM in the presence of Th1 and Th2 cytokines and 4) To determine the role of tubercie bacilli stimulated pleural mesothelial cell derived cytokines in the regulation of CCR2 receoptor on peripheral blood monocytes (PBMC) and pleural niacrophages (PM) in vivo and in
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vitro. Understanding the mechanism of regulation of CCR2 receptor by Th1 and Th2 cytokines may help us discern the pathophysiology of pleuro-pulmonary tuberculosis seen in patients with AIDS and may help develop therapeutic modalities that augment host-defense responses. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTERIAL MACROPHAGES
CELL
WALL
RECOGNITION
BY
Principal Investigator & Institution: Smith, Kelly D. Pathology; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2001; Project Start 01-JUN-2000; Project End 31-MAY-2003 Summary: Mycobacterium tuberculosis is a leading cause of death by infectious disease in the world. Mycobacterial cell wall components are potent stimulators of the immune system, and have long been as adjuvants. The host systems that recognize these components are poorly defined, but several studies implicate CD14, a component of LPS recognition pathway that utilizes Toll- like receptor-4 (TLR4). TLRs appear to be early and important inducers of tumor necrosis factor-alpha (TNFalpha). TNFalpha is a critical factor for protective immunity to tuberculosis. We hypothesize that innate recognition of mycobacterial cell wall components by TLRs is important for host defense. TLRs have a signaling pathway that has similarities to the IL-1 receptor pathway, and include MyD88 as a proximal component. Using murine macrophage cell line RAW 264.7, we have identified three cell wall fractions that stimulate MyD88dependent TNFalpha production. This implies TLR-dependent recognition of mycobacterial cell wall components. The mechanism of stimulatory ligand recognition by TLRs is unknown and may require direct or indirect involvement of CD14. Phagocytosis of mycobacterium involves another set of receptors including the complement and mannose receptors. Complement opsonization increases the infectivity of mycobacteria in vitro. In addition, phagocytic receptors recognize mycobacterium and potentially activate an overlapping set of signaling pathways. This suggests that cross- talk may exist between phagocytic and inflammatory signaling pathways. The aims of this proposal are to 1) identify the specific mycobacterial cell wall components recognized by TLRs, 2) identify the corresponding receptors that are responsible for MyD88-dependent recognition of mycobacteria, 3) characterize mycobacterial cell wall ligand-recognition by TLRs, and 4) characterize the consequences of complement opsonization and TLR- dependent recognition on phagocytosis of mycobacteria. Understanding the innate recognition of mycobacteria will greatly advance our understanding of tuberculosis, point to possible sites of host genetic variation that alter responses to M. tuberculosis, and disclose fundamental principles of innate immunity in infectious and inflammatory diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTERIAL TRANSMISSION
DETERMINANTS
OF
CAVITATION
&
Principal Investigator & Institution: De Jong, Bouke C. Medicine; Stanford University Stanford, CA 94305 Timing: Fiscal Year 2002; Project Start 18-SEP-2002; Project End 31-AUG-2006 Summary: (provided by applicant): Dr. de Jong has prior international research experience and recently completed her clinical training in infectious diseases at Stanford University. In order to foster her transition into an international research scientist, she
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now proposes to combine didactic training in epidemiology with a hypothesis driven project designed to better understand the determinants of tuberculosis transmission. This work will be conducted in a well-established research setting in the Gambia and mentored by faculty with complementary expertise. Over three consecutive summers she will obtain a Masters degree in epidemiology at the Netherlands Institute for Health Sciences. This curriculum provides a comprehensive survey of the principles and practices of epidemiologic research and is specifically structured to be spread over this period and integrated with an independent research project. The research project that she will pursue tests the hypothesis that genetic differences between mycobacterial strains contribute to variable rates of pulmonary cavitation and person- to- person transmission. It is well established that host and environmental factors greatly impact the clinical manifestations and consequences of infection with M. tuberculosis. However, little is known about the consequences of differences between mycobacterial strains. The combination of expertise the candidate has gained to date working in a molecular epidemiology laboratory and the clinical epidemiologic lessons that she will learn in the classroom will provide her the opportunity to formally address this issue in the field. Specifically, she will spend at least two years in The Gambia quantifying the propensity of clinical isolates of M. tuberculosis to cause cavitation and be transmitted to household contacts. Subsequently, at Stanford University, she will use a DNA microarray based comparative genomic approach to seek genetic correlates of these bacterial propensities. While it would be presumptuous to promise that these activities will have a major impact on the global burden of tuberculosis, it is likely that the proposed project will serve as a vehicle for her transition into an independent physician scientist focused on international translational clinical investigation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTERIAL GENES THAT ENHANCE INTRACELLULAR GROWTH Principal Investigator & Institution: Lakey, David L. Nursing; University of Texas Hlth Ctr at Tyler 11937 Us Highway 271 Tyler, TX 75708 Timing: Fiscal Year 2001; Project Start 18-APR-2000; Project End 30-JUN-2001 Summary: Dr. David Lakey completed a Pediatric and Adult Infectious Diseases fellowship at Vanderbilt University Medical Center. He spent 2 years in Dr. Douglas Kernodle's laboratory, studying expression of mycobacterial genes in E. coli. Dr. Lakey received 1 of only 3 fellowships awarded by the Pediatric Infectious Disease Society and was the 1998 Grant Liddle Scholar, given to the best research fellow in the Vanderbilt University Department of Medicine. Dr. Lakey has a superb grasp of molecular microbiology. His ability to think independently, use novel approaches to solve problems, and interact with other scientists make him an excellent candidate for a career in biomedical research. The UT Health Center at Tyler has a long-term commitment to excellence in tuberculosis research, and 5 NIH-funded investigators there are currently engaged in research in mycobacterial disease. The institution is committed to Dr. Lakey's career development and has provided him with a fully equipped laboratory and support staff. Dr. Peter Barnes, an accomplished investigator in tuberculosis, will serve as Dr. Lakey's mentor, and an Advisory Committee of 6 other experienced scientists will provide guidance to Dr. Lakey. Dr. Lakey will devote 90 percent effort to this proposal. The research project is to identify genes of M. tuberculosis that contribute to enhanced mycobacterial growth in macrophages. This information will facilitate development of attenuated M. tuberculosis strains as vaccines, as well as design of drugs for treatment and prevention of tuberculosis. Our specific aims are: 1) To establish methods to
Studies 95
quantitate mRNA expression of intracellular mycobacteria, using competitive RT-PCR; 2) To evaluate differential gene expression of laboratory and clinical M. tuberculosis strains during intracellular growth, using competitive RT-PCR, differential display-PCR and mycobacterial microarrays. We will identify genes that are differentially expressed by M. tuberculosis strains that grow at different rates in macrophages; 3) To determine the functional effects of differentially expressed genes identified in aim 2 on intracellular mycobacterial growth. Candidate mycobacterial genes associated with rapid intracellular growth will be expressed by E. coli and by M. tuberculosis H37Ra to assess their effects on intracellular growth. In addition to this research project, Dr. Lakey will take didactic courses offered by the Master's Program in Biotechnology, and participate in other educational activities within and outside of the institution. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTERIAL GENES, ANTIGENS, AND VACCINES Principal Investigator & Institution: Bloom, Barry R. Dean; Immunology/Infections Diseases; Harvard University (Sch of Public Hlth) Public Health Campus Boston, MA 02460 Timing: Fiscal Year 2001; Project Start 01-APR-1986; Project End 31-MAY-2006 Summary: (provided by the applicant): Tuberculosis and HIV/AIDS represent the major infectious causes of death in the world, and tuberculosis (TB) is the attributable cause of death in a third of AIDS patients in Africa. Drug resistance to TB is emerging in Europe and Asia, drug treatment regimens are long and expensive and compliance is limited. For these reasons, we propose to bring a multidisciplinary approach, joining molecular genetics and immunology, to developing safe and effective live attenuated vaccines against TB. Since peak age of disease is 15-25y, we believe a live attenuated vaccine that induces long enduring immunological memory will provide the most useful protection against disease. In previous work we have developed tools to genetically manipulate slow growing mycobacteria, including the capability of creating specific deletion mutants to attenuate virulent M tuberculosis. One aim is to test the hypothesis that M tuberculosis represents a better vaccine candidate than BCG, to create and test auxotrophic mutants, growth mutants and persistence mutants of M. tuberculosis for safety and immunogenicity in mice. A second is to determine the optimal duration of growth of vaccine strains in vivo for the development of immunological memory responses, particularly through the use of regulated promoters. This will also allow us to elucidate similar requirements for producing tissue damage. Our final aim remains to understand the immunological mechanisms of protection against experimental tuberculosis, particularly exploring the role of innate responses mediated by the Toll-like receptor family, the minimum epitope and antigen requirements for protection, and the possible role of cytotoxic T-lymphocytes (CTL) in protection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
•
Project Title: MYCOBACTERIAL SULFATION PATHWAYS Principal Investigator & Institution: Bertozzi, Carolyn R. Professor; Chemistry; University of California Berkeley Berkeley, CA 94720 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 30-MAR-2006 Summary: (provided by applicant): Sulfated are central mediators of extracellular traffic and cell-cell communication in humans. The enzymes that install and remove sulfate esters, sulfotransferases and sulfatases, respectively, are now appreciated as major
96 Tuberculosis
contributors to human health and disease. By contrast, the roles of sulfated sugars and the associated enzymes in bacteria remain relatively unexplored. Mycobacterial pathogens have been declared a global emergency by the World Health Organization, particularly in regard to the deadly synergy of Mycobacterium tuberculosis with AIDS, but also due to the emergence of drug-resistant strains. In Mycobacteria, several sulfated molecules have been identified. These include a sulfated glycolipid, SL- 1 that has been implicated as a virulence factor for M. tuberculosis. Another sulfated carbohydrate, part of a glycopeptidolipid, has been detected in a drug resistant strain of M. aviurn isolated from an AIDS patient. Recently, the complete genome sequences of M. tuberculosis, M. avium, and M. smegmatis have become available, enabling the search for genes that participate in sulfation pathways. We have identified an extensive family of sulfotransferases and sulfatases from the completed genomes of these three Mycobacteria. The enzymes may be critical determinants of Mycobacterial virulence and potential targets for anti-Mycobacterial therapy. Through a collaborative effort, our laboratories (Prof. Carolyn Bertozzi and Prof. Lee Riley, UC Berkeley) have initiated a program aimed at the genetic and biochemical characterization, and small molecule inhibition of the sulfotransferases from M. tuberculosis and M. smegmatis. In addition, we have identified several sulfatases that have considerable similarity to mammalian carbohydrate sulfatases, suggesting a role for these enzymes in host/pathogen interactions. Finally, in order to define the sulfur incorporation pathways of Mycobacteria, we have begun the characterization of enzymes involved in the early stages of cysteine biosynthesis. The aims of this proposal are threefold: (1) to determine the functions of the carbohydrate sulfotransferases in M. tuberculosis and M. smegmatis using genetic, biochemical and chemical approaches; (2) to investigate the involvement of bacterial sulfatases in host/pathogen interactions; and (3) to define the sulfur assimilation pathway of M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TUBERCULOSIS
MYCOBACTERIOPHANGE
INTERACTION
WITH
M
Principal Investigator & Institution: Riska, Paul F. Medicine; Suny Downstate Medical Center 450 Clarkson Ave New York, NY 11203 Timing: Fiscal Year 2001; Project Start 15-JUL-1998; Project End 30-JUN-2003 Summary: This training program is designed so that the investigator-candidate can develop the necessary skills to become an independent physician- scientist. The program builds on the skills and interests attained in the previous three years of a postdoctoral training position, during which the candidate adapted a novel molecular genetic tool for application in the clinical realm. Specifically, mycobaceriophages, viruses which infect Mycobacterium spp. including M. tuberculosis, were designed to express a reporter gene encoding the firefly light-emitting enzyme luciferase upon infection of a viable mycobacterium. This technology, which can be used to rapidly detect M. tuberculosis as well as determine its drug susceptibilities, is much needed to combat the resurgence of tuberculosis associated with the AIDS epidemic. The current application aims to investigate the genetic mechanisms underlying this technology in order to facilitate rational improvements in the systems's performance, while solidifying the candidates facility in the techniques of molecular genetics. The specific aims are to identify phage and host factors which influence the efficiency of the detection scheme. Specifically, the virus has certain features--its lytic phenotype and its shutdown of host protein synthesis which are unfavorable in its adopted role as a producer of receptor genes, yet are required for its effective propagation as a reagent. Cloning the
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determinants of these two processes will allow their rational regulation. The mycobacterial receptor for phage is an important parameter for both sensitivity and specificity of phage infection; its expression should correlate with the spectrum and degree of infectability of related mycobacteria, some of which are nonpathogens. Knowledge of receptor regulation will help optimize the conditions for performing phage infections, and perhaps allow selective detection of pathogens. The results of this work will produce a superior diagnostic reagent with an extended range of applications, and will likely provide valuable information in the biology of mycobacteriophage and mycobacteria. At the conclusion of the training period, the candidate investigator will have had extensive exposure to the methods of microbiology and molecular biology, and the experience to establish an independent research program in an academic setting, with a close connection to clinical medicine. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INITIATION
MYCOBACTERIUM
TUBERCULOSIS
AND
REPLICATION
Principal Investigator & Institution: Madiraju, Murty V. Associate Professor; Biochemistry; University of Texas Hlth Ctr at Tyler 11937 Us Highway 271 Tyler, TX 75708 Timing: Fiscal Year 2003; Project Start 01-AUG-1997; Project End 31-JAN-2008 Summary: (provided by applicant): Human tuberculosis caused by Mycobacterium tuberculosis is the most prevalent and deadly bacterial infectious disease worldwide. This problem is compounded by the emergence of strains of M. tuberculosis that are resistant to one or more anti-tuberculous drugs. Following initial infections, M. tuberculosis frequently enters a latent or dormant state for extended periods and subsequently, under appropriate conditions or following immune suppression, revives, multiplies and causes a secondary infection. DNA replication constitutes an important step in the exit from latency. The development of novel therapeutic agents to control M. tuberculosis infections in HIV infected patients as well as other individuals is severely hindered by our limited understanding of the initiation and regulation of M. tuberculosis DNA replication and its coordination with other events in cell cycle. Initiation of DNA replication is believed to be triggered when DnaA, the putative initiator protein, interacts with oriC or origin of replication. Although oriC is essential for survival, some clinical strains of M. tuberculosis appear to tolerate major deletions and IS6110 insertions in their oriC, thereby raising questions as to how these clinical strains replicate their genome. Our research proposal focuses on understanding the replication initiation process in M. tuberculosis. Specifically, we propose to inactivate oriC, dnaA individually and together by homologous recombination in an attempt to determine whether replication in M. tuberculosis can proceed from alternate origins, and if so whether dnaA function is required for such replication. The interactions of DnaA with replication origins and consequences of these interactions will be investigated using biochemical and genetic approaches. To begin identifying the factors that could potentially affect DnaA activity, a proteomic approach combining twodimensional gel electrophoretic separations of proteins with subsequent identification of protein spots by matrix-assisted laser ionization desorption/ionization mass spectrometry, will be used. Defining the molecular events involved in the initiation and regulation of replication is an essential prerequisite for developing defined systems for identifying novel antimycobacterial compounds, and thereby preventing the development of potentially lethal infections of M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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•
Project Title: MYCOBACTERIUM TUBERCULOSIS CELL WALL VIRULENCE FACTORS Principal Investigator & Institution: Dorman, Susan E. Medicine; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2005 Summary: (provided by applicant): Tuberculosis (TB) kills approximately 3 million people per year. An important characteristic of M. tuberculosis is its ability to survive within human macrophages, and mycobacterial cell wall components are plausible mediators of this effect. However, little is known about the role of specific cell wall components in either mycobacterial survival within host macrophages or modulation of macrophage responses to mycobacterial infection. In our laboratory, the recent generation of a M. tuberculosis transposon-mediated mutant library provides a unique opportunity to systematically investigate these issues. Our overall objective is to understand the role of M. tuberculosis cell wall components in the pathogenesis of TB. Our hypothesis is that M. tuberculosis cell wall components modulate macrophage immune responses, and that alteration of the cell wall will affect macrophage responses to mycobacteria. The specific aim of this R03 proposal is to characterize human macrophage responses to a) infection with M. tuberculosis transposon-mediated mutants predicted to have alterations in the cell wall, and b)stimulation with cell wall fractions from those mutants. Mutants with disruptions in genes known or predicted to be involved in biosynthesis of sulfolipids, lipoarabinomannan, mycolic acids, and phthiocerol dimycocerosate will be used. Host cell cytokine production, chemokine production, apoptosis, IFN gamma-mediated responses, and toll-like receptor-mediated responses will be characterized, and mycobacterial survival and growth within macrophages will be determined. This research will lead to a better understanding of how mycobacterial cell wall components affect bacteria-host cell interactions, and will facilitate identification of new anti-tuberculosis drug targets and vaccine strategies. If cell wall components contribute to M. tuberculosis-induced immune suppression, then evaluation of appropriate mutant strains as vaccines would be warranted. These studies will also serve as a foundation for development of rapid in vitro screening systems to evaluate the effects of other M. tuberculosis genes or cellular components on targeted host immune responses. Finally, new information from the proposed self-contained pilot studies will serve as the foundation for future comprehensive dissection of macrophage response pathways affected by specific cell wall components. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MYCOBACTERIUM TUBERCULOSIS IN VIVO GENE EXPRESSION Principal Investigator & Institution: Graham, James E. Medicine; Vanderbilt University 3319 West End Ave. Nashville, TN 372036917 Timing: Fiscal Year 2001; Project Start 17-SEP-2001; Project End 31-AUG-2006 Summary: (provided by applicant) Mycobacterium tuberculosis is a pathogen that is able to adapt to a variety of different environments encountered during the progressive course of human infection. An ability to inhibit maturation of the macrophage phagosome initially created an ideal environment for bacterial growth, allowing colonization of the body. when deprived of this favorable environment by the hosts's own tissue-damaging immune response, infection often fails to progress, and bacilli enter an nonreplicating latent state, having reached a degree of equilibrium with the host. Changes in the host over time may then allow bacteria to resume replication, leading to further tissue destruction and extracellular growth to high titers. This project
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will identify bacterial genes that are specifically expressed by M. tuberculosis during adaptation to these in vivo environments. A new method developed specifically for examination of mycobacterial mRNAs expressed in infected host cells and tissues (SCOTS) has so far identified 9 M. tuberculosis genes which are expressed in response to growth within cultured human macrophage phagosomes. The first aim of the proposed work is to make bacterial strains specifically inhibited in expression of these genes and evaluate their ability to survive and grow in cultured human macrophages. The second aim is to identify additional M. tuberculosis genes that are differently expressed by tubercle bacilli in another environment that bacilli normally encounter during the natural course of human infection. Bacterial genomic array hybridization with cDNAs obtained by SCOTS will be used to analyze global mRNA expression patterns in bacilli recovered from patient sputum samples, providing insight into the physiology and metabolism of the microbe during active growth in the human lung. Our third aim is to extend analysis of differential bacterial gene expression to a C57BL/6 mouse model of host interaction, facilitating studies of both active and latent types of infection in a genetically defined host. Bacterial cDNA already obtained by SCOTS from tubercle bacilli growing in cultured mouse macrophages and from infected mouse lung tissues will be compared by array hybridization to cDNA from tubercle bacilla growing in human macrophages and lung tissues. A limited number of M. tuberculosis genes commonly expressed in response to these mammalian host interactions will then be evaluated for their contributions to virulence in this animal model. Understanding the roles of such differentially expressed genes will further define host-pathogen interactions in human disease, and allow development of new tools to reduce the enormous global impact of tuberculosis on mankind. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTERIUM TUBERCULOSIS;INDUCED MACROPHAGE SIGNALLING Principal Investigator & Institution: Imboden, John B. Professor; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 31-AUG-2003 Summary: Adapted from the applicant's abstract): Tuberculosis (TB) is the most common fatal infectious disease in the world, and remains a threat to health in the United States. While improved case finding and access to treatment have reduced the incidence of TB in the United States, further improvements in TB control and therapy depend on better understanding of the pathogenesis of TB. Macrophages are essential for defense against infections, and are central to the pathogenesis of TB. When macrophages encounter most bacteria, they phagocytose and kill them. In contrast, macrophages phagocytose, but do not kill M. tuberculosis, even when they are stimulated with IFN gamma. Recent experiments in the PI's laboratory reveal that one means that M. tuberculosis uses to evade killing by macrophages is to block the signal transduction pathway initiated by interferon gamma. The PI has found that infection of macrophages with M. tuberculosis blocks several macrophage responses to IFN gamma, and has found that this disruption of signalling reduces transcriptional activation of IFN gamma-responsive genes at a distal step in the signalling pathway. M. tuberculosis infection of macrophages causes release of one or more soluble factors that inhibit IFN gamma signaling in uninfected macrophages. TGF-beta, IL-4, IL-6, IL-10, and prostaglandin E2 cannot account for this phenomenon. The PI proposes to identify the component of M. tuberculosis that initiates the inhibition of IFN gamma signaling. One hypothesis to be tested is that infection of macrophages by M. tuberculosis induces
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a repressor that binds specific DNA elements in the promoter region of interferon gamma-responsive genes. Finally, the PI will purify the soluble factor present in the conditioned medium from infected cells that inhibits interferon gamma signaling in uninfected macrophages. The proposed experiments will enhance the understanding of the pathogenesis of tuberculosis, and will provide insight essential for developing effective approaches to enhancing the protective immune response to M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MYCOBACTOCID, A NOVEL ANTITUBERCULOSIS DRUG Principal Investigator & Institution: Cynamon, Michael H. Consultant; Natural Drug Science, Llc 48 S Franklin Turnpike Ramsey, NJ 07446 Timing: Fiscal Year 2001; Project Start 01-SEP-2001; Project End 30-JUN-2002 Summary: (provided by applicant):Tuberculosis (TB) poses significant risk to mankind with nearly 8 million people contracting TB and 3 million people dying from it each year. A TB epidemic is becoming more dangerous because of the emergence of multidrug resistant tuberculosis and the lack of effective new drugs. The main goal of Natural Drug Services is to develop and commercialize a new anti-tuberculosis compound, Mycobactocid (MCB). MCB belongs to a new class of chemical compounds, has low toxicity in mice and is effective against strains of tuberculosis resistant to the known anti-tuberculosis agents. The specific aims of this project include:1) evaluation of the anti-tuberculosis activity of MCB in standard, well-characterized in vitro and in vivo models and, 2) compare it to other regimens of treatment. Previous studies indicate that MCB has potent specific activity against M. tuberculosis, low toxicity, and is effective against multi-drug resistant strains. Further study of MCB will facilitate commercialization of this novel class of compounds for treatment of tuberculosis. PROPOSED COMMERCIAL APPLICATION: Tuberculosis claims three million lives a year world-wide and reached epidemic proportions in the US, with outbreaks of drugresistent strains in several cities. The compound under investigation. Mycobactocid, has high specific activity against M. tuberculosis, low toxicity and is active against the resistant strains of M. tuberculosis. The specific goal of this project is to develop preclinical data to facilitate evaluation in human clinical trials and ultimately lead to a commercial product. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MYCOTHIOL BIOSYNTHESIS AND METABOLISM AS TB DRUG TARGETS Principal Investigator & Institution: Fahey, Robert C. Chemistry and Biochemistry; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, CA 92093 Timing: Fiscal Year 2003; Project Start 01-AUG-2000; Project End 31-JUL-2004 Summary: (provided by applicant): Tuberculosis is now second behind AIDS, as the World's most deadly microbial infection. However, a major fraction of AIDS patients die of mycobacterial infections, including TB. The TB problem is aggravated by the growing prevalence of drug-resistant TB, and especially multi-drug resistant (MDR) TB which cannot be treated with the front-line antibiotics for Mycobacterium tuberculosis. It is therefore important that targets be identified for development of new drugs for treatment of MDR TB. Suitable target enzymes should have biochemical functions essential for mycobacteria but with no similar function in mammals making it likely that drugs can be developed that will not lead to adverse reactions in humans. They should have well-defined assays suitable for screening of potential drugs. The proposed
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research elucidates the biochemistry associated with the production and utilization of the antioxidant thiol known as mycothiol. Mycothiol is produced only by mycobacteria, and other actinomycetes, and is not found in animals. The key genes for mycothiol biosynthesis have recently been identified and provide important potential new drug targets. Studies of MSH-deficient mutants indicate that mycothiol metabolism is involved in protecting against oxidative damage and in the detoxification of antibiotics, including one first-line TB drug. Although not essential for the laboratory culture of the model organism Mycobacterium smegmatis, current evidence suggests that mycothiol may be required for survival of M. tuberculosis in an oxygen rich environment. The present studies will determine the extent to which mycothiol is essential for survival of M. tuberculosis, will define the biochemistry involved in the first key step of mycothiol biosynthesis, and will determine how the biosynthesis of mycothiol is regulated. Methods used include new analytical and enzyme assays developed in these laboratories as well as established protocols in biochemistry and molecular biology. The results obtained will provide a key test of the suitability of mycothiol biosynthesis as a target for new TB drugs and will elaborate the biochemistry of a novel class of thiol important to a broad class of soil microorganisms, including most antibiotic producing bacteria. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NANOPARTICLE DELIVERY SYSTEM FOR ANTITURBERCULOSIS DRUG Principal Investigator & Institution: Heifets, Leonid B. Director; National Jewish Medical & Res Ctr and Research Center Denver, CO 80206 Timing: Fiscal Year 2003; Project Start 01-MAY-2003; Project End 30-APR-2005 Summary: (provided by applicant): The U.S. Department of State recently invited us to collaborate with Russian Research Center of Molecular Diagnostics and Therapy (RDCMDT) in the evaluation of nanoparticles as drug delivery vehicles for antituberculosis drugs. While the project by RDCMDT is funded through the International Science and Technology Center (ISTC), funding for the portions of the project to be performed at National Jewish must be funded from U.S. domestic sources. Nanoparticles are small (Submicron) colloidal particles in which different drugs can be entrapped or adsorbed, and their usefulness as an advanced drug delivery system has been demonstrated in many areas. They can be stored without refrigeration for extended periods, and may enable targeting of anti-tuberculosis drugs to macrophages, and the intracellular compartments of macrophages in which Mycobacterium tuberculosis resides. We hypothesize that formulations of existing antituberculosis drugs within nanoparticles modified to traffic to intracellular compartments bearing M. tuberculosis will result in a reduction of the minimal inhibitory concentrations (MIC) relative to the free drugs. Our preliminary data, the only data on anti-TB drugs, suggest that the MICs for streptomycin and isoniazid were reduced by 4.5-7 fold. Dr. Geuelperina at RDCMDT has already begun encapsulation of ethambutol, pyrazinamide, capreomycin, amikacin, kanamycin, ethionamide, levofloxacin, cycloserine, moxifloxacin, vancomycin, and cefoxitin. These drugs, if efficiently encapsulated, will be tested in a macrophage model of M. tuberculosis infection at National Jewish Medical and Research Center in Denver. We will test the hypothesis that increasing the intracellular accumulation, particularly in phagosomes harboring M. tuberculosis will result in enhanced efficacy, as measured by reduced growth and viability of intracellular organisms. We will characterize the accumulation of each formulation by labelling the drugs and following their routes of uptake when the particles are opsonized by serum proteins, when opsonization is
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inhibited by coating the particles with polyethylene glycol, and when the nanoparticles are targeted to specific cell surface receptors via modification with transferrin or mannoside clusters. Distribution of the drugs within the cells over time will also be monitored by immunostaining of the drug molecules within the cells at increasing intervals following uptake. The immunostaining will be made quantitative by creating standard curves for each drug concentration, such that concentrations within particular cellular compartments can be estimated. Finally, we will correlate viability of intracellular M. tuberculosis with drug exposure at the sub cellular level, and determine which formulations are most effective at directing drug accumulation in the cellular compartments harboring M. tuberculosis. Successful completion of this project will provide us with the most promising formulations of encapsulated anti-TB drugs which will create a basis for further collaboration with RDCMDT in preclinical development, including animal models of efficacy, pharmacokinetics, and toxicology. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NATAL-COLUMBIA CLINICAL AIDS/TB TRAINING PROGRAM Principal Investigator & Institution: Abdool Karim, Salim S. Deputy Vice-Chancellor of Research; University of Natal Durban 4041, South Africa Durban, Timing: Fiscal Year 2002; Project Start 22-SEP-2002; Project End 21-SEP-2003 Summary: (provided by applicant): South Africa is currently experiencing one of the worst H IV epidemics in the world and tuberculosis is the most common opportunistic infection associated with advancing HIV disease and AIDS. The recent, substantial increase in numbers of people co-infected with HIV and tuberculosis is exacerbating the existing tuberculosis crisis in South Africa. Building on the longstanding collaborative relationships between the University of Natal and Columbia University with respect to research and training in HIV and tuberculosis through the existing Fogarty AITRP, HVTN and HPTN, we propose to develop a collaborative program in clinical, operational and health services research and training to fill an important training gap in the local response to the HIV and tuberculosis epidemics in South Africa. In order to expand the existing collaboration between the two institutions and the South African Medical Research Council we propose to: i. develop a common vision and understanding of the training program between the US and South African partners; ii. identify the specific research training opportunities and required infectious diseases resources in both the US and South Africa; iii. establish the structure, policies and procedures for the comprehensive ICOHRTA-AIDS/TB co-operative agreement; iv. develop integrated links with other training programs such as the CU-SA Fogarty AITRP; and v. prepare an institutional development plan for the next 10 years demonstrating how ICOHRTA resources will enable the University of Natal to achieve its long-term goals in infectious diseases. The continuum of training concept that has evolved through the CU-SA Fogarty AITRP for HIV and tuberculosis basic science, public health, behavioral and ethics research training where Fellows do coursework in the US and conduct their research in South Africa will be applied to this proposed training program for building clinical, operational and health services research capacity. Training opportunities at Columbia University through the Division of Infectious Diseases in the context of providing clinical care include: consultation rounds for patients on the HIV/TB inpatient care, a unit dedicated to the management of HIV infection and the diagnosis and management of tuberculosis, and augmented by a core conference schedule. The following collaborative projects will provide research opportunities for Fellows in South Africa: HPTN 046, HPTN 035, HPTN 043, HVTN 040, numerous operational tuberculosis projects and new initiatives such as the proposed
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CIPRA which includes an acute infection and natural history study in a clade C population and strategies to integrate antiretroviral therapy with the tuberculosis directly observed short course therapy program. This planning grant will facilitate the development of an ICOHRTA training program which addresses South Africa's priority needs and the institutional development plan of the University of Natal. It optimally utilizes the Columbia University training resources and strong linkages with existing NIH funded clinical research and other Fogarty training programs through building on a well-established, vibrant collaboration involving Columbia University, the University of Natal, the South African Medical Research Council and potentially the NIH Clinical Center. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NOVEL COMPOUNDS FOR IMPROVED TREATMENT OF TUBERCULOSIS Principal Investigator & Institution: Owens, Albert H. Member; Fasgen, Inc. 5210 Eastern Ave Baltimore, MD 21224 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2004 Summary: (provided by applicant): In 2000, the U.S. had over 16,000 cases of infectious tuberculosis and an estimated 10-15 million latent cases. Despite prolonged treatment regimens with significant side effects, reports of drug resistant TB in 43 states, and an estimated $345 million market opportunity, few new drugs to treat TB are in development. FASgen's SBIR Phase I goal is to choose a lead anti-tuberculosis candidate from 6 molecules active in vitro against Mycobacterium tuberculosis. Phase I specific aims are: 1) resupply candidate compounds and determine purity and chemical and biological stability; 2) determine minimum inhibitory concentrations (MICs) against a panel of mycobacteria in vitro; 3) determine in vitro cytotoxicities (lC50s) against Vero cells; 4) determine maximum tolerated dose (MTD) and dose limiting toxicity in mice for compounds with selectivity index (MTD/IC50) >10; 5) test the best compound in a murine inhalation model of TB; 6) verify that the MTD is not substantially different in immunocompromised (beige) mice; 7) test whether the best anti-tuberculosis compound is also active against M. avium in an immunocompromised mouse model; 8) repeat/confirm efficacy in animal models. In SBIR phase II, the lead compound will undergo safety/toxicology testing in animals and Phase I/Il safety and Phase II efficacy testing in man, most likely against multidrug resistant TB. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: NOVEL DELIVERY OF A NEW VACCINE FOR TUBERCULOSIS Principal Investigator & Institution: Pereira, Diane; Mycos Research, Llc 217 Racquette Dr, #6 Ft. Collins, CO 80524 Timing: Fiscal Year 2003; Project Start 01-FEB-2003; Project End 30-NOV-2003 Summary: (provided by applicant): Tuberculosis is responsible for the death of over 3.3 million people each year, which is more than any other infectious agent. The current vaccine for tuberculosis is the BCG vaccine. BCG has been proven safe and effective at protecting young children, it is relatively inexpensive to produce, and it requires only a single immunization. Unfortunately, protecting adults from pulmonary disease with the BCG vaccine has been variably efficacious. As a result, the effort to produce a novel and efficacious vaccine for tuberculosis has intensified over the last decade. In the mouse model, the combination of a TH1 stimulating adjuvant and a M. tuberculosis derived recombinant fusion construct (Corixa Corporation) has resulted in a approximately 2 log
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decrease in bacterial burden following an aerogenic challenge with M. tuberculosis (approaching the level seen with the BCG vaccine). This vaccine formulation has also been effective in the guinea pig and cynomolgus monkey models of tuberculosis. This application proposes to investigate the ability of a transcutaneous immunization (TCI) to deliver this vaccine formulation to mice. The effectiveness of TCI has been demonstrated in pre-clinical and clinical trials and has been shown to induce both serum and mucosal humoral responses as well as cellular immune responses. The success of this novel delivery method will be evaluated by its ability to induce the development of a TH1 immune response and to provide a significant level of protection following a low dose aerosol challenge with M. tuberculosis. If successful, the advantages of transcutaneously delivering a vaccine for tuberculosis would be the lack of a needle injection, the simplicity of administration (especially in developing countries where tuberculosis is endemic), and the potential for using a lower dose of specific antigen, which would decrease the cost of vaccination. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NOVEL INHIBITORS OF M. TUBERCULOSIS RNA POLYMERASE Principal Investigator & Institution: Lynch, Anthony S.; Cumbre, Inc. 1502 Viceroy Dr Dallas, TX 75235 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 14-APR-2004 Summary: (provided by applicant): Previous efforts at Cumbre Inc. have resulted in the discovery, characterization and optimization of a novel class of inhibitors of bacterial RNA polymerase The overall objective of this project is to establish the SAR of the Cumbre series as inhibitors of the RNA polymerase derived from M. tuberculosis, the causative agent of TB. These studies will involve the preparation and reconstitution of recombinant core and holoenzyme forms of M. tuberculosis RNA polymerase and the adaptation of current plate and gel format assays for the characterization of the mechanism of action of inhibitors of the M. tuberculosis enzyme. In addition, the Cumbre Inc. inhibitor series will be assessed for antibacterial activity against a panel of Mycobacterium species with the goal of establishing a strategy wherein future synthetic chemistry efforts may be directed toward the optimization of the series for activity against M. tuberculosis. Finally, additional studies will be focused toward establishing whether there may be any therapeutically relevant benefits of combining the Cumbre inhibitor series with rifampicin, a known inhibitor of bacterial RNA polymerase, which is clinically approved and currently used for the treatment of TB. Preliminary data obtained in Escherichia coli suggests that some mutations conferring resistance to rifampicin cannot be combined with mutations conferring resistance to the Cumbre series, and vice versa. Hence, the frequency of resistance to either agent may be reduced when the two classes of inhibitors are administered together in a single regimen. The results of the combined Phase I studies should provide a platform of data by which an informed decision can be made as to whether the Cumbre series has potential for future development as a novel antitubercular agent. Phase II studies would thereafter be addressed toward systematic optimization of the series as an antitubercular agent. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: NOVEL MECHANISM OF INNATE DEFENSE IN MURINE TB Principal Investigator & Institution: Kornfeld, Hardy; Professor; Medicine; Boston University Medical Campus 715 Albany St, 560 Boston, MA 02118 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-JUL-2004
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Summary: We discovered that M. tuberculosis (Mtb) infection causes apoptosis of primary human alveolar macrophages (AMphi) in vitro by a tumor necrosis factor-alpha death signaling pathway. Apoptotic AMphi are present in bronchoalveolar lavage of tuberculosis (TB) patients and in TB lung biopsies. We found that virulent Mtb strains downregulate apoptosis of host AMphi, and we have shown that apoptosis is linked to microbicidal processing. Based on these observations, we hypothesize that lung Mphi apoptosis constitutes a newly recognized mechanism of innate immunity in TB. TB is a major co-infection in AIDS, contributing to increased morbidity and mortality. With declining adaptive immunity in AIDS, the relative importance of innate immunity may be augmented. Our research plan proposes novel use of the murine aerosol TB model to define the role of lung Mphi apoptosis in disease susceptibility and resistance in vivo. The specific aims address three key issues: 1, we will characterize the kinetics and distribution of lung Mphi apoptosis following aerosol Mtb infection; 2, we will examine the relationship between lung Mphi apoptosis and host resistance in experiments comparing Mtb strains of high and low virulence, common inbred mouse strains that are innately susceptible or resistant to TB, wildtype and knockout mice with targeted deletion of genes regulating apoptosis, and Mtb transduced to express apoptosis regulating genes; 3, we will evaluate the impact of lung Mphi apoptosis on cytokine expression and the pattern of leukocyte recruitment to the lung in TB, and it's contribution to the development of delayed type hypersensitivity. These studies will provide a detailed analysis of early events in murine TB, and will be the first to fully characterize lung Mphi apoptosis in vivo and to define it's contribution to the integrated host response to infection. Our model may reflect a mechanism broadly involved in the defense against a variety of other intracellular pathogens (such as Legionella) of immediate relevance to the HIV-infected host. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NUTRIENT TRANSPORT MECHANISMS IN MYCOBACTERIA Principal Investigator & Institution: Connell, Nancy D. Associate Professor and ViceChair for r; Microbiol & Molecular Genetics; Univ of Med/Dent Nj Newark Newark, NJ 07103 Timing: Fiscal Year 2001; Project Start 01-JUL-1993; Project End 31-MAY-2005 Summary: (Adapted from the Applicant's Abstract): Mycobacteria are extremely important human pathogens. Their unusual cell wall structure and slow growth rate make them difficult to study from the standpoint of basic bacterial physiology. Mycobacteria have evolved into facultative intracellular parasites, capable of surviving with the phagocytic vacuole of the macrophage. It is likely that the ability of mycobacteria to acquire nutrients within the macrophage vacuole is tightly linked to intracellular survival and, therefore, to virulence. Knowledge of nutrient transport mechanisms for virulent mycobacteria will contribute directly to the design of novel therapeutic strategies and the development of new vaccines. The PI has isolated several mutants of Mycobacterium bovis BCG and M. tuberculosis which are deficient in transport of amino acids and peptides. In this competing continuation proposal, the PI will use classical and molecular genetic techniques and macrophage infection technology to address four specific aims: (1) to isolate and/or construct mutants of BCG and M. tuberculosis defective in transport and metabolism of arginine and oligopeptides; (2) to characterize transport of substrates by mutant and wild type BCG and M. tuberculosis; (3) to isolate and characterize transport regulatory mutants of BCG and M. tuberculosis; and (4) to examine the survival and growth characteristics, and nutrient transport activities, of mutant mycobacteria within mouse and human
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macrophages. These experiments will lead to a more complete understanding of the vacuolar environment which is the preferred ecological niche for mycobacteria within a mammalian host, and the nutrient uptake strategies employed by virulent mycobacteria to survive and grow in that intracellular environment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PHARMACOKINETICS OF ISONIAZID/RIFABUTIN--HIV RELATED TB Principal Investigator & Institution: Sterling, Timothy R.; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: The treatment of tuberculosis in persons co-infected with HIV using standard rifampin-based regimens is generally successful. However, rifampin activates the hepatic cytochrome p450 enzyme system, resulting in a 40-90% decrease in serum concentrations of the HIV-1 protease inhibitors, a key component of highly active antiretroviral therapy. Rifabutin ishighly active against M. tuberculosis, yet is a lesspotent inducer of the cytochrome p450 enzyme system than rifampin. The CDC Tuberculosis Trials Consortium study, "Treatment of HIV-related tuberculosis using a rifabutin-based regimen" (USPHS Study 23--JCCI RPN 99-01-26-01), is evaluating the efficacy of an intermittent TB treatment regimen that includes rifabutin. Although the pharmacokinetics of isoniazid and rifabutin in immunocompetent individuals are sufficient to result in effective therapy without drug toxicity, there are concerns that malabsorption and diarrhea in HIV-infected persons will affect the pharmacokinetics of both drugs. The purpose of this study is to determine the proportion of patients with HIV-related tuberculosis who have abnormal pharmacokinetics of isoniazid and rifabutin. This study will also determine risk factors for abnormal pharmacokinetics of isoniazid and rifabutin, and evaluate the correlation between the pharmacokinetic parameters of isoniazid and rifabutin and the therapeutic efficacy and toxicity attributed to these medications. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PHOSPHOLIPASES IN THE PATHOGENESIS OF M. TUBERCULOSIS Principal Investigator & Institution: Parker, Sarah K. Microbiology; University of Colorado Hlth Sciences Ctr Uchsc at Fitzsimons Aurora, CO 800450508 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 30-APR-2007 Summary: (provided by applicant): The applicant, Dr. Sarah Parker, will conduct research on phospholipases in the pathogenesis of Mycobacterium tuberculosis. Though M. tuberculosis kills three million people per year, 450,000 of whom are children, the mechanisms of its success are not well understood. Evidence suggests that phospholipase A2 (PLA2), which is described here for the first time in M. tuberculosis, and phospholipase C (PLC) are surface exposed components of the mycobacterial cell wall. These phospholipases degrade phospholipids to fatty acids and other inflammatory mediators, and Dr. Parker hypothesizes that they contribute to the intracellularsurvival or extracellular pathogenesis of M. tuberculosis. She postulates that the degradation of hostsurfactant and membranes by M. tuberculosis PLC and PLA2 contributes to virulence by a number of possible mechanisms: facilitation of bacillary entry into host cells, production of inflammatory mediators, alteration of the phagosomal environment, and/or acquisition of nutrition in the form of fatty acids. Dr. Parker has designed her specific aims to address each of these possible contributions of
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PLAand PLC to M. tuberculosis pathogenesis. Currently she has a partial PLC deletion mutant in M. tuberculosis. Once she has purified PLA2 from M. tuberculosis, she will use the partial amino acid sequence to identify the pla2 gene. She will construct phospholipase C, A2 and C + A2 deletion mutants, and they will be bacteriologically and biochemically characterized. Phospholipid degradation and utilization by these strains will be investigated in varied nutritional environments, including using pulmonary surfactant as a carbon source. In a human macrophage model of infection, she will analyze PLC and PLA2 contributions to bacillary macrophage entry, trafficking and survival. The research project is designed to result in Dr. Parker becoming a successful and independent M. tuberculosis investigator. Not only will she learn many basic and state-of-the-art techniques, she will receive didactic training to compliment her research. She will spend 15% of her time honing her clinical skills as an assistant professor in Pediatric Infectious Diseases. Dr. Parker is committed to research in M. tuberculosis, and it holds a special significance to her because of her life experiences. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PLANNING GRANT FOR ICOHRTA AIDS/TB PROGRAM IN INDIA Principal Investigator & Institution: Pande, Jitendra N.; All-India Institute of Medical Sciences Ansari Nagar New Delhi, Timing: Fiscal Year 2002; Project Start 22-SEP-2002; Project End 21-SEP-2003 Summary: (provided by applicant): Both AIDS and TB are very large health problems in India, The objective of this project is to develop a plan for the coming ten years to strengthen integrated and multidisciplinary research and training at the All India Institute of Medical Sciences (AIIMS), New Delhi, India in the fields of HIV/AIDS and tuberculosis. The University of California in Los Angeles (UCLA) will be the partner institution in the U.S, in this collaboration. Research, training and capacity building needs are truly multidisciplinary and will encompass clinical medicine, drug and vaccine trials, behavioral and social sciences, operational research and health services research. This will include cost-effective strategies for prevention, diagnosis and management of tuberculosis and HIV/AIDS and associated complications, including opportunistic infections. The project envisages training of several categories of health professionals including clinicians, laboratory scientists, research nurses, social scientists, psychologists, biostatisticians, and health care administrators. It will consolidate partnership with other stakeholders in India including Ministry of Health, Directorates of National Programs for Tuberculosis and AIDS, research organizations such as Indian Council of Medical Research (ICMR) and its institutions (Tuberculosis Research Center, Chennai, National AIDS Research Institute, Pune), Department of Biotechnology of the Ministry of Science and Technology, etc. Leadership in key Departments at AIIMS have been identified and agree to participate. Similar steps have been taken at UCLA. Multiple linkages already exist between faculty at AIIMS and UCLA. Administrative support has been obtained at the highest levels. Preliminary meetings took place in New Delhi, February 2002. Key Faculty from AIIMS will visit UCLA, the FIC and other NIH institutes and attend major relevant research meetings in the U.S. between June 2002 and February 2003. Several UCLA Faculty will visit AIIMS. Planning will proceed from June 2002 with repeated opportunities for input and critique by involved faculty at both institutions. This will complement existing or planned U.S, programs including FIC, CIPRA, ICTTRI and international CFAR activities, Current plans for training at UCLA and courses, workshops and cooperative projects at AIIMS in 2002 and 2003 will continue. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: POPULATION BASED INVESTIGATIONS OF TUBERCULOSIS Principal Investigator & Institution: Alland, David; Associate Professor of Medicine; Montefiore Medical Center (Bronx, Ny) Bronx, NY 104672490 Timing: Fiscal Year 2001; Project Start 01-MAY-2000; Project End 04-JAN-2002 Summary: (adapted from applicant's abstract): The rational design and implementation of the next generation of therapies and vaccines against Mycobacterium tuberculosis will require a thorough understanding of the mechanisms of antibiotic resistance and bacterial pathogenesis as they apply to human infections. In this proposal, populationbased genetic studies of human specimens will be used to determine the clinical consequences of mutations in genes associated with bacterial antibiotic resistance and virulence. Previous population based genetic studies of M. tuberculosis have been limited in scope due to the difficulty and expense of large scale DNA sequencing or DNA chip analysis. The applicants have developed molecular beacon PCR assays which are expected to allow them to rapidly and accurately screen large numbers of samples for specific genetic mutations with the accuracy of a single base pair. They will screen for the presence of larger insertions and deletions in DNA sequence using a modified slot blot cross- hybridization approach. They propose to use novel transducing phage techniques to induce homologous recombination and gene substitution in M. tuberculosis. These techniques and more established methods will be used to study clinical M. tuberculosis isolates acquired by three large scale epidemiological studies underway in Arkansas, San Francisco, and Orizaba, Mexico. DNA sequence analysis will be combined with classical and molecular epidemiology to study the mutational events that lead to resistance to isoniazid (INH), to determine the distribution of specific mutations in susceptible and resistant isolates, and to discover other mechanisms of isoniazid resistance that develop in clinical strains. Transducing phage assays will be used to uncover and characterize additional resistance mutations. A similar approach will be applied to investigate infectivity and virulence in clinical M. tuberculosis strains. The complete genomic sequence differences between the laboratory strain H37Rv, and a highly virulent clinical strain CSU093, have recently become known. Studies of these sequence variations in clinical strains will be used to determine the associations between specific mutations and the phenotypes of infectivity and virulence. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: POPULATION BASED MOLECULAR EPIDEMIOLOGY OF TB Principal Investigator & Institution: Hopewell, Philip C. Professor of Medicine; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 01-APR-1993; Project End 31-JUL-2006 Summary: (provided by applicant): Since January 1991 we have been using genotyping ot Mycobacterium tuberculosis together with conventional epidemiological approaches to elucidate the distribution and dynamics of tuberculosis in San Francisco. During this time we have refined and validated molecular epidemiological methods and applied these methods in a systematic series of studies that have been used to guide interventions tailored to the prevailing epidemiological circumstances. This study will extend our previous population-based, molecular epidemiologic studies of tuberculosis in support of the broad objective of eliminating tuberculosis in San Francisco that is caused by the transmission of Mycobacterium tuberculosis in San Francisco. This objective can be measured only by long-term application of molecular epidemiological methods. In addition, we propose to contribute to this objective by utilizing our detailed
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understanding of the dynamics of tuberculosis in San Francisco to examine genetic factors in both host and microbe that are associated with transmission of M. tuberculosis and progression of tuberculosis infection to clinical tuberculosis. In the proposed studies we will be combining state-of-the-art molecular epidemiology with recent advances in molecular biology, genomics, and computational biology in the setting of an effective tuberculosis control program to address some of the major current impediments to the elimination of the disease. The specific aims have been divided into four closely related components, intended to examine the interrelationships between clinical and epidemiological features of tuberculosis and human host and microbial genetic events in a setting wherein findings can be translated quickly to tuberculosis control efforts. The specific aims are divided as follows: 1) Identification and evaluation of tuberculosis control strategies; 2) Quantification of exposure and transmission; 3) Identification of host gene expression responses that distinguish susceptible and resistant persons; 4) Identification of mycobacterial factors associated with various outcomes following exposure to infectious tuberculosis. The components of these aims are all related to elucidating the factors related to transmission of M. tuberculosis and directed toward providing the scientific basis for measures designed to prevent transmission. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PRO-APOPTOTIC TUBERCULOSIS VACCINE Principal Investigator & Institution: Kernodle, Douglas S. Associate Professor; Medicine; Vanderbilt University 3319 West End Ave. Nashville, TN 372036917 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2007 Summary: (provided by applicant): A major hurdle in the development of effective vaccines against pathogens that reside within macrophages, including Mycobacterium tuberculosis, is how to deliver antigens in a manner that stimulates a protective cellular immune response. Recent investigations involving antisense mutants of M. tuberculosis that have diminished production of iron-cofactored superoxide dismutase (SOD) show that they are attenuated, induce strong CD4+ and CD8+ T-cell responses in mice, and exhibit promising activity as a vaccine prototype. These effects appear to be related to an unmasking of the innate immune responses normally inhibited by SOD, which is a prominent extracellular enzyme of M. tuberculosis and other pathogenic mycobacteria. The enhanced innate host immune responses presumably permit apoptosis-associated cross-presentation of microbial antigens via MHC Class I pathways to induce strong adaptive CD4+ and CD8+ T-cell responses, in contrast to the current vaccine for tuberculosis, BCG, which exhibits a predominant CD4+ T-cell response and minimal CD8+ T-cell responses. The goals of the current proposal are first, to characterize the cellular and cytokine responses in the lung observed early after infection with SODdiminished M. tuberculosis, as rapid pulmonary interstitial infiltration with mononuclear cells undergoing apoptosis appears to be a process unique to the SODdiminished strains that is not observed during infection with either virulent M. tuberculosis or BCG. This should define the conditions under which antigen crosspresentation occurs in vivo, yielding information that may be useful for a variety of vaccines. The second goal is to construct non-reverting SOD-diminished mutants of H37Rv and BCG by replacing the wild-type SOD allele with mutant alleles, some of which encode enzymatically less efficient mutants of SOD. This should yield a SODdiminished vaccine candidate that is stable and safe enough for administration to man. The third goal is to determine the optimal level of SOD production for maximal vaccine efficacy and the immune correlates of protection. Diminishing the production of factors
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produced by intracellular pathogens that inhibit macrophage apoptosis is a strategy for making new vaccines that achieve MHC Class I antigen presentation. This should have implications not only for tuberculosis but also for other infectious diseases in which CD8+ T-cell responses are a critical component of a protective immune response. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PULMONARY IMMUNE DEFENCES AGAINST M. BOVIS-BCG INFECTION Principal Investigator & Institution: Fulton, Scott A. Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-JUL-2005 Summary: Dr. Fulton is an Instructor at Case Western Reserve University School of Medicine in the Division of Infectious Diseases. As a fellow, Dr. Fulton was supported by an institutional training grant supported by the NIAID and a training grant from the American Lung Association. He has been responsible for setting up a new animal model to study the immune response to tuberculosis and chosen to continue studying tuberculosis at CWRU because of its long and productive interest in in immunity to tuberculosis. The scholarly environment was initiated by Dr. J. Ellner who is the current PI for a multi-center collaborative Tuberculosis Research Unit. Dr. Fulton's mentors, Drs. W. H. Boom and F. P. Heinzel are interested in cellular immune responses to complex intracellular pathogens and have had significant success in training physician scientists. Other members include Dr. Z. Toossi, Dr. R. Wallis, Dr. C. Hirsch and Dr. R. Silver who contribute to the intellectual environment. Additional formal and informal interactions with scientists in Geographic Medicine (Dr. J. Kazura, Dr. C. King, Dr. E. Pearlman) will foster Dr. Fulton's training environment. Dr. Fulton now proposes addditional specialized training with the goal of becoming an independent physician scientist. He has set up a mouse model of aerogenic M. bovis-BCG infection as a model of human disease since M. bovis-BCG grows in the lung and elicits protective T cell responses that eradicate 99 percent of the bacteria. 1 percent of bacteria persist in a steady state infection characterized by granuloma formation and immune surveillance. In contrast, viulent M. tuberculosis leads to progressive infection. Since human pulmonary immune responses can be only partially and indirectly studied, animal models have provided a means for studying the unique susceptibilty of the lung. To analyze immune responses that characterize the susceptibility of the lung to mycobacteria, three aims are proposed: Aim 1. To characterize the expression of chemokines and cytokines within the bronchoalveolar and lung microenvironments during early, maximal, and late immune activation phases of aerogenic M. bovis-BCG and M. tuberculosis infection. Aim 2. To characterize mycobacterial antigen presentation and T cell activation within the bronchoalveolar and lung microenvironments during early, maximal and late immune activation phases of aerogenic M. bovis-BCG and M. tuberculosis infection. Aim 3. To characterize the early pulmonary immune responses to aerogenic M. bovis-BCG and M. tuberculosis during local and systemic immunomodulation of cytokine and chemokine expression. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PUTATIVE MYCOBACTERIAL REGULATORY FACTOR Principal Investigator & Institution: Godfrey, Henry P. Professor; Pathology; New York Medical College Valhalla, NY 10595 Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2002
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Summary: (Adapted from the Applicant's Abstract): The recent increase in the incidence of tuberculosis (TB) in the United States (due at least in part to the HIV epidemic) has focused renewed interest on this disease and the physiology of its causative organism. Regulation of gene expression in pathogenic mycobacteria such as M. tuberculosis is incompletely characterized. The investigators have identified a 36 nucleotide sequence, rpt-1, occurring both as single units and as 2-, 3- or 4-unit tandem repeats in 30 M. tuberculosis sequences. Sequences similar to rpt-1 were also found to occur in M. bovis BCG and M. leprae, suggesting a broader role of repetitive DNA elements in regulating mycobacterial gene expression. rpt-1 sequences occurred upstream and in the same orientation to annotated ORFs in 27 of 30 cases; in the remaining cases, rpt-1 was located between two ORFs read in opposite directions. Among the genes preceded by rpt-1 sequences were a putative DNA helicase, a putative transcriptional regulator and several putative response regulators. Tandem repeats of rpt-1 units were separated by 20-23 intervening nucleotides, close to the number of nucleotides comprising two turns of the DNA helix so that tandem rpt-1 sequences lie on the same side of the DNA helix. The regulatory role of rpt-1 or its ability to interact with mycobacterial proteins is completely unknown. The investigators' preliminary studies using electrophoretic mobility shift assays and radiolabeled rpt-1 probes indicated that rpt-1 bound specifically to distinct protein(s) in cleared BCG cytosolic lysates. Unexpectedly, specific DNA-binding activity for rpt-1 was also present in nuclear extracts from eukaryotic HeLa and MCF-7 human breast cancer cell lines. The existence of mycobacterial DNA recognition sites in eukaryotic transcription factors could have important implications for pathogenesis of TB and other chronic intracellular infections. The long-term goal of this project is to characterize the role of regulators of mycobacterial gene expression in the pathogenesis of TB. The present project will test the hypothesis that rpt-1 is important for regulating mycobacterial gene expression. The Specific Aims of the project are: 1) characterize nucleotide motifs involved in binding of rpt-1 to mycobacterial cytosolic proteins; 2) characterize the protein(s) binding to rpt-1 biochemically using microsequencing and cloning and immunochemically; and 3) examine the role of rpt-1 as a regulatory element in M. tuberculosis gene expression under various environmental conditions and in reporter gene fusions preceded by rpt-1 sequences. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: RAPID DIAGNOSTICS FOR TB Principal Investigator & Institution: Kritski, Afranio; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: Rapid and accurate diagnosis of tuberculosis (TB) is the essential first step in the successful treatment and control of the disease in the individual and the community. In addition, rapid identification of drug- resistant TB is critical for tailoring effective regimens, particularly in areas with emerging drug-resistant TB. Current diagnostic tests for TB are cumbersome, expensive, slow and may lack sensitivity. Novel tests that provide an accurate result and that are affordable and feasible for studies of innovative diagnostic methods for detecting Mycobacterium tuberculosis in clinical specimens from clinical settings in a developing country, and to evaluate novel assays for the rapid diagnosis of drug- resistant TB. The purpose of this project is to develop and evaluate novel, rapid methods for the identification of M. tuberculosis and drug resistance from clinical specimens in a developing country setting. We will utilize the patient population of the University Hospital in Rio de Janeiro to perform pilot studies of candidate tests in patients suspected of having TB. Candidate tests will be evaluated in well-characterized
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patients for sensitivity, specificity, predictive value, and time required to provide results in comparison with conventional techniques. Candidate tests that show promise in the hospital setting will then be field tested in patients suspected of having TB at community clinics at the sites of our other trials (Project 1 and 2 of this proposal). Candidate tests to be developed and evaluated include: 1) C18-Carboxypropylbetaine (CB-18) specimen processing for enhancement of identification of M. tuberculosis by smear and in standard solid and radiometric media assays, as well as in nucleic acid amplification tests; 2) multiantigen serologic assays using lipid antigens of M. tuberculosis to distinguish active diseases from latent or absent infection; 3) solid phase amplification assays (e.g., Lineprobe (LIPA]) for rapid identification of both M. tuberculosis and specific resistance mutations; 4) use of immunocapture of BrdUlabeled mycobacterial DNA with PCR amplification to diagnose TB and identify phenotypic resistance after in vitro exposure to anti-mycobacterial drugs; and 5) use of redox dyes such as Alamar blue and triphenol tetrazolium to determine rapidly MICs of M. tuberculosis to first line anti-mycobacterial drugs. Identification of novel diagnostic tests that can provide a reliable and rapid diagnosis of TB and of drug resistance will contribute substantially to improving TB control globally. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: RATIONAL INHIBITION OF INHA, A TUBERCULOSIS DRUG TARGET Principal Investigator & Institution: Tonge, Peter J. Associate Professor; Chemistry; State University New York Stony Brook Stony Brook, NY 11794 Timing: Fiscal Year 2001; Project Start 01-MAY-1999; Project End 30-APR-2003 Summary: (Adapted from Applicant's Abstract) The development of multidrug-resistant strains of Mycobacterium tuberculosis and the emergence of tuberculosis as a major opportunistic infection in HIV- infected people are linked to the worldwide increase in tuberculosis. InhA, the NADH-dependent enoyl-ACP reductase from M. tuberculosis, is the target for the antitubercular drug isoniazid. The enzyme is involved in the biosynthesis of fatty acids and enzyme inhibition interferes with the synthesis of mycolic acid, a critical component of the mycobacterial cell wall. Currently 20% of M. tuberculosis strains are resistant to isoniazid due primarily to mutations in the InhA protein or mutations in the metabolic pathway for isoniazid activation. Consequently, InhA is a bona fide target for the development of novel antitubercular agents. The objective of this proposal is to provide detailed information on the mechanism of InhA with the knowledge that this information will be critical to the design and development of drugs that are effective against both sensitive and drug-resistant strains of M. tuberculosis. Initial efforts are focused on using conjugated substrate analogs to probe the mechanism of InhA. Raman and infrared spectroscopy will provide precise information concerning the geometry and electronic structure of substrates, substrate analogs and cofactors bound to InhA. The vibrational studies will elucidate whether the enzyme promotes catalysis by activating the ground state of the substrate or cofactor to hydride transfer. Site-directed mutagenesis, in concert with the spectroscopic studies, will be used to identify and quantitate the role of specific amino acid residues in substrate activation and transition state stabilization. Structural information on the enzyme- bound ligands will also be provided by NMR spectroscopy using transferred NOE methods and X-ray crystallography. This information will be combined with studies into the molecular basis for isoniazid action to drive the development of novel InhA inhibitors that are effective against both sensitive and drug-resistant strains of M.
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tuberculosis. Finally, the antimycobacterial activity of InhA inhibitors will be tested directly against cultures of M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: REACTIVATION TUBERCULOSIS IN A/J MICE Principal Investigator & Institution: Jagannath, Chinnaswamy; Associate Professor; Pathology and Lab Medicine; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) One quarter of the world's population is infected with M. tuberculosis resulting in approximately 2.9 million deaths each year. Reactivation tuberculosis is the major cause of adult tuberculosis today although the mechanisms that predispose to reactivation are complex and enigmatic. Since animal models are useful for understanding pathogenesis of tuberculosis, we performed a series of studies to standardize murine models of acute, chronic and reactivation tuberculosis by varying the mouse strain, route and dose of infection. We then found that A/J mice were highly susceptible while C57Bl/6 mice were relatively resistant to progressive disease when infected either i.v. or via aerosol routes. A/J mice were unable to form granulomas in lungs and their macrophages were defective in killing MTB. Unlike C57Bl/6 mice, A/J mice also underwent an early and near uniform reactivation of tuberculosis following the Cornell model. A/J mice have a deletion in the gene encoding for Complement C5 which in intact mice yields the C5a anaphylatoxin, a known regulator of cytokine and chemokine synthesis of macrophages. Therefore, in this investigation, we will examine the hypothesis that the lack of C5a compromises the immune responses in mice allowing the reactivation of tuberculosis through the following aims. Specific Aim I will investigate whether the deletion in C5 gene affects the synthesis of cytokines (TNF alpha, IL1-beta and IL-6), prevents macrophage activation and thereby macrophage mediated killing of MTB in A/J mice. Specific Aim II will investigate the effects of the deletion in C5 gene to the secretion of chemokines by MTB infected A/J macrophages and evaluate whether they are important in causing influx of immune cells into the lungs and formation of granulomas. Specific Aim III will characterize histological, cytokine and chemokine responses of lungs in A/J mice to determine the type of immune response (Th1 vs Th2) that dominates during the reactivation of tuberculosis. These studies are anticipated to enhance our understanding on putative mechanisms that precede the reactivation of tuberculosis in the lungs of mice and ultimately help us to develop better strategies to prevent tuberculosis in man. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: REGULATION OF HOST RESPONSE GENES IN PATHOGENESIS OF TB Principal Investigator & Institution: Roman, Jesse R. Division Director; Medicine; Emory University 1784 North Decatur Road Atlanta, GA 30322 Timing: Fiscal Year 2001; Project Start 10-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death worldwide. Our inability to control the spread of this disease and the absence of new effective chemotherapeutic agents are due in part to the limited knowledge about host genes that control granuloma formation and other aspects of the host's response to this pathogen in lung. One host response considered important in tuberculosis is that of tissue remodeling which is characterized by alterations in
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extracellular matrix expression and degradation. Tissue remodeling is responsible for the development of Mtb- mediated fibrosis, bronchiectasis, and cavitation. Although these processes are often considered late manifestations of pulmonary tuberculosis, mounting evidence suggests that the genes involved in the control of tissue remodeling (TR genes) are expressed very early in lung after Mtb infection, and are involved in other key processes including leukocyte recruitment and granuloma formation. Consistent with this, we have demonstrated that: 1) Mtb Erdman bacilli and isolated cell wall components of Mtb induce the expression of TR genes encoding for extracellular matrices, matrix-degrading proteases, and pro-fibrotic growth factors in vitro (i.e., human monocyte/macrophages) and in vivo (C57BL/6 mice). In mice, the induction of TR genes correlated both spatially and temporally with the inflammatory response. 2) The induction of TR genes in monocyte/macrophages by Mtb occurs via receptormediated protein kinase pathways and requires the induction of specific transcription factors (e.g., AP-1). 3) The injection of trehalose-6,6'-dimycolate (previously called mycobacterial cord factor) or live Mtb Erdman strain into mice with knockout mutations in a TR gene (Matrix Metalloproteinase-9) resulted in increased inflammation and granuloma formation. Together, this information suggests that the interaction between Mtb and host cells triggers TR gene expression; in turn, the products of TR genes play important roles in the host response to Mtb. The overall goal of this application is to identify the TR genes that are differentially expressed in pulmonary tuberculosis and study their function. This will be accomplished in 3 specific aims designed to: 1) Identify host TR genes differentially expressed in vitro in human monocyte/macrophages after infection with Mtb using High Density Oligonucleotide Array or HDOA. 2) Identify the TR genes differentially expressed in the lungs of infected mice and in the lungs of humans with pulmonary tuberculosis using HDOA. 3) Determine the function of specific TR genes identified in Specific Aims I and II by infection of mice with gene knockout mutations. We propose to study the function of 3 (at the most 4) TR genes. We will begin exploring the function of 2 TR genes encoding for matrix metalloproteinases which we have demonstrated to be differentially expressed in Mtb-infected lungs and for which knockout animals are already available in our laboratory. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ROLE OF HTRA-LIKE SERINE PROTEASES IN TB PATHOGENICITY Principal Investigator & Institution: Morin, Paul M. Microbiology and Immunology; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2001; Project Start 01-APR-2001 Summary: (provided by the applicant): Two billion people are currently infected with the infectious organism, M tuberculosis, which causes tuberculosis. Understanding TB pathogenicity is critical for the development of more effective treatment and vaccines that could change the global emergency. This research proposal will use novel genetic tools to create specific TB mutants that will be used to elucidate the role of HtrA-like serine proteases in M tuberculosis pathogenicity. HtrA serine proteases have been shown to be associated with virulence in many organisms including Salmonella, Yersinia, and Brucella, and are induced by exposure to heat shock and oxidative and osmotic stresses. By using a novel phage delivery system, allelic exchange will be performed in TB to create mutants with deletions in the HtrA-like serine protease genes: htrA, pepA, and Rv0983. These 3 TB mutants will be characterized in vitro for their growth characteristics, thermosensitivity, and ability to survive in cultured macrophages. TB serine protease mutants will also be characterized in vivo by infecting C57BL/6 mice and BALB/c SCID mice to determine if mutants are attenuated in the
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murine model. HtrA mutants in other organisms have been shown to act as vaccines by protecting mice against a lethal challenge with the wildtype organism. BALB/c mice will be vaccinated with TB serine protease mutants and challenged with a virulent wildtype strain of TB. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ROLE OF MMPL TRANSPORTERS IN M. TUBERCULOSIS VIRULENCE Principal Investigator & Institution: Cox, Jeffery S. George Williams Hooper Foundation; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 15-SEP-2001; Project End 31-AUG-2006 Summary: (provided by applicant) Tuberculosis (TB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. For years, the identification of new drug targets has been hampered by the intractability of the bacillus to genetic analysis. Now with the advent of powerful genetic tools, combined with well-established mouse infection models, we have isolated novel M. tuberculosis mutants with lesions in individual genes that are required for normal growth during acute infection. Our initial results have led us to the hypothesis that M. tuberculosis influences host- pathogen contacts by utilizing the MmpL family of transporters to secrete biologically active lipids to the surface of the mycobacterial cell and ultimately into infected host cells. The studies proposed here give us the opportunity to test this model and thus understand the molecular details host- pathogen interactions critical during this stage of M.tuberculosis infection. Specifically, we will study a subset of Mmpls that are required for disease and identify the host-pathogen interactions mediated by these virulence molecules. We will determine the mechanism of transport of the cell wall lipid phthiocerol dimycocerosate (PDIM) by MmpL7 and seek to understand why this molecule is important for lung specificity of M. tuberculosis. Furthermore, we will identify the molecules transported by the other MmpL proteins identified by our genetic screens and determine their role in pathogenesis. Finally, we will determine if these molecules serve distinct roles in modifying the host for the benefit of the bacterium. Because members of the MmpL family of transporters are highly homologous to one another and to MmpL proteins of other mycobacterial pathogens, understanding the common mechanisms of their function may lead to the development of inhibitors that could be useful for treating a broad range of infectious diseases. The results from these studies will direct our long-term plans to understand the role secreted lipids play in the struggle between M. tuberculosis and the host. Ultimately, by understanding tuberculosis pathogenesis at the molecular level, we hope to aid in the discovery of new therapies to combat and eradicate this persistent infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ROLE OF MMPL4 IN MYCOBACTERIUM TUBERCULOSIS PATHOGENESIS Principal Investigator & Institution: Skoble, Justin; George Williams Hooper Foundation; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 01-JUL-2001
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Summary: (provided by the applicant): Mycobacterium tuberculosis infects nearly one third of the world?s population and is a resurgent health hazard. In order to identify new targets for antimicrobial chemotherapies, it is critical to understand the molecular mechanisms of M tuberculosis pathogenesis. Cox et al. have recently performed a signature tagged mutagenesis to identify genes required for M. tuberculosis virulence in mice. One of the genes identified in this screen is mmpL4, which is predicted to encode a member of a family of large membrane proteins that act as lipid transporters. We have demonstrated that disruption of mmpL4 results in M. tuberculosis strains that are attenuated for growth in mice and that this growth defect is rescued in IFN-g -/mice. Thus, we hypothesize that MmpL4 secretes a lipidic virulence factor into host cells that either inhibits IFN-g signaling or an IFN-g-regulated effector function. We propose to use biochemical methods to identify the molecule(s) secreted by MmpL4 and study the effects of this molecule on the immune response of the host. In order to understand the role of MmpL4 and its secretion substrate, we will compare the host-parasite interaction of wild-type and mmpL4 mutant bacteria using macrophage and mouse models of infection. We will determine whether MmpL4 is required for survival in activated macrophages or whether MmpL4 is required for proper IFN-g signaling. Finally, to identify other components of the MmpL4 pathway, we will disrupt genes in the putative mmpL4 operon and determine whether they are required for virulence. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: SCREEN FOR ISOPRENOID PRODUCTION IN M. TUBERCULOSIS Principal Investigator & Institution: Brown, Mark J.; Echelon Biosciences, Inc. 675 Arapeen Dr, Ste 302 Salt Lake City, UT 84108 Timing: Fiscal Year 2003; Project Start 15-MAY-2003; Project End 14-NOV-2003 Summary: (provided by applicant): Isoprenoid compounds form a large ubiquitous class of natural products that fulfill a wide variety of essential cellular functions in all living organisms. In eukaryotes, isoprenoid compounds are synthesized by a mevalonate (MVA) dependent pathway. However, in many bacteria, these compounds are synthesized by an alternative, MVA-independent route whose first committed intermediate is 2-methylerythritol 4-phosphate (MEP). Both pathways converge at isopentenyl diphosphate (IPP) and subsequent steps are similar in all organisms. Since the MEP pathway is absent in mammals, it is considered an attractive target for the development of novel antibiotics. The pathogen, M. tuberculosis belongs to the MEP pathway class of organisms. The applicants will prepare a transgenic bacterial E. coli host cell whose genome contains disruptions in a first endogenous gene in the MEP pathway and a second endogenous gene which is located downstream of the first gene in the MEP pathway. A transgene from M. tuberculosis that functionally replaces the disrupted downstream gene will be cloned into the cell. The growth of the host cell in the presence of test agent is then compared to a control culture to determine the activity of the test agent. Where the test agent renders the bacterial host cell nonviable on media containing a chemical supplement to relieve the 1-deoxy-D-xylulose-5-phosphate (DXP) block and viable on the media containing MVA indicates that the test agent has the potential to be an effective antibacterial against M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: STEROL/HOPANOID BIOSYNTHESIS: AN ANTI-TB DRUG TARGET Principal Investigator & Institution: Crick, Dean C. Microbiology, Immunology & Pathology; Colorado State University Fort Collins, CO 80523
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Timing: Fiscal Year 2001; Project Start 15-JUN-2001; Project End 31-MAY-2005 Summary: (provided by applicant): Multi-drug resistant tuberculosis is increasing in prevalence worldwide; therefore, a greater understanding of the basic biochemistry of Mycobacterium tuberculosis is of utmost importance. Analysis of the M. tuberculosis genome suggests that there may be a biosynthetic pathway analogous to eukaryotic sterol synthesis in this organism. Preliminary evidence indicates that the M. tuberculosis genome encodes enzymes with structural homology to several eukaryotic sterol synthesis enzymes including farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, oxidosqualene cyclase and lanosterol 14a-demethylase. It has been shown that both the M. tuberculosis farnesyl diphosphate synthase and lanosterol 14ademethylase are functional as well as structural homologs of the eukaryotic enzymes. More importantly, commercial anti-fungal drugs that are known inhibitors of sterol synthesis (specifically oxidosqualene cyclase and lanosterol 14a-demethylase) effectively inhibit the growth of M. tuberculosis in culture. It is hypothesized that M. tuberculosis synthesizes cyclic isoprenoid compounds, perhaps sterols or hopanoids, which are essential to the viability of the organism. Therefore, the specific aims of this proposal are to: 1) identify and characterize cyclic isoprenoid compounds in M. tuberculosis. 2) isolate, enzymatically characterize and determine the essentiality of the sterol synthesis homologs expressed by M. tuberculosis. 3) identify and characterize the active site of the oxidosqualene cyclase homolog. The identification of a sterol/hopanoid biosynthetic pathway in M. tuberculosis and characterization of relevant enzymes represents a novel approach to the identification of previously unsuspected antituberculosis drug targets. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: STIGMA AND TUBERCULOSIS IN HAITIAN POPULATIONS Principal Investigator & Institution: Coreil, Jeannine; Community and Family Health; University of South Florida 4202 E Fowler Ave Tampa, FL 33620 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2006 Summary: (provided by applicant): Tuberculosis is a public health problem of global magnitude. In the U.S. its victims are primarily the poor, foreign immigrants and persons with AIDS. Efforts to control the disease are severely handicapped by the effects of social stigma, and further compounded by issues of race, social class, ethnic stereotypes, immigrant status and HIV co-infection. We will investigate the social dynamics of stigma in relation to TB in two populations particularly affected by all of these issues, Haitians in the U.S. and in Haiti. The study design will enable important comparisons across national settings that highlight the differential effects of politicaleconomic context, as well as comparisons of stigma in a traditional public health clinic and an innovative culturally competent clinic. It will identify differences in stigma dynamics for active disease compared to latent infection, and will measure the impact of stigma on adherence to preventive therapy. Results of the study will enlarge our understanding of the role of social context on stigma enactment, and will help improve illness management strategies. An innovative study design applies the methodology of cultural epidemiology in an interactive quantitative-qualitative approach to developing locally valid measures of illness concepts and behavior. The approach combines the strengths of ethnography with traditional epidemiologic research. We will conduct an ethnography of TB stigma, a cross-cultural epidemiologic study, and a community trial of adherence to preventive therapy. Study sites are Broward County, Florida, and Leogane, Haiti. The methodology has been tested and refined in a broad range of geographic settings and illness problems, including tuberculosis. The research team brings ample qualifications to implement the proposed study, including preliminary
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studies of stigma and TB among Haitians in South Florida that led to the development of a culturally competent Haitian community clinic. The project has strong support from the Florida Bureau of Tuberculosis Control and Immigrant Health, the Broward County Health Department, Haitian Physicians Abroad and St. Croix Hospital in Haiti. Parallel cultural epidemiologic studies of illness-related stigma are being planned for several other countries. This will provide an opportunity to integrate the Haitian study with a larger, multi-country collaboration. However, this application is designed as a standalone project with independent research aims. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: STRUCTURE/FUNCTION RELATIONSHIP FOR ANTIBODIES TO M. TUB Principal Investigator & Institution: Glatman-Freedman, Aharona; Pediatrics; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2004 Summary: Our overall aim is to develop a novel approach to the prevention and therapy of Mycobacterium tuberculosis infections. We have generated murine monoclonal antibodies that recognize surface antigens of M. tuberculosis and identified one of them as protective in a mouse model. The aims of this project are: 1) to define the relationship between antibody isotype and biological efficacy; 2) to establish the mechanism(s) of antibody-mediated protection (or enhancement) in M. tuberculosis infection; 3) to study the interaction of monoclonal antibodies with cell-mediated immunity. The experimental approach will be: to generate switch variants of the protective monoclonal antibody; test the ability of these reagents to prolong survival and decrease organ mycobacterial burden in a mouse model; study the mechanisms by which they exert their biological functions using in-vivo and in-vitro studies; and study their interaction with cell-mediated immunity using in-vivo and in-vitro studies. This study will help us understand the structural characteristics required for useful antibodies to M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: SURFACTANT AND TUBERCULOSIS PATHOGENESIS Principal Investigator & Institution: Ferguson, J Scott. Internal Medicine; University of Iowa Iowa City, IA 52242 Timing: Fiscal Year 2001; Project Start 01-AUG-1998; Project End 31-JUL-2003 Summary: (Adapted from applicant's abstract) Tuberculosis is the leading cause of death due to an infectious disease in the world. As a pulmonary pathogen, the initial interaction between the causative bacterium Mycobacterium tuberculosis (M.tb) and the human host is in the lung where surfactant, epithelial cells, and alveolar macrophages are located. This initial interaction between these components of the alveolus and M.tb dictate the outcome of infection. The understanding of this process is limited, but critical in this disease. This five year award will provide insight into the pathogenesis of tuberculosis, specifically in the role that surfactant components play in the early host response to this host-adapted microorganism. The specific aims are to: characterize the binding of surfactant protein D (SP-D) to virulent and attenuated M.tb strains, and their major surface components, and to determine the impact of this binding on bacterial agglutination; determine if SP-D alone or in conjunction with surfactant protein A (SPA), and the major surfactant phospholipid, dipalmitoyl phosphatidylcholine (DPPC), influences the phagocytosis of M.tb by macrophages; determine if SP-D, SP-A, and
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DPPC influence the intracellular survival of M.tb in the macrophage, and to examine whether this is the result of altered cellular responses such as phagosome-lysosome fusion or the oxidative burst; and, determine if SP-D, SP-A, and DPPC influence the M.tb-pulmonary epithelial cell interaction. These specific aims will be accomplished using in vitro techniques available at the primary laboratory and through collaborative efforts. The candidate is a fellow in the Division of Pulmonary, Critical Care, and Occupational Medicine. Through an intense course of didactic and laboratory study, he will define the essential role that surfactant components play in the host defense response to M.tb using methods of microbiology, immunology, biochemistry, molecular biology, and cell physiology. He will determine the role that SP-A, SP-D, and DPPC, have in regulating the immune response of macrophages and epithelial cells to this intracellular pathogen. The proposed work will be performed at the University of Iowa, a well-known leader in biomedical research. The work to be performed encompasses the Division of Infectious Diseases and the Division of Pulmonary, Critical Care, and Occupational Medicine, as well as various departments from the College of Medicine. Laboratory facilities are at the Iowa City Veterans Administration Center, and the University of Iowa. The candidate, mentors, advisory committee, and collaborators will work closely to achieve the goals of this proposed award: to define critical interactions between the human pathogen M.tb and the host, and for the principal investigator to become an accomplished independent biomedical investigator. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: T TUBERCULOSIS
CELL
RESPONSE
TO
CDI-RESTRICTED
LIPIDS
IN
Principal Investigator & Institution: Moody, David B.; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 01-MAR-2001; Project End 28-FEB-2006 Summary: (provided by the applicant): Mycobacterium tuberculosis has infected one third of all humans, resulting in 2-3 million deaths annually and increasing rates of coinfection with the human immunodeficiency virus (HIV). The main impediment to eradication of tuberculosis relates to the ability of M. tuberculosis to persist long term intracellularly within host tissues. Cellular immune responses, particularly activation of Th1 T cells, are crucial for killing intracellular mycobacteria and successfully resolving tuberculosis infections. Although most studies have evaluated mycobacterial protein antigen for activation of T cells, it is now known that group 1 CD1 molecules (CD1a, CD1b, CD1c) mediate T cell activation by mycobacterial glycolipids, including two classes of glycolipid antigen discovered by the applicant's group, glucose monomycolate (GMM) and mannosyl phosphodolichol (MPD). Preliminary studies indicate that CD1restricted T cells that recognize MPD and GMM are detectable in the peripheral blood of human subjects infected with M. tuberculosis, but not naive controls, indicating that M tuberculosis infection generates glycolipid-specific T cell responses in vivo. T cells use clonally variable T cell receptors to specifically recognize several mycobacterial glycolipids without crossreactivity. T cell recognition of mycobacterial glycolipids is generally specific for the carbohydrate structure of the antigens, including a product of glycosylation reactions that is produced during intracellular growth within host tissues. Now the applicant proposes to measure polyclonal T cell responses from naive and M tuberculosis infected humans to the major classes of purified mycobacterial glycolipids typical of extracellular and intracellular growth. We will use purified CD1-presented antigens as well as mycobacterial glycolipids that are specifically upregulated during intracellular growth to measure human lymphocyte responses during the first year after
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infection. Antigen-specific lymphocytes will be detected using proliferation assays, antibody-capture cytokine ELISA (elispot) and glycolipid-loaded CD1 -tetramers. Glycolipid-specific T cells will be characterized with regard to restriction by CD1 isoforms, dependence on prior infection and expression of cell surface markers of immunological memory. These studies will determine whether human infection by M tuberculosis generally results in acquired T cell responses that are specific for mycobacterial glycolipids expressed during intracellular growth. Determination of the immunodominant glycolipid targets of the human T cell response during natural tuberculosis infection will provide crucial information for development of CD1presented glycolipids as immunomodulatory agents, including vaccines. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TARGET FOR MEFLOQUINE IN MYCOBACTERIA Principal Investigator & Institution: Bermudez, Luiz E. Associate Professor; California Pacific Med Ctr-Pacific Camp Pacific Campus San Francisco, CA 94115 Timing: Fiscal Year 2001; Project Start 01-APR-2001; Project End 31-MAR-2003 Summary: (provided by applicant): Infections caused by mycobacteria are responsible for severe morbidity and mortality. Mycobacterium tuberculosis and Mycobacterium avium are intracellular pathogens that infect both healthy individuals and immunocompromised patients. M. avium are usually resistant to conventional antituberculosis therapy, and with the few drugs shown to have anti-M. avium activity in humans, such as the new macrolides, treatment or prolonged prophylaxis of disseminated disease selects resistant mutants after a course of monotherapy. In addition, multiple outbreaks of multi-drug resistant M. tuberculosis have created clinical challenges for hospital and community management of patients. The goal of this proposal is to be focused and apply new strategies to identify and characterize the targets of mefloquine, a drug just recognized to have activity against mycobacteria. We have found that mefloquine has in vitro activity against both M. avium and M. tuberculosis and is borderline bactericidal against M. avium organisms in mice (we have not tested the activity in vivo against M. tuberculosis). Because mefloquine can achieve tissue concentrations 80-fold greater than serum levels, and mycobacteria survive intracellularly and have a long half-life, this class of compound has potential to become part of anti-mycobacterial regimen. Furthermore, mefloquine is active against M. avium strain resistant to macrolides, quinolones, isoniazid, ethambutol and rifampin, suggesting a novel mechanism of action. Therefore, we believe that determining the biochemical target of mefloquine in mycobacteria can lead the way to developing compounds with even more potent activity. The results thus far obtained with mefloquine suggest that it is the first very active drug identified against mycobacteria in years. Specifically, we plan to: (1) clone the mefloquine resistant-determinant using resistant mutants. In addition, by using a M. avium promoter library cloned in a reporter construct (green fluorescent protein), we plan to determine the pathways in the bacterium that are inhibited or stimulated when M. avium is exposed to mefloquine. This work, focused on an active anti-mycobacterial compound, has the potential to unveil new target(s) in both M. avium and M. tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TARGETING M. TUBERCULOSIS ALANINE LIGASE FOR DRUG DESIGN Principal Investigator & Institution: Barletta, Raul G. Veterinary & Biomedical Scis; University of Nebraska Lincoln 14Th and R Sts Lincoln, NE 68588
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Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2004 Summary: (Provided by the applicant): Mycobacterium tuberculosis causes a serious chronic infection in human beings. M. tuberculosis, along with Mycobacterium avium, is a major opportunistic pathogen of AIDS patients. Although generally susceptible to antimycobacterial agents, multi-drug resistant strains of M. tuberculosis have emerged, underlying the need for new therapeutic agents. Peptidoglycan is the backbone of the mycobacterial cell wall, and drugs that inhibit its biosynthesis cause a bactericidal effect due to cell lysis. D-alanine is a required component of the mycobacteriai peptidoglycan. Thus, those biosynthetic enzymes involved in the synthesis and incorporation of Dalanine are attractive targets for new drug development, especially because these enzymes are not found in mammalian hosts. The terminal D-alanyl-D-alanine dipeptide of the peptidoglycan side chain is an essential component for this process and its synthesis is catalyzed by the enzyme D-alanyl-D- alanine synthetase, usually denominated D-alanine ligase (Ddl). Unfortunately, the specific characteristics of the M. tuberculosis enzyme have not been fully characterized, nor the essentiality of the gene has been elucidated. In this context, our hypothesis for the proposed project is that Dalanine ligase plays an essential role in M. tuberculosis physiology and is a useful target for drug design. To test this hypothesis, we plan to: 1) Overexpress, purify, and characterize biochemically the M. tuberculosis Ddl enzyme; and 2) Test the essential role of Ddl enzyme in M. tuberculosis physiology. These studies are expected to provide basic knowledge on key enzymes involved in the pathway of peptidoglycan biosynthesis in mycobacteria. Most importantly, we will obtain information on the physiological essentiality and biochemical parameters of the Ddl enzyme necessary to develop assays for the screening and testing of candidate compounds. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TB LAM AND AG--STRUCTURES CORRELATED WITH BIOLOGY Principal Investigator & Institution: Chatterjee, Delphi; Associate Professor; Microbiology, Immunology & Pathology; Colorado State University Fort Collins, CO 80523 Timing: Fiscal Year 2001; Project Start 01-JUN-1996; Project End 30-JUN-2003 Summary: (Adapted from the Applicant's Abstract): Important physiological and immunopathogenic features of mycobacteria including slow growth, acid fastness, resistance to drug permeation, and immunological persistence have been attributed to the presence of lipoarabinomannan (LAM) and arabinogalactan (AG) in the cell wall. This competitive renewal of Al 37139 is built on success in the initial funding period of determining structural characteristics of truncated LAM from laboratory induced ethambutol (Emb) resistant M. smegmatis and LAM and AG from clinical isolates of M. tuberculosis resistant to Emb. To take the next step in fully understanding the cell surface of M. tuberculosis and its manifestation in bacterial physiology and pathology, it is proposed to determine the entire structures of LAM and AG, to characterize LAM and AG in a panel of Emb resistant M. tuberculosis isolates, and, in collaboration, to determine and precise structures of LAM needed for CD1 T-cell and macrophage mannose receptor recognition. Although some structural features of LAM and AG have been delineated, key issues, including the structure of the arabinan regions of both polymers, and the site of the arabinan attachments to the hexose backbones of each polymer remain unknown. To obtain the complete primary structure of these two 100 glycosyl residues glycans, novel cell wall degrading enzymes will be used to prepare fragments whose structures will be determined by state-of-the-art nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. The enzymes include seven
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novel arabinanases and galactofuranases secreted by a Cellulomonas species and an endo alpha-1,6 mannosidase from Bacillus circulans. NMR will focus on the analysis of fully 1 3C labeled polysaccharides and polysaccharide fragments by 2-D and 3-D techniques. Mass Spectrometry will feature a novel Q-TOF-tandem electrospray mass spectrometer to obtain sequence information. Techniques based on SDS-PAGE, endoarabinanase cleavage, 1-D-NMR, and MS will be used to efficiently characterize LAM and AG from our panel of Emb resistant M. tuberculosis. The structural studies will directly aid the immunological studies by providing a wealth of LAM related molecules needed to decipher the structural characteristics necessary for recognition by T-cells and macrophages. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TECHNOLOGY FOR NEW TUBERCULOSIS ANTI-INFECTIVES Principal Investigator & Institution: Cunningham, Philip R. Associate Professor; Biological Sciences; Wayne State University 656 W. Kirby Detroit, MI 48202 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): A genetic system was developed in Escherichia coil that uses combinatorial genetics to identify mutations in ribosomal RNA (rRNA) drug targets that might lead to antibiotic resistance. Recently, the 16S RNA from Mycobacterium tuberculosis was substituted for E. coil 16S RNA in this system but the construct produced inactive ribosomes when expressed in E. coll. Hybrid 16S rRNAs containing the 5' and central domains from E. coil and the 3' major and minor domains from M. tuberculosis, however, produce active ribosomes in E. coil This suggests that nucleotide differences in the 5' and/or central domain of M. tuberculosis 16S RNA are responsible for loss of function when expressed in E. coll. Absence of function in 30S subunits composed entirely of M. tuberculosis 16S rRNA is probably due to the inability of a nucleotide(s) in M. tuberculosis 16S RNA to interact with an E. coil 30S ligand(s). The goal of this project is to develop genetic technology for the isolation of new anti-infectives that address the issue of drug resistance in M. tuberculosis. Two aims are proposed: (1) The nucleotides in M. tuberculosis rRNA responsible for loss of function in E. coil will be identified and (2) the M. tuberculosis 30S ligand(s) required for expression of M. tuberculosis 16S RNA in E. coil will be identified and cloned. Coexpression of M. tuberculosis 16S RNA and the ligands should produce functional ribosomes containing M. tuberculosis 16S RNA in E. coil Drug resistance in M. tuberculosis is due primarily to chromosomal mutations in the drug targets. Multi-drug resistance appears to occur through sequential accumulation of such mutations. For target-site mutations to be clinically significant, the mutated target must retain most of its biological activity since loss of function decreases the fitness and virulence of the pathogen. This is especially so for functional regions of rRNA, which are critical for protein synthesis. Successful completion of this project will provide a technology to develop novel anti-infectives that recognize all possible functional forms of the target, even if not yet found in nature, and are therefore unlikely to be susceptible to the development of resistance through target modification. Once developed, this technology will allow the use of rRNA genes from other microbial pathogens in designing new antiinfectives. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: THE COMPLETE GENOME SEQUENCE AND ANALYSIS OF M SMEGMATIS Principal Investigator & Institution: Fleischmann, Robert D. Investigator; Institute for Genomic Research Rockville, MD 20850 Timing: Fiscal Year 2001; Project Start 01-APR-2000; Project End 31-MAR-2003 Summary: The completion and annotation of the DNA sequence from two strains of M. tuberculosis, the laboratory strain H37Rv and the virulent clinical isolate CDC1551 will provide the mycobacteria and tuberculosis research communities with an unprecedented resource for investigating the infectivity, virulence, colonization, pathogenicity, and the mechanisms of drug- sensitivity and resistance of M. tuberculosis. Despite the abundance of information provided by the genomic sequence, the exceedingly slow growth rate and highly virulent nature of the organism make working with M. tuberculosis and its genetic manipulation formidable tasks. A great deal of effort has gone into developing M. smegmatis, a fast-growing non-pathogen, as a genetic surrogate for M. tuberculosis. Through these efforts, it is possible for the mycobacterial researcher to utilize M. smegmatis in much the same manner as E. coli is used in the greater microbiological community. Obtaining the complete genome sequence of M. smegmatis will represent a milestone in mycobacterial research. In addition to providing invaluable information for continuing its development as a genetic system for recombinant methodologies, the organism provides a valuable model for studying unique aspects of mycobacterial biology, including cell wall biosynthesis, regulation of growth-rate, drug-sensitivity, protein secretion, gene regulation, etc. The Institute for Genomic Research has developed a cost effective and efficient approach to microbial genome sequencing that has been used to sequence and assemble seven microbial genomes. The genome of M. smegmatis strain mc2155 will be sequenced using this whole genome shotgun strategy. Small and large insert random libraries will be prepared and a sufficient number of random shotgun sequences to reach 8-fold coverage will be obtained. Assembly of the genome will be done using the version 2.0 of the TIGR Assembly software and closure will be obtained with a variety of PCR based techniques developed at The Institute. Annotation of the genome will employ a variety of computer techniques for identifying open reading frames and identifying proteins of known function through similarity searches against curated databases. Additional analyses will identify features such as exported proteins, tRNA and rRNA genes, repeated sequences, promoter sequences and ribosome binding sites. The data will be made available to the research community through the TIGR Microbial Database on the World Wide Web (www.tigr.org). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: THE TUBERCULOSIS
DYNAMICS
OF
GRANULOMA
FORMATION
IN
Principal Investigator & Institution: Kirschner, Denise E. Associate Professor; Microbiology and Immunology; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant) The goal of this proposal is to explain the formation of granuloma in infection with Mycobacterium tuberculosis. Understanding granuloma formation and function will elucidate the primary immune mechanism for controlling tuberculosis infection. Our goal is to simulate the process of granuloma formation on a spatio-temporal scale and present the results in a time-lapse movie format. This will
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yield an interactive tool to study the role of specific immune elements in granuloma formation and function. Development of a virtual model of human infection will allow for integration of the plethora of chemokine, cytokine, cellular influx information and other relevant immunological factors, as generated by experimental systems. To this end, powerful techniques (e.g., microarrays) are available for obtaining comprehensive gene expression data. Using these methods to survey expression within the granulomas of non-human primates and mice will enable us to determine which immunological mediators are involved in granuloma formation, what the timing of their expression is in the formation, and their location within the granuloma. Further studies will indicate which cell-types are expressing which mediators. Our specific aims are to: (1) Identify the temporal and spatial expression of host immune elements participating in granuloma formation using gene expression tools in murine models of tuberculosis (2) Identify the temporal and spatial expression of host immune elements participating in granuloma formation using gene expression tools in murine models of tuberculosis (3) Determine the dynamics of granuloma formation and function in humans using mathematical models of the granuloma response in tuberculosis. Through this unique approach, the interaction of multiple factors that control the formation of the granuloma will be defined. Key parameters governing these interactions will be identified. The ability to synthesize the data generated by the experiments in the models allows for an understanding of the dynamics of granuloma formation as more than the sum of its parts. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: THE EFFECT OF ART ON RATES AND TRANSMISSION OF TB Principal Investigator & Institution: Bekker, Linda-Gail; Univ of Witwaterstrand Johannesburg, Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2007 Summary: Mycobacterium tuberculosis is the commonest pathogen leading to fatal opportunistic disease in sub-Saharan Africa. South Africa is one of 5 countries with HIVpositive TB > 300 cases/100,000This proposal aims at demonstrating the benefits of antiretroviral therapy on TB in the community of Masiphumelele. Because the major cause of morbidity and mortality in HIV-1 infected adults and children is TB, safeguarding the household from the disease is a public health priority Project hypothesis: The introduction of ART in HIV-positive members of a community will have an impact on the TB morbidity and mortality in both HIV negative and positive members. Masiphumelele, a well circumscribed community of 10,000 in Cape Town where 600 adults, 100 children and 60 neonates will be treated with ART (Project 1 and 2 of this CIPRA). In addition it is our hypothesis that ART will change transmission patterns of tuberculosis in this community. Specific Aims: 1. To measure the impact of community-based antiretroviral therapy (ART) on active tuberculosis case rates, TB hospitalization rates and tuberculosis death rates, of the community residents, both HIV infected and un-infected. 2. To measure the impact of ART on the proportion of active TB in HIV infected individuals due to reactivation of latent infection versus reinfection. 3. To measure the impact of ART on transmission patterns of TB within the community. Is there a reduction of cluster sizes of M. tuberculosis? 4. To measure the contribution of HIV infected individuals to the transmission of TB. 5. To type the strains of M. tuberculosis cultured from the community using restriction length polymorphism (RFLP) to determine if the introduction of ART will change the transmission patterns of tuberculosis qualitatively as well as quantitatively in this community. 6. To use a geographic information system (GIS) and RFLP database for the Masiphumelele village,
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identifying TB transmission locations. 7. To measure the ongoing transmission of TB using annual tuberculin skin tests (TST) in cohorts of local school children and correlate it with the successful use of ART by. the reduction in AIDS related deaths, opportunistic infections and hospitalizations. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: THE ROLE OF THE GRANULOMA IN M. TUBERCULOSIS INFECTIONS Principal Investigator & Institution: Russell, David G. Professor and Chairman; Microbiology and Immunology; Cornell University Ithaca Office of Sponsored Programs Ithaca, NY 14853 Timing: Fiscal Year 2001; Project Start 30-SEP-1995; Project End 31-MAR-2006 Summary: M. tuberculosis demonstrates an extraordinary penetrance of the human population. Much of its success is liked to its ability to persist within the host, which is dependent on the formation of granulomas, or localized infections that support bacterial persistence without overt disease. The granuloma fulfills functions ambivalent to the host because although it limits spread of the infection it also provides a haven for the bacterium from the more extreme rigors of the host immune response. Dr. Russell proposes to extend his existing studies into the life cycle stages of M. tuberculosis by studying how both the host and the bacterium initiate and maintain the granuloma in both the murine and human infections. The specific aims of the proposal are: 1. Characterization of the bacterial factors that induce and modulate granuloma development. M. tuberculosis synthesize and release 7 major lipids these lipids induce granuloma-like structures in mice and stimulate a pro-inflammatory response in macrophages in culture. These lipids will be identified structurally and their biological activities delineated. 2. Elucidation of the roles of host cytokines, chemokines and their receptors in the biology of the granuloma. The ability of the cell wall lipids to induce granuloma in non-immune and immune mice will be determined and compared with bacterial granulomas. Bacterial lipid granulomas will be dissected in an in vivo model that mixes labeled macrophages from relevant knock-out mice with the lipid-bearing particles and the data compared to immunohistological analysis of murine tuberculosis granulomas. The PI will also develop an in vitro cell migration model to determine the cytokines and chemokines responsible for recruitment of macrophages to the infection foci. 3. Examination of human alveolar macrophages from BAL cells from tuberculosis patients. These studies will encompass functional characterization of BAL macrophages for phagocytosis, vacuolar acidification and cellular responsiveness. The PI will also examine the cytokine/chemokine profiles of these cells as well as whether or not they contain mycobacterial cell wall constituents. The BAL cells will also be examined in cell migration assays based on the result from the murine granuloma model described in aim # 2. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TRAINING AND RESEARCH IN HIV PREVENTION IN RUSSIA Principal Investigator & Institution: Merson, Michael H. Dean of Public Health; Epidemiology and Public Health; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2001; Project Start 30-SEP-1998; Project End 31-MAY-2003 Summary: This proposal is an "Administrative Supplemental Budget Request" to the Fogarty International Center (FIC) for expanded training within the Yale AITRP to
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further build in-country research and public health capacity in Transitional Case Management (TCM) of cases of active Mycobacterium tuberculosis infection in Russian prisoners at the time of their release. The training and research program that we propose has the following objectives: 1) To identify two psychologists or sociologists from the Departments of Psychology and/or Sociology at St. Petersburg State University and one junior physician currently working at the Tuberculosis Institute I St. Petersburg, Russia who will receive targeted training in; infectious disease epidemiology at Yale University School of Public Health; Transitional Case Management of prisoners at the time of their release from prison; direct observed therapy (DOTS). To provide these junior physician/scientists with re-entry grants for conducting research in St. Petersburg concerning identifying cost-effective practical methods for assuring that these recently discharged prisoners will be properly placed in an after-care situation providing adequate treatment for their Mycobacterium tuberculosis infection. This proposal links five institutions; Yale University School of Public Health; the CT Prison Association; the Tuberculosis Institute in St. Petersburg; the Departments of Psychology and Sociology at St. Petersburg State University; and the Center for Sociology, Psychology and Law Research, an NGO in St. Petersburg with experience in aftercare for individuals recently released from prison. These five institutions will collaborate in a training and research program to introduce a system of Transitional Case Management (TCM) of active cases of tuberculosis in prisoners immediately upon release from a prison in St. Petersburg, Russia. This three-year program will conduct an open search in the Departments of Psychology at St. Petersburg State University (SPSU) for two psychologists and/or sociologists and in the Tuberculosis Institute for one junior physician who will each spend 12 months in the United States; 7 months at Yale for graduate training in infectious disease epidemiology; 4 months in the Translational Linkage to the Community (TLC) Program at the Connecticut Prison Association for on-the job training in translational use management of prisoners with anti-tuberculosis treatment. Following this 12 month stay in the United States; each Fogarty Transitional Case Management Scholar will return to St. Petersburg to begin research work on a subject related to transitional case management of prisoners released with active TB. This work will be supported by his/her Re-entry Grant. A senior scientist(s) at SPSU or the TI and a senior scientist(s) at Yale will provide appropriate mentorship. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TRANSDERMAL TEST FOR ACTIVE TUBERCULOSIS Principal Investigator & Institution: Nacy, Carol A. Chief Executive Officer; Sequella, Inc. 9610 Medical Center Dr, Ste 200 Rockville, MD 20850 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 29-SEP-2002 Summary: (provided by applicant):This proposal seeks to establish a development program to transition an experimental Transdermal Tuberculosis Skin Test into a commercial product. This technology has already demonstrated a remarkable ability to specifically identify AFB+ TB patients by adminstering a small amount of a TB protein (MPB64) to the skin and monitoring for an erythemic skin response. PPD+ symptomless individuals are non-reactive to the protein. The MPB-64 Transdermal Patch was applied to sixty-two patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and 28 non-TB but tuberculin-positive controls. The results were read at 72h. The sensitivity of the Transdermal Patch was 87.8 percent, efficacy 92.9 percent, and specificity 100 percent. The thirteen TB patients who had each completed 6 months of TB chemotherapy showed different reactions to MPB64 patch test: those who had completed chemotherapy less than 4 months before testing were positive; 50 percent of
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patients who completed chemotherapy 5 months prior were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. The current version of the technology is a research grade patch. This proposal will outline a development pathway to improve product packaging, ease of use and product stability. Initial efforts will be to characterize a unique animal model for use in examining component materials and technologies. Our specific aims are 1. Validate an animal model to test variables in the formulation and transdermal delivery of the skin test antigen, 2. Establish the most cost effective source of MPB64, 3. Establish the best packaging / delivery technology for the patch, 4. Establish animal test procedures for toxicity, product release and to test product stability. Phase II aims include optimizing protein production and manufacturing scale up, and performance of clinical trials for product registration. PROPOSED COMMERCIAL APPLICATION: The proposed commercial application would be to develop a patch test for diagnostic screening of active cases of tuberculosis by using transdermal delivery of MPB64 protein. The patch test could be used in place of other screening tests, such as the PPD skin test or the AFB smear, as a means of diagnosing patients with active tuberculosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TRANSMISSION OF PAUCIBACILLARY AND DRUG RESISTANT TB Principal Investigator & Institution: Schoolnik, Gary K. Professor; Medicine; Stanford University Stanford, CA 94305 Timing: Fiscal Year 2003; Project Start 01-JUN-1994; Project End 31-JUL-2004 Summary: Considerable efforts are being expended to implement "Directly Observed Therapy, Short course" (DOTS) globally. However, several key assumptions about the basic biology of tuberculosis transmission, upon which this strategy is based, remain unresolved. Results obtained from our current ICIDR project and other work in which we are engaged have lead us to hypothesize that, in the context of a well functioning DOTS based program, 1) relying solely on sputum microscopy (AFB smear) for diagnosis provides a biased underestimate of TB epidemiology, 2) that patients who are AFB smear negative, but culture positive (termed "paucibacillary") contribute significantly to disease transmission 3) that there are concerning rates of ongoing transmission of drug resistant M. tuberculosis, and 4) insight gained from the availability of the complete genomic sequence of M. tuberculosis can be exploited to provide clinically and epidemiologically relevant understanding of M. tuberculosis population genetics. We now propose to use "state of the art" molecular epidemiologic techniques to test these hypotheses. These results will have immediate implications for tuberculosis control programs in many tropical countries. In addition, the availability of fully characterized strains from this study will allow us to examine the nature and consequence of genetic diversity within populations of M. tuberculosis and begin to exploit this information for improved genotyping and diagnostic systems. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TUBERCLE BACILLI BINDING TO HOST CELLS: VACCINE DESIGN Principal Investigator & Institution: Hall-Stoodley, Luanne; Ligocyte Pharmaceuticals, Inc. 920 Technology Blvd, Ste C Bozeman, MT 59715
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Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-MAR-2004 Summary: (Provided by Applicant) Three million people die each year from tuberculosis (TB) in spite of the use of the existing anti-mycobacterial antibiotics and the BCG vaccine. Clearly, a better vaccine and better ways to treat TB are needed. The purpose of this STTR Phase I feasibility study is to evaluate Mycobacterium tuberculosis-binding interactions with several human cell types and pathogen recognition molecules. Pathogens typically gain entry to a host tissue by using cell-tocell recognition and attachment mechanisms. Conversely, the innate immune system recognizes many common motifs in microbial cell walls. These motifs are present in M. tuberculosis and bacilli-host binding interactions are increasingly being identified for M. tuberculosis. The experimental aim of the proposed study is to functionally evaluate M. tuberculosis binding interactions with human host molecules and cells under shear conditions that more accurately simulate physiological conditions in the lung. By exploring adhesion events we anticipate the discovery of novel molecular targets that could be used to develop better therapies or an improved vaccine. Blocking these targets may prevent infection by abrogating initial attachment by M. tuberculosis to permissive host cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TUBERCULOSIS EDUCATION AND CONTROL PROGRAM Principal Investigator & Institution: Blumberg, Henry M. Medicine; Emory University 1784 North Decatur Road Atlanta, GA 30322 Timing: Fiscal Year 2000; Project Start 30-SEP-1993; Project End 31-AUG-2004 Summary: The resurgence of tuberculosis has had a marked impact upon the Emory University School of Medicine community and Grady Memorial Hospital (GMH), a major teaching affiliate. GMH provides care to a large number of indigent minority patients with and at risk for tuberculosis. Currently, Atlanta has the highest rate of TB in the U.S. We propose to establish the Emory Tuberculosis Education and Control Program which will take advantage of a number of unique resources in our area (Emory University School of Medicine (EUSM) and School of Public Health, the Emory AIDS Training Network (EATN), GMH, the Centers for Disease Control and Prevention (CDC), Georgia Institute of Technology) in developing a model program for education, applied research, and TB control to help reduce the rate of TB in metropolitan Atlanta and the state of Georgia. Our first objective is to implement a coordinated competency based program of instruction for medical students and house staff in the principles and practice of preventing, managing, and controlling TB. Our curriculum expansion will be based on Knowles' principles of androgogy. Design, implementation, and evaluation will follow Green and Kreuter's PRECEDE-PROCEED analysis for health educational activities. Included in our model program will be a computer based learning component; the introduction of a problem based learning module on TB; summer research projects for medical students in the control of TB, applied research, or basic research. A second major aim of our program is to increase awareness and improve the knowledge base of community physicians. This effort will be done in collaboration with other groups such as the EATN and the American Lung Association through conferences, symposia, and a mini-residency program. We also plan to develop A Clinician's Guide to Tuberculosis in Georgia that can serve as a primary reference for practicing physicians. Applied research in the control and prevention of TB, both in the hospital and community is the third major objective of our program. Study of the efficacy of control measures to prevent nosocomial transmission of TB will be an important focus. We also plan a major expansion of screening and prevention efforts.
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Our fourth objective is to reduce the incidence of tuberculosis in Atlanta. This will require efforts to enhance communication and coordination of TB control activities with local and state health departments; EUSM and GMH; Morehouse School of Medicine; and the Division of TB Elimination at CDC. This will be accomplished in part by the establishment of a computerized network between these different groups that have responsibility for the management and care of TB patients. The establishment of a computerized network/TB database which will allow sharing of information between medical providers and the ability to track patients through the health care system should lead to improved surveillance and control of TB in our community and state. Implementation and evaluation of an intensive education program for TB patients at GMH, including one-to-one counseling and low-literacy teaching aids will also be a component of this objective. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TUBERCULOSIS IMMUNITY IN YOUNG CHILDREN Principal Investigator & Institution: Lewinsohn, Deborah A.; Oregon Health & Science University Portland, OR 972393098 Timing: Fiscal Year 2003; Project Start 15-MAY-2003; Project End 30-APR-2007 Summary: (provided by applicant): Tuberculosis (TB) is one of the most important causes of infectious morbidity and mortality worldwide. Young children are more likely to contract infection with and develop severe disease from the causative agent Mycobacterium tuberculosis (Mtb). These clinical observations likely reflect fundamental differences in the immune systems of young children and adults. Critical differences relevant to TB immunity include the propensity for infants and young children to develop TH2-type CD4+ T cells in response to immunogens, deficiencies in the development of TH1-type T cells in response to pathogens, and deficiencies in macrophage and dendritic cell (DC) function. We propose to systematically define, in young children (less than or equal to 10 years of age), these important differences relevant to the successful containment of Mtb infection. The specific aims are: 1) To determine if severity of disease following Mtb infection in young children is associated with TH2-type Mtb-specific immunity, and conversely, if absence of disease following Mtb infection is associated with TH 1-type Mtb-specific immunity. 2) To determine if immunologic immaturity is associated with the development of TH2-type Mtb-specific immunity following Mtb infection, and conversely if immunologic maturity is associated with the development of TH 1-type Mtb-specific immunity following Mtb infection. 3) To evaluate aspects of innate immunity relevant to Mtb infection by characterizing the phenotype and function of macrophages and DC from cord blood derived from healthy neonates in comparison to macrophage and DC function in healthy adults. These studies may contribute to a more complete understanding of TB immunity in young children, and hence 'facilitate the development of an improved TB vaccine for this vulnerable population. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TUBERCULOSIS POLICY IN LOS ANGELES, 1870-1940 Principal Investigator & Institution: Abel, Emily K. Health Services; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024 Timing: Fiscal Year 2003; Project Start 01-SEP-2003; Project End 31-AUG-2006 Summary: (provided by applicant): This project seeks to make three important contributions to the history of tuberculosis in the United States during the late
130 Tuberculosis
nineteenth and early twentieth centuries. First, it examines tuberculosis in a major metropolitan area, which has received little attention from medical historians. Although several historians have shown how the medical, social, and cultural context of tuberculosis changed over time, most restrict their purview to the East Coast. The disease had a very different meaning in a region that promoted itself as uniquely healthy. Second, the project broadens the understanding of the relationship between tuberculosis and immigrant groups. The association with tuberculosis had an even more devastating impact on Mexicans in Los Angeles than on the European immigrants in the East Coast, whom medical historians previously have examined. Reports of high rates of tuberculosis among Mexicans hardened the perception that they were "illegal aliens" and contributed not only to the campaign to restrict their immigration in the 1920s but also to their ex
