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., 1960Lupus: 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: 0-597-83626-4 1. Lupus-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 lupus. 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 LUPUS........................................................................................................ 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Lupus .......................................................................................... 15 E-Journals: PubMed Central ..................................................................................................... 114 The National Library of Medicine: PubMed .............................................................................. 118 CHAPTER 2. NUTRITION AND LUPUS ............................................................................................ 253 Overview.................................................................................................................................... 253 Finding Nutrition Studies on Lupus ......................................................................................... 253 Federal Resources on Nutrition ................................................................................................. 262 Additional Web Resources ......................................................................................................... 262 CHAPTER 3. ALTERNATIVE MEDICINE AND LUPUS ..................................................................... 265 Overview.................................................................................................................................... 265 National Center for Complementary and Alternative Medicine................................................ 265 Additional Web Resources ......................................................................................................... 284 General References ..................................................................................................................... 288 CHAPTER 4. DISSERTATIONS ON LUPUS ....................................................................................... 289 Overview.................................................................................................................................... 289 Dissertations on Lupus .............................................................................................................. 289 Keeping Current ........................................................................................................................ 292 CHAPTER 5. CLINICAL TRIALS AND LUPUS .................................................................................. 293 Overview.................................................................................................................................... 293 Recent Trials on Lupus .............................................................................................................. 293 Keeping Current on Clinical Trials ........................................................................................... 313 CHAPTER 6. PATENTS ON LUPUS .................................................................................................. 315 Overview.................................................................................................................................... 315 Patents on Lupus ....................................................................................................................... 315 Patent Applications on Lupus ................................................................................................... 339 Keeping Current ........................................................................................................................ 347 CHAPTER 7. BOOKS ON LUPUS ...................................................................................................... 349 Overview.................................................................................................................................... 349 Book Summaries: Federal Agencies............................................................................................ 349 Book Summaries: Online Booksellers......................................................................................... 354 The National Library of Medicine Book Index ........................................................................... 360 Chapters on Lupus ..................................................................................................................... 361 Directories.................................................................................................................................. 363 CHAPTER 8. MULTIMEDIA ON LUPUS ........................................................................................... 365 Overview.................................................................................................................................... 365 Video Recordings ....................................................................................................................... 365 Bibliography: Multimedia on Lupus.......................................................................................... 366 CHAPTER 9. PERIODICALS AND NEWS ON LUPUS ........................................................................ 369 Overview.................................................................................................................................... 369 News Services and Press Releases.............................................................................................. 369 Newsletters on Lupus ................................................................................................................ 374 Newsletter Articles .................................................................................................................... 374 Academic Periodicals covering Lupus........................................................................................ 380 CHAPTER 10. RESEARCHING MEDICATIONS................................................................................. 381 Overview.................................................................................................................................... 381 U.S. Pharmacopeia..................................................................................................................... 381 Commercial Databases ............................................................................................................... 382
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Researching Orphan Drugs ....................................................................................................... 383 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 387 Overview.................................................................................................................................... 387 NIH Guidelines.......................................................................................................................... 387 NIH Databases........................................................................................................................... 389 Other Commercial Databases..................................................................................................... 392 The Genome Project and Lupus ................................................................................................. 392 APPENDIX B. PATIENT RESOURCES ............................................................................................... 397 Overview.................................................................................................................................... 397 Patient Guideline Sources.......................................................................................................... 397 Associations and Lupus ............................................................................................................. 411 Finding Associations.................................................................................................................. 415 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 417 Overview.................................................................................................................................... 417 Preparation................................................................................................................................. 417 Finding a Local Medical Library................................................................................................ 417 Medical Libraries in the U.S. and Canada ................................................................................. 417 ONLINE GLOSSARIES................................................................................................................ 423 Online Dictionary Directories ................................................................................................... 426 LUPUS DICTIONARY.................................................................................................................. 429 INDEX .............................................................................................................................................. 537
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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 lupus is indexed in search engines, such as www.google.com or others, a nonsystematic 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 lupus, 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 lupus, 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 lupus. 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 lupus, 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 lupus. 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 LUPUS Overview In this chapter, we will show you how to locate peer-reviewed references and studies on lupus.
The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and lupus, 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 “lupus” (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: •
Prevalence of Infective Endocarditis in Patients with Systemic Lupus Erythematosus Source: JADA. Journal of the American Dental Association. 130(3): 387-392. March 1999. Summary: Compared with the general population, patients with systemic lupus erythematosus (SLE) have an increased prevalence of functionally impaired cardiac valves due to the presence of Libman Sacks lesions. These lesions may place patients with SLE at risk of developing infective endocarditis (IE). This article reports on a study that featured a retrospective chart review undertaken to determine the association between SLE with valvulopathy and IE. The authors reviewed the records of 361 patients from two health care facilities who had the diagnostic code of SLE. Of the 275 records that met the 1982 revised American Rheumatism Association criteria for SLE, 51 (18.5 percent) were for patients who had a clinically detectable heart murmur that resulted in echocardiography being performed. Nine (3.3 percent) of the 275 patients
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had a clinically significant valvular abnormality, three (1.1 percent) had a potentially significant valvular abnormality, and one (0.4 percent) had a history of IE that was diagnosed two years before her diagnosis of SLE was made. The findings suggest that 18.5 percent of this group of patients with SLE had a clinically detectable heart murmur that would require further investigation to determine its significance. Furthermore, between 3.3 and 4.4 percent of the study population had cardiac valve abnormalities that potentially required antibiotic prophylaxis before certain dental procedures. However, the authors identified no cases that demonstrated an association between IE and diagnosed SLE. The authors conclude that dentists should query their patients with SLE about their cardiac status and consult with the patient's physician if the cardiac status is unknown. Patients with confirmed valvular abnormalities should receive antibiotic prophylaxis for designated bacteremia producing dental procedures. 1 table. 55 references. (AA). •
Systemic Lupus Erythematosus: Recognizing its Various Presentations Source: Postgraduate Medicine. 97(4): 79, 83, 86, 89-90, 92-94. April 1995. Contact: Available from McGraw-Hill, Inc. 1221 Avenue of the Americas, New York, NY 10020. (612) 832-7869. Summary: In this article, the author discusses the epidemiologic and pathogenetic factors of systemic lupus erythematosus (SLE) and describes the many manifestations of the disease. Symptoms discussed include skin signs, musculoskeletal manifestations, cardiovascular involvement, renal findings, pulmonary features, gastrointestinal symptoms, neuropsychiatric disease, hematologic features, immunologic factors, and systemic manifestations. The author discusses probably causative factors, including genetic predisposition, complement deficiencies, persistence of antigen, drugs, and environmental factors. The author also presents a brief overview of current treatment options for SLE. 3 figures. 1 table. 19 references.
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Shedding New Light on Lupus Source: American Journal of Nursing. 94(11): 26-32. November 1994. Contact: Available from AJN Subscription Department. P.O. Box 50480, Boulder, CO 80322-0480. (800) 627-0484 or (303) 447-9330. Summary: In this article, the authors bring nurses up to date on systemic lupus erythematosus (SLE) and its management. Topics include a description of the disease and how it affects the body; the wide variety in course and presentation of the disease; the criteria for classifying lupus; childbearing and contraception for women with SLE; treatment options, including medications, rest, exercise, proper nutrition, and stress management; teaching patients about medications; counseling patients about stress; and the role of family and other support groups. Readers can qualify for continuing education credits with the posttest at the end of the article. 1 figure. 1 table. 9 references.
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Lupus Erythematosus: Considerations for Dentistry Source: JADA. Journal of American Dental Association. 129(3): 330-339. March 1998. Summary: Lupus erythematosus (LE) is a connective tissue disease that affects a number of organ systems. Patients with this condition can experience several other serious conditions, including bleeding, infection, endocarditis, adrenal insufficiency, and mucocutaneous disease; these conditions can affect the provision of dental care. In this article, the authors describe considerations for managing dental treatment in patients
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with LE. Topics include pathogenesis of LE, different forms of LE, diagnosis and laboratory evaluation of the disease, clinical manifestations, and dental management considerations for each of the complications of LE. The authors emphasize that, before undertaking the dental care of a patient with LE, a dentist must consider the patient's immunosuppressed status, as well as the risks of adrenal insufficiency, hematologic disease, renal insufficiency and endocarditis; then, the dentist must diagnose and manage the various oral and dental manifestations of the disease. One sidebar summarizes recommendations for pre-dental care, during care, and post dental care for the treatment of patients with LE. 2 figures. 3 tables. 54 references. (AA-M). •
Progress in the Treatment of Proliferative Lupus Nephritis Source: Current Opinion in Nephrology and Hypertension. 9(2): 107-115. 2000. Contact: Available from Lippincott Williams and Wilkins. P.O. Box 1600, Hagerstown, MD 21741. (800) 638-3030 or (301) 223-2300. Fax (301) 223-2400. Website: www.currentopinion.com. Summary: Lupus nephritis (kidney inflammation associated with systemic lupus erythematosus, or SLE) is often well developed at the time of diagnosis. This article reviews progress in the treatment of proliferative lupus nephritis. High dose corticosteroids are universally accepted as the initial approach to the control of severe inflammation in the kidney. Long term disease control and the minimization of iatrogenic (physician caused) risk usually require adjunctive therapies that target the more fundamental immunoregulatory disturbances of lymphoid cells. Of the available cytotoxic drugs, cyclophosphamide is currently among the most effective, although it cannot be considered ideal in terms of efficacy or toxicity. New prospects for the treatment of proliferative lupus nephritis include novel immunosuppressive agents (e.g., mycophenolate, cyclosporine, fludarabine), combination chemotherapy (e.g., cyclophosphamide plus fludarabine), and sequential chemotherapy (e.g., cyclophosphamide followed by azathioprine), immunological reconstitution using intensive cytoreductive chemotherapy (with or without stem cell rescue), and co stimulatory molecule inhibition. Gene therapy remains an attractive prospect, but its feasibility clearly depends on the further definition of lupus promoting genes and the availability of methods to establish stable expression of disease corrective genes in the appropriate lymphoid cells. 3 figures. 83 references.
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Treatment of Lupus Nephritis Source: Seminars in Nephrology. 20(3): 265-276. May 2000. Contact: Available from W.B. Saunders Company. Periodicals Department. 6277 Sea Harbor Drive, Orlando, FL 32887-4800. (800) 654-2452. Summary: Patients with lupus nephritis pose a therapeutic challenge and stimulate investigation of innovative treatment strategies. This article reviews those current and potential strategies that may optimize management of lupus nephritis. The clinical presentations of lupus nephritis can vary from asymptomatic hematuria (blood in the urine) or proteinuria (protein in the urine) to acute nephritic or nephrotic syndromes and from rapidly progressive glomerulonephritis to insidious chronic renal insufficiency. Although patient survival and renal function outcomes have improved over the last 4 decades, contemporary immunosuppressive regimens are not consistently effective and often require extended courses (resulting in negative drug effects and toxicity). Several strategies are under investigation to induce remissions more rapidly and to reduce the risk of long courses of cytotoxic drug therapy. The
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combination of pulse methylprednisolone and pulse cyclophosphamide may be more effective than pulse cyclophosphamide alone for patients with relatively severe proliferative lupus nephritis. A particularly vigorous strategy employs immunoablative cyclophosphamide, with or without stem cell rescue. Several studies of sequential immunosuppressive therapy are in progress. It is anticipated that long term toxicities can be lessened by substituting various maintenance agents (e.g., azathioprine or mycophenolate mofetil) after initial cyclophosphamide therapy has induced a renal responses. Innovative approaches (e.g., costimulatory blockade) offer the hope of more effective treatments without the risks of contemporary regimens. 2 figures. 2 tables. 88 references. •
Natural History and Treatment of Lupus Nephritis Source: Seminars in Nephrology. 19(1): 2-11. January 1999. Contact: Available from W.B. Saunders Company. Periodicals Department. 6277 Sea Harbor Drive, Orlando, FL 32887-4800. (800) 654-2452. Summary: Renal involvement occurs in most patients with systemic lupus erythematosus (SLE). This article discusses the natural history and treatment of lupus nephritis. Contemporary therapeutic regimens for immunosuppression and for the treatment of hypertension, hyperlipidemia, infections, and seizures have likely contributed to improvements in the prognosis of these patients over the past four decades. Corticosteroids usually ameliorate the manifestations of lupus nephritis but achieve less complete and sustained remissions than cytotoxic drugs. Among the cytotoxic drugs, pulse cyclophosphamide has one of the best profiles of efficacy and toxicity. Because each episode of lupus nephritis exacerbation results in cumulative scarring, atrophy, and fibrosis, the authors recommend continued maintenance treatment for 1 year beyond the point of complete remission of proliferative lupus nephritis. Studies are in progress to determine whether innovative treatment strategies will enhance efficacy and minimize toxicity associated with cytotoxic drug therapies. Lupus membranous nephropathy poses a lower risk of renal failure, but persistent nephrotic syndrome confers risks of cardiovascular events; this form of lupus nephritis is usually treated with less intensive regimens of corticosteroids, cytotoxic drugs, or cyclosporine. The prognosis and overall success of treatment for lupus nephritis seem to vary widely among geographically and racially diverse populations. The causes for the apparently worse prognosis and poorer responses to treatment of lupus nephritis in African American patients are currently unexplained and require further study. Until such data are available, caution is clearly warranted in extrapolating evidence, particularly about the prognosis and effects of treatment among different populations of patients with lupus nephritis. 4 figures. 2 tables. 85 references. (AA).
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Lower Urinary Tract Symptoms in Patients with Sjogren's Syndrome and Systemic Lupus Erythematosus Source: International Urogynecology Journal. 11(2): 84-86. 2000. Contact: Available from Springer-Verlag New York Inc. 175 Fifth Avenue, New York, NY 10010. (212) 460-1500. Fax (212) 473-6272. Summary: Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE) are autoimmune diseases which have many similarities with interstitial cystitis (IC), a urinary bladder disease with unknown etiology. This article reports on a survey studying the occurrence, severity, and nature of lower urinary tract symptoms among patients with SS or SLE. The results showed that these patients have significantly more
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urinary complaints, especially irritative bladder symptoms, than age and sex matched controls. The authors studied 36 patients with SS, 85 patients with SLE, and 121 controls. In these groups, 25 percent, 29 percent, and 66 percent, respectively, were free of urinary symptoms. The prevalences of mild symptoms were 61 percent (SS), 62 percent (SLE), and 27 percent (control group); and severe symptoms 14 percent (SS), 9 percent (SLE), and 7 percent (control group). SS and SLE patients with urinary complaints reported mostly urinary frequency (27 percent of SS and 62 percent of SLE patients) and suprapubic pain (36 percent of SS and 34 percent of SLE patients). The most common symptom in the control group was stress urinary incontinence. The frequency of lower urinary tract problems in patients with SS and SLE supports the concept that autoimmune disorders also have bladder manifestations. 2 tables. 21 references. •
Advances in the Treatment of Lupus Nephritis Source: in Coggins, C.H. Hancock, E.W., Eds. Annual Review of Medicine: Selected Topics in the Clinical Sciences, Volume 45. Palo Alto, CA: Annual Reviews Inc. 2001. p. 63-78. Contact: Available from Annual Reviews Inc. 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303-0139. (800) 523-8635. Fax: (415) 855-9815. PRICE: $47. ISBN: 0824305450. Summary: Systemic lupus erythematosus (SLE) is an autoimmune disease that leads to the formation and deposition of immune complexes throughout the body, which are pathogenic (causing disease) for SLE. Different forms of glomerulonephritis (inflammation of the filtering units of the kidney) can occur in patients with SLE and can contribute significantly to the associated morbidity (illness and complications) and, ultimately, mortality (death) from the disease. Over the past two decades, there have been significant strides in the understanding of the disease and in treatments that attempt to control the formation and deposition of anti-DNA auto-antibodies and immune complexes, as well as the subsequent inflammatory cascade mediated through various cellular and humoral pathways leading to progressive renal (kidney) damage and end stage renal disease (ESRD). This article reviews the current understanding of the pathogenesis and treatment of lupus nephritis in its various stages and discusses the experimental and human data regarding some of the potential newer forms of therapy. The authors discuss data regarding the use of steroids, azathioprine, cyclophosphamide, cyclosporine A, mycophenolate mofetil, gammaglobulin, plasmapheresis, LJP 394, flaxseed oil, bindarit, anti-CD-40 ligand, and CRLA41g. The authors conclude that the long term morbidity and mortality for patients with lupus nephritis (LN) has improved markedly over the past two decades. This is due in part to the addition of newer adjunctive therapies to control blood pressure and intraglomerular pressure, reduce proteinuria (protein in the urine), and manage hyperlipidemia (high levels of fats in the blood). 89 references.
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Efficacy of Mycophenolate Mofetil in Patients with Diffuse Proliferative Lupus Nephritis Source: New England Journal of Medicine. 343(16): 1156-1162. October 19, 2000. Summary: The combination of cyclophosphamide and prednisolone is effective for the treatment of severe lupus nephritis (kidney inflammation associated with systemic lupus erythematosus or SLE) but has serious adverse effects. This article reports on a study that investigated the efficacy of mycophenolate mofetil in patients (n = 42) with proliferative lupus nephritis. The authors compared the efficacy and side effects of a regimen of prednisolone and mycophenolate mofetil given for 12 months (group 1) with
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those of a regimen of prednisolone and cyclophosphamide given for 6 months, followed by prednisolone and azathioprine for 6 months (group 2). Of the patients in Group 1 (n = 21), 81 percent had a complete remission, and 14 percent had a partial remission, as compared with 76 percent and 14 percent, respectively, of the 21 patients in Group 2. The improvements in the degree of proteinuria (protein in the urine) and the serum albumin (protein levels in the blood) and creatinine concentrations were similar in the two groups. One patient in each group discontinued treatment because of side effects. Infections were noted in 19 percent of the patients in Group 1 and in 33 percent of those in Group 2. Other adverse effects occurred only in group 2; they included amenorrhea (23 percent), hair loss (19 percent), leukopenia (10 percent), and death (10 percent). The rates of relapse were 15 percent in Group 1, and 11 percent in Group 2. The authors conclude that for the treatment of diffuse proliferative lupus nephritis, the combination of mycophenolate mofetil and prednisolone is as effective as a regimen of cyclophosphamide and prednisolone followed by azathioprine and prednisolone, with similar levels of toxicity. 2 figures. 4 tables. 15 references. •
Hepatitis-Lupus Connection Source: Seminars in Liver Disease. 11(3): 234-240. August 1991. Summary: This article investigates the connection between chronic active hepatitis (CAH) and systemic lupus erythematosus (SLE). Topics discussed include a definition of both conditions; a review of the literature; the pathologic aspects; the serologic aspects including DNA and nuclear antigens, smooth muscle antigens, atypical serologic reactivities in CAH, and the liver-kidney microsomal antigen; the immunogenetic aspects, including the HAL-B8-DR3 association, complement alleles, and the Gm allotype markers; and pathogenetic aspects. The author concludes that autoimmune hepatitis and SLE are diseases with some similar serologic expressions, as judged by formation of antinuclear antibodies (ANA), but the specificities of these ANAs differ. 66 references.
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Systematic Lupus Erythematosus: Dental Hygiene Management Source: Case Studies in Periodontal Management. 1(2): 1-4. August 1995. Summary: This article presents a case of a male patient with systemic lupus erythematosus (SLE), whose periodontal status was affected by several factors relating to the SLE. SLE is an autoimmune disease of unknown origin affecting the connective tissues and various organs in the body. The patient presented with severely decayed teeth and advanced periodontal disease. The oral health plan was developed in conjunction with the patient's physician, dental and dental hygiene faculty members, and a dental and dental hygiene student. Topics include the clinical features and oral manifestations of SLE; the medical and dental history of the case client; the diagnosis and treatment plan; the implementation phase, including patient education; evaluation of the dental hygiene treatment; complications to care; and modifications suggested for future care of this patient. 'Before' and 'after' pictures of the patient's teeth and gingiva are provided. 3 figures. 1 table.
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End-Stage Renal Disease and Systemic Lupus Erythematosus Source: American Journal of Medicine. 101(1): 100-107. July 1996. Summary: This article reports on a literature review to provide an overview of the course of systemic lupus erythematosus (SLE) following the onset of end-stage lupus nephropathy, regarding clinical and serological manifestations, survival on dialysis, and
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renal transplant outcomes. Results showed that there is a tendency for decreased clinical and serological lupus activity following the onset of end-stage renal disease (ESRD). Survival of lupus patients on dialysis is no different from that of non-SLE dialysis patients, and is better than that of several other rheumatic diseases. Following renal transplantation, there is no difference in patient or graft survival in lupus versus nonlupus patients. Like their nonlupus counterparts, SLE transplant patients do better with living relative grafts or regimens containing cyclosporin A or both. Transplantation during an acute exacerbation of SLE is controversial and may increase the risk of poor outcomes. 4 tables. 59 references. (AA-M). •
Reliability of Histologic Scoring for Lupus Nephritis: A Community-Based Evaluation Source: Annals of Internal Medicine. 119(8): 805-811. 1993. Summary: This article reports on a research study undertaken to determine the reliability of the National Institutes of Health (NIH)-modified semiquantitative histologic scoring system for lupus nephritis. Five pathologists, all experienced in reading renal biopsy specimens, assessed 25 specimens that had been obtained from patients with a clinical diagnosis of systemic lupus erythematosus and showed diffuse proliferative glomerulonephritis. Biopsy specimens were scored independently and blindly by pathologists for components of nephritis chronicity and activity. Reliability was measured by percentage agreement, intraclass correlation coefficient or kappa statistic, and individual reader effect on the group arithmetic mean. The results show that, in a nonreferral setting, the NIH-modified scoring system for lupus nephritis is only moderately reproducible. The authors stress that, if this system is used to predict renal outcome, it may result in erroneous predictions of risk for renal failure and response to therapy. 2 figures. 5 tables. 39 references. (AA-M).
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Systemic Lupus Erythematosus: Emerging Concepts. Part 1: Renal, Neuropsychiatric, Cardiovascular, Pulmonary, and Hematologic Disease Source: Annals of Internal Medicine. 122(12): 940-950. June 15, 1995. Summary: This article reports on a review of advances and controversies in the diagnosis and management of systemic lupus erythematosus with visceral involvement (renal, neuropsychiatric, cardiopulmonary, and hematologic disease). The authors reviewed more than 400 English language articles in the medical literature. They note that recent debates pertaining to lupus nephritis have focused on the value of kidney biopsy data and the role of cytotoxic drug therapies. Many studies have shown that estimates of prognosis are enhanced by consideration of clinical, demographic, and histologic features. For patients with severe lupus nephritis, an extended course of pulse cyclophosphamide therapy is more effective than a 6-month course of pulse methylprednisone therapy in preserving renal function. They conclude that the optimal duration and intensity of cytotoxic therapy remain undefined. 3 figures. 3 tables. 124 references.
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Renal Vascular Lesions in Lupus Nephritis Source: Medicine. 76(5): 355-368. September 1997. Contact: Available from Lippincott Williams and Wilkins. 227 East Washington Square, Philadelphia, PA 19106. (800) 638-6423.
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Summary: This article reports on a study of a series of 169 kidney biopsies performed between 1980 and 1994 in 132 patients with lupus nephritis (LN). The biopsies were performed to obtain a comprehensive clinical and histologic description of the intrarenal vascular lesions in LN and, more specifically, to clarify two incompletely resolved issues: first, to outline the clinical manifestations associated with the different types of renal vascular lesions and the prognostic significance of each; second, to better understand the so-called lupus vasculopathy (also called noninflammatory renal microangiopathy, renal angiitis, and other names). The terms used suggest that blood clotting and endothelial lesions are involved; however, the research reported in this article does not support these mechanisms. The authors favor the hypothesis that lupus vasculopathy could in fact be due to formation of immunoglobulin microvascular casts. The authors call for a better description of the clinical significance of these renal vascular lesions in LN, with particular attention to lupus vasculopathy. The most common vascular lesions were nonspecific sclerotic changes, found in 37 percent of the biopsies; the other common vascular lesions were immunoglobulin microvascular casts (24 percent of biopsies). Vasculitis and thrombotic microangiopathy were rare lesions (2.4 percent and 0.6 percent of cases, respectively). The authors conclude that, taken as a whole, their data confirm that the presence of active and severe forms of diffuse proliferative LN (WHO class IV) carries a worse prognosis compared with the other forms of LN. The long term renal survival of patients with class IV LN was significantly worse than that of patients with other forms of LN, with a 10 year renal survival of 70 percent compared with 85 percent, respectively. However, the data do not support the conclusions of some previous studies that the presence of intrarenal vascular lesions is a marker of poor renal prognosis in LN. 4 figures. 8 tables. 43 references. (AA-M). •
Maternal and Fetal Complications in Pregnant Women with Systemic Lupus Erythematosus Source: American Journal of Kidney Diseases. 17(2): 123-126. February 1991. Summary: This article reviews recent studies that provide important insights into maternal complications in patients with systemic lupus erythematosus (SLE) established before onset of pregnancy. Exacerbations or relapse occur during the course of pregnancy and immediately postpartum in 25 to 60 percent of pregnancies. However, the likelihood of increased clinical activity of SLE during pregnancy is influenced by signs of activity present at onset of pregnancy. The introduction of an assay for anticardiolipids has led to a new concept for the pathogenesis of autoimmune disease, namely immune-related thrombosis. Recent studies suggest that this mechanism may play an important role in clinical episodes in SLE, involving late fetal death and maternal arterial and venous thrombosis. 2 tables. 21 references. (AA-M).
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Systemic Lupus Erythematosus: A Review for Dental Professionals Source: Journal of Dental Hygiene. 72(2): 35-40. Spring 1998. Summary: This article reviews, for dental professionals, systemic lupus erythematosis (SLE), a chronic, autoimmune disorder of unknown etiology with an annual incidence of 2 to 8 cases per 100,000 adults. Symptoms include fevers, arthralgias, and a characteristic rash over the cheeks and nose. More serious manifestations involve the cardiac, renal, and central nervous systems. Due to the valvular damage associated with SLE, prophylactic antibiotic premedication is recommended prior to invasive dental procedures. Drug therapy consists of nonsteroidal anti-inflammatory agents, antimalarials, and corticosteroids. The appropriate dental management of these patients requires an understanding of the etiology, clinical manifestations, current treatment
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recommendations, and psychological aspects of the disease. The authors note that the clinical course of systemic lupus erythematosus is unpredictable, marked by exacerbation and remission. During periods of disease flare-up, patients with SLE may find dental treatment taxing. Adjusting the dosage of corticosteroid medications may be necessary to prevent adrenal crisis. The pain and fatigue associated with the disease may require temporary postponement of elective procedures. The psychological condition of the SLE patient must also be considered when planning treatment. 3 figures. 1 table. 28 references. (AA-M). •
Lupus: High-Stakes Dx, Broad Treatment Options Source: Patient Care. 32(4): 105-106, 109-110, 112, 115-116, 118, 123. February 28, 1998. Summary: This journal article explains for health professionals the diagnosis and management of systemic lupus erythematosus (SLE). This chronic autoimmune disease is more common in women than in men, and its severity can vary markedly among patients. Disease flares can be followed by periods of remission. Diagnosis is based on the medical history, a physical examination, and selected laboratory tests. Symptoms may include a malar rash, arthritis, mouth sores or nasal ulcers, swollen hands or ankles, weight loss, fever, hair loss, and chest pain. Laboratory studies are important to the diagnosis, and the fluorescent antinuclear antibody test and the rheumatoid factor test are commonly performed. The anti-DNA and the anti-Sm antibody tests are considered diagnostic markers, and a classification system developed by the American College of Rheumatology can be used as a guide. Treatment should begin as early in the disease course as possible. Nonsteroidal anti-inflammatory drugs and antimalarials are the most commonly prescribed agents for milder manifestations, while more severe ones require corticosteroids and cytotoxic drugs. Also, patients should be counseled on lifestyle changes that may help counter some of their nonspecific symptoms. Once the treatment plan has been established, patients with active disease should visit their primary care physician every 3 to 6 weeks and their rheumatologist once or twice a year. Although the prognosis has improved, physicians need to be aware of the various complications of SLE and how to prevent them. 1 figure, 1 table, and 6 references.
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Revisiting Autoantibody Profiles in Systemic Lupus Erythematosus Source: Journal of Rheumatology. 24(2):297-302; 1997. Summary: This journal article for health professionals describes a study that reexamined the use of autoantibody profiles to gain information about the pathogenesis of systemic lupus erythematosus (SLE). Sera of 68 patients with SLE were assayed for autoantibodies by ELISA or immunoprecipitation. Specificities were grouped into sets, including double stranded (ds) deoxyribonucleic acid (DNA) or histone, U1 RNP or Sm, Ro or La, ribosomes, Ku, Ki, and others. An analysis was also performed of reported SLE autoantibody profiles. Results indicate that the prevalences of autoantibody sets included the following: dsDNA or histone, 59 percent; U1 RNP or Sm, 40 percent; Ro or La, 41 percent; ribosomes, 4.4 percent, Ku, 4.4 percent; and Ki, 2.9 percent. On average, autoantibody positive patients had two to three autoantibodies and about two to three autoantibody sets. This finding is consistent with a retrospective analysis of past studies. Data further support a model in which global immune dysregulation in SLE leads to organ-specific autoimmunity against particular ubiquitous autoantigens. 35 references, 2 figures, and 3 tables. (AA-M).
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Predicting Complications From Lupus Source: Arthritis Today. 22-23; March-April 1998. Summary: This journal article for individuals with arthritis, doctors may soon have a test to predict which individual with lupus is at risk for lupus nephritis. The test helps determine which type of Fc gene individual with lupus has. One variant of this gene produces receptors that bind well to antibodies, and the other leads to receptors that leave unacceptable numbers of antibodies in circulation. Research has shown that most of the individuals with lupus have two copies of the poorly binding variant. Other studies have shown that many lupus patients with kidney disease also have two copies of the inefficiently binding gene.
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Cutaneous Manifestations of Rheumatic Diseases: Lupus Erythematosus, Dermatomyositis, Scleroderma Source: Dermatology Nursing. 10(2): 81-95. April 1998. Summary: This journal article presents nurses and other health professionals with information, which is part of a continuing education series, on recognizing and managing the cutaneous manifestations of rheumatic diseases. It discusses the classification, diagnosis, clinical features, and management of skin disease seen in patients with lupus erythematosus (LE), dermatomyositis (DM), and scleroderma/systemic sclerosis. The cutaneous manifestations of LE can be divided into those that are histologically specific and those that are not. Each LE-specific skin disease produces a particular type of skin lesion. Patients with LE are photosensitive, so they should protect their skin from sun exposure. In addition, patients with LE may be treated with topical and intralesional corticosteroids, antimalarials, nonimmunosuppressive anti-inflammatory drugs, and immunosuppressives. A cautious approach should be used with regard to surgery. DM is characterized by skin lesions and a histopathologically specific pattern of skeletal muscle inflammation. Patients should use sunscreens, and they may be treated with topical antipruritics, antihistamines, antimalarials, prednisone, and other drugs. Scleroderma, which has two forms, is characterized by thickened, hardened, leather-like skin. The initial manifestation of localized scleroderma is asymmetrical circumscribed indurated plaques on the truck or proximal extremities that are often surrounded by a halo of violaceous skin. Systemic sclerosis, the second form, usually begins with Raynaud's phenomenon. The same moisturization and antipruritic measures described for DM should be used for scleroderma: that is systemic antibiotics, vasodilators, anticoagulants, immunosuppressive drugs, and various investigational approaches. 8 figures, 7 tables, and 21 references.
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Lupus: Mysterious Disease Holds Its Secrets Tight Source: Science. 296: 689-691. April 26, 2002. Summary: This journal article provides health professionals and people who have lupus with information on lupus research. In people who have this disease, the immune system goes awry and turns its cell killing forces against the host. Symptoms can range from mild to severe, and they flare and recede over time. Damage can affect almost any organ in the body. The majority of patients are women, and African American women are three times as likely to get lupus as white women. The peak incidence is between the ages of 15 and 40, so estrogen has long been considered a key risk factor. Although scientists have proposed numerous causes, they agree that, while environmental factors are important, just as critical are inherited genetic traits that make a person's immune
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system susceptible to dysregulation. Animal studies suggest several ways this interaction between environment and genetics might lead to chronic disease. One pathway is a hyperactive immune system that responds too aggressively to foreign stimuli and fails to turn off when it should. A second pathway involves a deficient rather than a hyperactive immune system. Some researchers argue that people develop lupus because they have flaws in complement, a multistage immune response that helps clear dead material from the body. Researchers have found several environmental agents that seem to trigger lupus, including exposure to sunlight, certain prescription drugs, viruses, and bacteria. Immunologic research is focusing on the role of anti-DNA antibodies in lupus, while molecular biological research is seeking genes that may be involved in its etiology. Several biotechnology companies have launched clinical trials testing novel proteins for lupus therapy. The article also includes information on a new source of funding for lupus research. 3 figures. •
Neonatal Lupus Erythematosus Source: Dermatology Nursing. 14(3): 157-160. June 2002. Summary: This journal article provides nurses with information on the pathogenesis, clinical manifestations, diagnosis, and treatment of neonatal lupus erythematosus (NLE). Although the exact incidence of NLE is unknown, it appears to occur in about 1 in 20,000 live births and can affect all ethnic groups. The pathogenesis of NLE involves the anti-Ro and anti-La antibodies, but their presence does not necessitate clinical disease. The most common manifestations of NLE are distinctive, nonscarring skin lesions and congenital heart block (CHB). Most NLE infants will have lesions on the face and scalp. Extremity lesions are also common. Half of NLE neonates will have CHB, and the heart block is often complete. The mycocardium can also be affected in NLE. Other extracutaneous findings include hepatic disease, thrombocytopenia, and neutropenia. The diagnosis of NLE should be suspected based on the maternal history of anti-Ro/La antibodies, a previous child with NLE, or autoimmune disease. A neonate who has cutaneous lesions, but with a questionable diagnosis, should have a skin biopsy taken. The treatment for the cutaneous lesions involves strict sun avoidance and use of mid to low potency topical steroids. There is no firm consensus regarding the management of CHB. Infants with NLE are at risk of developing other autoimmune diseases during childhood or adolescence. 3 figures and 18 references.
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Systemic Lupus Erythematosus: When Your Immune System Attacks You Source: Healthline. p. 16-20. November-December 2000. Summary: This journal article provides people who have systemic lupus erythematosus (SLE) with up to date information on the causes, manifestations, diagnosis, and treatment of this autoimmune disease. The article begins with a brief historical review of lupus and an explanation of the physiology of the immune system. SLE is caused by an combination of hereditary susceptibility, immune system deficiencies, and environmental triggers. It occurs mainly among young women in their childbearing years. The difference in the manifestation of lupus in men and women can be explained by the hormonal influences of estrogen. Lupus manifestations vary from one patient to another. Diagnosis is based on the presence of 4 or more of 11 criteria that the American Rheumatic Diseases Association published in 1982. Drugs used to treat lupus include nonsteroidal antiinflammatory drugs, steroids, immunosuppressive drugs, and antimalarial drugs. The article explains how these drugs work and presents their side effects. In addition, the article offers suggestions on living with lupus.
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Antiphospholipid (Hughes) Syndrome in Systemic Lupus Erythematosus Source: Rheumatic Disease Clinics of North America. 26(2): 331-348. May 2000. Summary: This journal article uses a question and answer format to provide health professionals with information on the clinical features, diagnosis, and management of antiphospholipid syndrome (APS) in systemic lupus erythematosus (SLE). APS occurs in approximately 30 percent of patients who have SLE. Clinical features and antiphospholipid antibody (aPL) specificities are similar between the primary and secondary forms of APS. Major features of APS include thrombosis, cytopenias, recurrent pregnancy loss, and cardiac valve lesions. The clinical course of the secondary syndrome is independent of the activity and severity of lupus, but the presence of APS worsens the prognosis. Lupus anticoagulant and anticardiolipin antibody tests confirm APS. Preliminary classification criteria consider that APS is present when one or more clinical and one or more laboratory criteria occur in the same patient. The differential diagnosis of APS includes autoimmune diseases, malignancies, drug induced disease, infectious diseases, and vasculitis. Treatment for APS remains empirical and directed at coagulation and immune mechanisms because of the limited amount of controlled prospective data. There is strong evidence that patients with aPL associated thrombosis are subject to recurrences and require prophylactic therapy. The treatment of choice for recurrent fetal loss is anticoagulation with heparin. 85 references. (AA-M).
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Lupus Erythematosus: 'Reading' the Skin for Diagnostic Clues Source: Consultant. 39(4): 1173-1176,1179-1180,1183-1184,1189-1190. April 1999. Summary: This journal article, the first in a three-part series on connective tissue diseases, provides health professionals with information on the classification, pathogenesis, diagnosis, treatment, and prognosis of the various types of cutaneous lupus erythematosus. The fundamental defect in lupus erythematosus occurs in the immune system. Cutaneous manifestations of lupus erythematosus offer clues to its diagnosis. The classic form of chronic cutaneous lupus erythematosus (CCLE) is characterized by discoid lesions with well-demarcated, erythematous, slightly infiltrated plaques and associated thick adherent scale with follicular plugging. Lesions can be localized or generalized, with involvement of the skin both above and below the neck. The classic subtypes of CCLE include hypertrophic lupus and lupus panniculitis. Both the papulosquamous and annular forms of subacute cutaneous lupus erythematosus (SCLE) may be distinguished from CCLE by the absence of thick adherent scale, follicular plugging, and atrophy. The lesions of acute cutaneous lupus erythematosus appear as either malar erythema or a generalized vesiculobullous or morbilliform eruption. Infants who have neonatal lupus erythematosus generally exhibit lesions similar to the papulosquamous and annular ones seen in SCLE; however, adherent scale, atrophy, and follicular plugging are occasionally observed. Useful diagnostic tests include hematologic profiles and immunofluorescent studies. Physicians should keep in mind that various drugs, such as procainamide, hydralazine, isoniazid, and methyldopa, can cause a lupus-like reaction. The management of cutaneous lupus erythematosus involves both topical and systemic therapy. The course of lupus erythematosus is highly variable. 10 figures, 5 tables, and 30 references. (AA-M).
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Treatment of Lupus Nephritis: A Work in Progress (editorial) Source: New England Journal of Medicine. 343(16): 1182-1183. October 19, 2000. Summary: Until the pathogenesis (development of disease state) of nephritis (kidney infection) due to systemic lupus erythematosus (SLE) is unraveled, optimal treatment
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for patients with this disease remains an elusive goal. This article outlines one option for treatment of lupus nephritis, serving as an introduction to a separate article in this issue of the Journal. The author first reviews the differing presentations of SLE, noting that in some patients the kidneys are not involved but in others, there is rapidly progressive destructive kidney disease. This difference may be due in part to genetic risk factors, to environmental factors (such as exposure to ultraviolet light, infectious pathogens, and silica dust), race, or socioeconomic factors. In general, the treatment of lupus glomerulonephritis depends on the severity of the disease. Intravenous cyclophosphamide is given, in addition to oral glucocorticoids, for the aggressive forms of the disorder. However, the adverse effects of these therapies have prompted the search for alternative treatments. The author then comments on the accompanying article which presents the results of a study in which patients with diffuse proliferative lupus nephritis were successfully treated with prednisolone and mycophenolate mofetil. The editorial author notes that there are several reasons for caution before generalizing these findings to other patients with proliferative lupus glomerulonephritis, notably underrepresentation of patients with poor prognosis and certain demographic characteristics. 10 references.
Federally Funded Research on Lupus The U.S. Government supports a variety of research studies relating to lupus. 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 lupus. 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 lupus. The following is typical of the type of information found when searching the CRISP database for lupus: •
Project Title: A TRANSGENIC MODEL FOR B CELL TOLERANCE AND AUTOIMMUNITY Principal Investigator & Institution: Erikson, Jan S. Associate Professor; Wistar Institute Philadelphia, PA 191044268 Timing: Fiscal Year 2001; Project Start 01-DEC-1991; Project End 30-NOV-2004 Summary: A hallmark of systemic lupus erythematosus (SLE), and murine models of lupus, is the presence of anti-double-stranded (ds) DNA Abs. Our goals are to understand how SLE-associated autoantibodies are regulated in healthy individuals and to identify the mechanisms underlying their expression in autoimmunity. The approach we have taken is to develop a transgenic (Tg) model using a heavy chain-only Tg
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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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(VH3H9) which can pair with endogenous light chains to generate a spectrum of antiDNA and non-DNA antibodies (Abs). The advantage of this model is that the development of anti-dsDNA B cells can be tracked in the context of a diverse B cell repertoire in non- autoimmune and autoimmune-prone backgrounds. Aim 1 of this competitive renewal is to compare the effects of distinct genetic mutations that are known to result in the production of SLE-associated autoantibodies on the phenotype and functional capacity of anti-dsDNA B cells. Specifically, lpr/lpr, gld/gld, and lyn-/mice will be studied. Furthermore, the regulation of dsDNA B cells will also be investigated in induced models of SLE. Using the VH3H9 Tg in MRL-lpr/lpr mice, we have identified changes in the developmental status and tissue localization of antidsDNA B cells that precede autoantibody production. Aim II is to understand the mechanisms behind these phenomena, with particular emphasis on identifying the role that defective Fas (lpr/lpr) plays in autoantibody expression. Aim III is to characterize the nature and significance of the CD4 T cells that co-localize with anti-dsDNA B cells in MRL-lpr/lpr mice. The novel aspect of the proposed studies is that, in the context of the VH3H9 Tg, we can follow the fate of anti-dsDNA B cells under diverse circumstances to begin to identify steps that lead to autoantibody production. Knowledge of the parameters that influence the production of autoantibodies may inspire more targeted therapy for SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ACE INHIBITORS AUTOANTIBODY PRODUCTION
IN
LUPUS
NEPHRITIS--TGFB
AND
Principal Investigator & Institution: Singh, Ram R. Associate Professor; Children's Hospital Med Ctr (Cincinnati) 3333 Burnet Ave Cincinnati, OH 45229 Timing: Fiscal Year 2001; Project Start 15-MAR-2001; Project End 28-FEB-2006 Summary: Angiotensin-converting enzyme inhibitors (ACEIs), such as captopril, are widely used to control hypertension in patients who have chronic renal disease. ACEIs improve renal function in patients with chronic renal disease, however, than would be expected from their suppression of hypertension. ACEI-induced improvement in renal function is associated with decreased renal TGF-beta expression and matrix deposition. We anticipate that ACEIs may have a similar effect on TGF-beta production, renal fibrosis and end stage renal disease in patients with lupus. However, because TGF-beta can inhibit T and B cell activation and auto-antibody productions, an ACEI-induced decrease in TGF-beta may exacerbate auto-antibody-mediated disease in lupus by enhancing auto-antibody production. Consequently, this proposal will explore potential therapeutic and damaging effects of ACEIs in SLE, inflammatory component of lupus nephritis, its continued presence enhances renal matrix deposition and fibrosis. To test this hypothesis we will: 1) evaluate autoantibody responses and renal disease in lupusprone mice treated with ACEIs; and 2) generate and characterize mice that have kidneyspecific deletion of the Tgfb1 gene. These mice will be used in future to determine the effect of TGF-beta deletion on lupus nephritis. Lupus-prone and control mice will be treated with captopril or a control anti-hypertensive agent; the effect on blood pressure, renal functions, renal histology, renal immune and collagen deposition will be determined. These changes will be correlated with TGF-beta expression in kidneys and spleens, and serum auto-antibodies. We will then generate mice that have renal-specific Tgfb1 gene deletion, and characterize their phenotype, specifically for any inflammatory changes in kidneys and other organs. The broad objectives of this proposal are to understand the role of TGF- beta in the pathogenesis of lupus nephritis, to explore how manipulation of in vivo TGF-beta can influence lupus, and to elucidate the mechanism
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and clinical utility of ACEIs in lupus. Delineation of pathways that cause matrix deposition in kidneys, but do not affect T and B cell activation, may lead to treatment strategies that improve end stage renal disease in SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ADAPTATION /COGNITIVE BEHAVIOR THERAPY FOR SLE Principal Investigator & Institution: Brown, Ronald T.; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2007 Summary: The purpose of this program is to enhance the adjustment and quality of life in adolescents with systemic lupus erythematosus (SLE). The theoretical rationale is based on the Investigators' program of research in the validation of models in pediatric psychology designed to predict adjustment and adaptation in children and adolescents with chronic illness. To accomplish this purpose, the first aim of the investigation is to assess the associations between adaptational processes and adjustment and healthrelated quality of life in female adolescents with SLE. A major hypothesis of the study aim is that adaptational processes including methods of coping, expectations of selfefficacy, and social support will account for a significant proportion of the variance in adjustment and health-related quality of life after controlling for demographic characteristics and disease parameters. It also is hypothesized that methods of coping, expectations of self-efficacy, and social support will independently mediate the association between severity of disease and adjustment and health-related quality of life. The second aim of the investigation is to conduct a controlled randomized manualizedbased trial designed to enhance adjustment and quality of life in female adolescents with SLE. The hypothesis of the study aim is that the manualized-based cognitivebehavioral intervention will lead to improvements on measures of adjustment and quality of life in comparison to an education-only control group and a no-contact control group. Finally, it is hypothesized that the benefits of the manualized intervention will persist over a six-month follow-up period. To accomplish the objectives of this program of research, female adolescents diagnosed with SLE ranging in age from 12 to 18 years, the majority of whom are African-American, will serve as participants. Data to bc obtained from the participants will include periodic assessments of pain, methods of coping, expectations of efficacy, social support, negative affecxtivity, perceptions of physical appearance, social competence, and health-related quality of life. In addition, caregivers and teachers also will report on the adolescents' general adjustment, while physicians will complete measures of disease severity. It is anticipated that the 147 patients will be randomized to the three arms of the intervention trial. The research study proposed here will add to the extant literature by validating mediational processes of adjustment and quality of life in adolescents with SLE and will also validate empirically a cognitive behavioral therapy intervention program that has been demonstrated in other research to alter maladaptive cognitions and increase the use of selective coping skills, thereby enhancing adjustment and quality of life for individuals with a chronic disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ALPHA-ACTININ /RENAL PATHOGENICITY OF ANTI-DNA ANIBODIES Principal Investigator & Institution: Putterman, Chaim; Assistant Professor; Yeshiva University 500 W 185Th St New York, NY 10033
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Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2007 Summary: Antibodies against double stranded (ds) DNA are a characteristic serologic hallmark of SLE. While it has been demonstrated that anti-dsDNA antibodies play a critical role in the pathogenesis of lupus nephritis, the mechanisms of injury are incompletely understood. Experimental evidence strongly suggests that a least some anti-dsDNA antibodies are pathogenic by virtue of their direct cross reactivity with renal antigen. We recently demonstrated that a pathogenic anti-dsDNA antibody (R4A) binds to a 100 kD protein expressed on the cell surface of a mesangial cell line derived from a lupus-prone MRL-lpr/lpr mouse, and that DNAse treatment of the lysate does not affect binding. Binding was greatly diminished in lysates of mesangial cell line derived from a non-autoimmune mouse, suggesting that antigen expression and/or availability at the level of the target orphan may be a factor in determining susceptibility to lupus nephritis. Following identification of the 100 kD protein bound by R4a as alpha-actinin, the binding of R4A to alpha-actinin was confirmed by Western blot, ELISA, immunofluorescence, and inhibition studies. High titers of anti-alpha-actinin antibodies were found in sera and kidney eluates of lupus mice with nephritis, and in sera of lupus patients. The goals of this proposal are to study if cross-reactivity with alpha-actinin may be an important determinant of the renal pathogenicity of some antiDNA antibodies, and if the expression of alpha-actinin is genetically regulated and modulated by gender and exposure to cytokines. We will determine if anti-alpha actinin antibodies are pathogenic and if they cross-react with dSDNA, by immunization of mice with alpha actinin and studying the anti alpha-actinin antibody response in normal and autoimmune mice. The molecular basis for the differential levels of antigen display of alpha-actinin between autoimmune and non-autoimmune mouse strains will be studies, and the effects of age, gender, and cytokines known to be present in lupus kidneys on alpha-actinin expression and antibody binding will be determined. Finally, we will identify the epitopes of alpha-actinin that are recognized by pathogenic anti-dsDNA antibodies to understand the generation of anti-alpha actinin antibodies, and determine the potential of alpha-actinin peptides in the treatment of acute lupus nephritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANALYSIS OF APOLIPOPROTEIN H IN LUPUS Principal Investigator & Institution: Kamboh, M Ilyas. Professor; Human Genetics; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2002; Project Start 01-JAN-1997; Project End 31-MAY-2007 Summary: (provided by applicant): The risk of coronary heart disease (CHD) in systemic lupus erythematosus (SLE) women is up to 50 times higher than in the general population. The conventional risk factors are insufficient to explain premature CHD in SLE patients. Compared to about 1-5 percent prevalence of antiphospholipid antibodies (APA) in the general U.S. white population, about 50 percent of the SLE patients are positive for APA. ApoH is a principal autoantigen for the production of APA in patients with autoimmune diseases. ApoH inhibits the in vitro uptake of oxidized low-density lipoprotein (oxLDL) by macrophages, but in the presence of APA it promotes the ihflux of oxLDL into macrophages. As the accumulation of oxLDL in macrophages is believed to initiate the atherosclerotic process, these findings suggest that apoH-mediated immune response in patients with autoimmune diseases, like SLE, may lead to atherosclerosis. In this renewal we propose to examine the joint roles of APA, antibodies to oxLDL (anti-oxLDL) and APOH genetic variation (known and discovered as part of this proposal) in relation to the occurrence of CHD in SUE and non-SLE patients. Our hypothesis is that individuals positive for APA and/or anti-oxLDL are prone to
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premature CHD and this susceptibility is modified by common genetic variation in the APOH gene. The objectives of the study will be achieved by fulfilling the five aims. Aim 1) identify and characterize naturally occurring common mutations in all exons, introns and the 3' region of the APOH gene by polymerase chain reaction (PCR), denaturing HPLC analysis and DNA sequencing in SLE and non-SLE CHD patients, and African blacks positive for APA. Aim 2) determine the prevalence and correlation between APA (anti-apoH, anticardiolipin, lupus anticoagulant) and anti-oxLDL in plasma samples from SLE patients and controls. Aim 3) determine the relationship between APOH genetic variation (data generated in Aim 1) and the occurrence of APA and anti-oxLDL (data generated in Aim 2). Aim 4) examine the relationship between APOH genetic variation (data generated in Aim 1) and the occurrence of subclinical cardiovascular events in SLE patients and with coronary atherosclerosis in non-SLE patients. Aim 5) perform in vitro mutagenesis and expression studies to express different apoH allelicisoforms to evaluate isoform-specific inhibition of LDL oxidation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTIBODIES WITH PROTHROMBINASE ACTIVITY IN LUPUS Principal Investigator & Institution: Thiagarajan, Perumal; Professor of Pathology & Medicine; Internal Medicine; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-OCT-2001 Summary: Prothrombin is the precursor of thrombin, the central enzyme in blood coagulation. Prothrombin binding autoantibodies from lupus patients are clinically associated with thrombosis, but the association is paradoxical, because conventional mechanisms of antibody action predict decreased thrombus formation (e.g., antibody mediated inhibition of prothrombin activation by factor Xa; accelerated prothrombin clearance). In Preliminary Studies, we identified a new and potent mechanism by which these antibodies can induce thrombosis, i.e. the catalytic cleavage of prothrombin. This proposal is based on the hypothesis that prothrombinase autoantibodies promote thrombus formation by generating thrombin-like activities from prothrombin, like factor Xa, the physiological activator of prothrombin. The turnover capability of prothrombinase antibodies suggests that they can exert substantially more potent biological effects than reversibly binding stoichiometric antibodies. The products of prothrombin processing, i.e., thrombin (or thrombin-like fragments) are also catalysts, which will further amplify the procoagulant effect of the prothrombinases compared to reversibly binding antibodies. The specific aims are: to determine the statistical correlation of prothrombinase activity to thrombosis in lupus patients; define the biochemical characteristics of the prothrombinase autoantibodies relevant to their potential clinical effects; clone catalytically efficient prothrombinase Fv constructs from lupus patients for mechanistic studies; determine the procoagulant effects of the antibody-generated prothrombin fragments using in vitro model systems; and, determine whether the Fv constructs administered to mice induce thrombosis. To these ends, the prothrombin cleaving activity of polyclonal IgG from lupus and normal subjects will be compared by electrophoretic, fluorimetric and radiometric methods; affinity purified anti-prothrombin antibodies will be analyzed to determine kinetic parameters, specificity, cleavage sites, cofactor requirements, and enzymatic activity of prothrombin fragments; recombinant prothrombinase Fv constructs will be isolated from phage display libraries by selection using prothrombin and chemically reactive antigen analogs reactive with serine protease-like catalytic sites found in autoantibodies; the ability of antibody-generated prothrombin fragments to mimic the procoagulant
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effects of thrombin on fibrinogen, coagulation factors V, VIII and XI, and platelets will be determined in vitro by measuring fibrin formation, cleavage and activation of the various coagulation factors and protease activated receptor 1 on platelet. The prothrombotic effects of prothrombinase Fv administered to mice will be determined by measuring depletion of circulating prothrombin, consumption of coagulation factors and enhanced occlusion of the femoral vein. These studies will permit assessment of the extent to which the prothrombinase autoantibodies contribute toward the hypercoagulable state in lupus. If our hypotheses are valid, our studies can be extended to ameliorating the thrombotic events in lupus via inhibition of the prothrombinase activity of the autoantibodies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTIC5 THERAPY OF LUPUS NEPHRITIS Principal Investigator & Institution: Holers, Michael; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, CO 800450508 Timing: Fiscal Year 2001 Summary: This is an investigator-initiated collaborative Phase II treatment study in which we will examine the hypothesis that treatment of patients with systemic lupus erythematosus (SLE) and active lupus nephritis with a blocking anti-human complement C5 monoclonal antibody will lead to objective improvement in renal disease parameters. The anti-C5 monoclonal antibody will lead to objective improvement in renal disease parameters. The anti-G5 monoclonal antibody will be provided by Alexion Pharmaceuticals. Several lines of investigation have supported the concept that C5 plays a central role in renal injury in antibody- mediated diseases such as SLE. While short term studies using a similar inhibitor have shown efficacy in patients with inflammatory complications of coronary artery bypass surgery, the proposed study represents the first application of this therapeutic strategy, chronic inhibition of complement C5 activation, to patients with autoimmune diseases. Patients enrolled in this double blinded, placebo controlled Phase II study will be those who have active but clinically stable nephritis and, thus, do not require immediate introduction of high dose cyclophosphamide or other cytotoxic drug therapy. Two patient groups, treated and untreated (vehicle control only as a placebo), will be studied. The primary outcome variable will be proteinuria. Secondary outcomes will include other measures of renal disease activity, other measures of lupus activity and measure of complement activation. Three Specific Aims will be pursued. Specific Aim #1. Determine the changes in renal disease activity that accompany short term treatment with an anti-C5 monoclonal antibody in patients with active lupus nephritis. Specific Aim #2. Identify changes in levels of complement activation fragments that accompany treatment with anti-c5 monoclonal antibody in patients with active lupus nephritis. Specific Aim #3. Assess these patients treated with an inhibitory anti-C5 monoclonal antibody for evidence of toxicity. This study is integrated into other components and goals of the Denver Autoimmunity Center itself in several ways. First, it utilizes a population of patients drawn from several sources in the Denver Autoimmunity Center. Second, it meets the goal of extending the use of complement inhibitors from animal models, which are being extensively studied here in the laboratory of the P.I. and others, into clinical trials in patients. Third, the analysis of the role of complement inhibitors as compared to cytokine inhibitors is a major component of the Basic Science Project #2 headed by Dr. William P. Arend. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Studies 21
•
Project Title: ANTICARDIOLIPIN PHOSPHOLIPIDS
ANTIBODIES
AND
OXIDIZED
Principal Investigator & Institution: Witztum, Joseph L. Professor; Medicine; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, CA 92093 Timing: Fiscal Year 2001; Project Start 01-JAN-1997; Project End 31-JUL-2005 Summary: Patients with the antiphospholipid antibody syndrome (APS) have autoantibodies to certain phospholipids (aPL) such as cardiolipin and/or the lupus anticoagulant and clinically experience recurrent venous or arterial thrombosis, history of fetal death and autoimmune thrombocytopenia. Increased aPL also appear to predict increased risk of stroke and myocardial infarction in otherwise healthy men as well. However, controversy exists about the target antigens of aPL, and even university laboratories cannot agree who has elevated aPL titers. In turn, clinical management is hampered by lack of an underlying hypothesis to explain why antibodies should form to such ubiquitous compounds as PL. We have developed the novel hypothesis that many aPL are directed against epitopes of oxidized PL (OxPL) and/or against covalent adducts of OxPL and associated PL binding proteins, such as beta2GPI. Our hypothesis suggests that states of enhanced lipid peroxidation, as occurs in inflammation or atherosclerosis, leads to oxidation of PL (such as in LDL or in membranes of apoptotic or dying cells) which creates neo self-determinants and immunogenic epitopes. The resultant autoantibodies can then target such neoepitopes in many tissues, and may have a variety of biological consequences. Cardiolipin (CL) is the most common PL used to test for aPL. We have shown that APS plasma bind exclusively to OxCL, or to OxCL adducts with beta2GPI, and not to native CL. We propose to further test our hypothesis by determining if antibodies to other OxPL are also present in sera from patients and mice with lupus- like syndromes. We will generate a panel of such aOxPL murine monoclonals from (NZWxBXSB) F1 males. Similar Fab and scFv antibodies will be generated from a human phage-display library. We will determine the epitopes to which they bind and their impact on in vitro and in vivo coagulation, with an emphasis on the Protein C pathway. We will treat lupus-prone mice with potent antioxidants to see if changes in aPL titers and/or other clinical parameters occur. Understanding the etiology of even some of the aPL should lead not only to development of more standardized assays, which should improve our ability to detect high risk individuals, but also to consideration of new therapeutic modalities for patients with aPL and APS (e.g. aggressive anti-inflammatory and/or antioxidant interventions). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTIGEN DRIVEN SELECTION/TOLERANCE: AUTOIMMUNITY TO DNA Principal Investigator & Institution: Marion, Tony N. Professor; Molecular Sciences; University of Tennessee Health Sci Ctr Health Science Center Memphis, TN 38163 Timing: Fiscal Year 2001; Project Start 01-JUL-1988; Project End 31-AUG-2005 Summary: (Adapted from the Investigator's abstract): Systemic lupus erythematosus is a systemic autoimmune disease in humans and genetically predisposed mice. Antibodies to a variety of cellular antigens, mostly nuclear in origin, have been detected in lupus sera from mice and humans; however, the autoantibody for which there is the most compelling evidence for pathological relevance is antibody to DNA. Anti-DNA antibodies deposit in kidneys either as immune complexes or by binding directly to glomerular structures and initiate glomerulonephritis. The immunological basis for the generation of anti-DNA autoantibody in mice and humans has been difficult to
22 Lupus
elucidate. The goal of the applicant's research on "Antigen Driven Selection and Tolerance in Autoimmunity to DNA" continues to be directed toward understanding how autoimmunity to DNA is initiated and sustained at the level of individual DNAspecific B cells in autoimmune (NZB x NZW) F1 mice. The applicant's research efforts since the last competitive review of this project have continued to support the hypothesis that autoimmunity to DNA is both initiated and sustained as a clonally selective, antigenic-specific immune response to DNA most likely in the form of DNAprotein complexes. The research has continued to focus on experiments to understand how the specificity and specificity maturation of the autoimmune anti-DNA antibody response within individual (NZB x NZW) F1 mice and the DNA-peptide induced immune anti-DNA antibody response in normal mice proceed. The results have provided new information about B cell selection in the autoimmune response to DNA and the V region structures necessary for that selection to occur. In the applicant's continuing research efforts to understand how autoimmunity to DNA is initiated, they will test the hypothesis that autoimmunity to DNA is initiated by antigen-specific B cell stimulation in the absence of peripheral B cell tolerance induced by extracellular DNA or nucleosomes. The specific experimental aims to be pursued in the research proposed will be to determine what role, if any, germinal centers play in the specificity maturation that generates high avidity autoantibodies to native DNA. Proposed experiments will also determine the role of soluble DNA or nucleosomes in maintaining immunological tolerance to DNA. The experimental systems designed to complete the proposed research will include the use of (NZB x NZW) F1 mice transgenic for expression of antiDNA antibodies. These mice have an interesting autoimmune phenotype that make them highly suited for experiments to test the hypothesis that autoimmunity to DNA in (NZB x NZW) F1 mice derives from antigen-selective B cell stimulation in the absence of effective peripheral tolerance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTI-SM B CELL REGULATION Principal Investigator & Institution: Clarke, Stephen H. Professor; Microbiology and Immunology; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, NC 27599 Timing: Fiscal Year 2003; Project Start 15-JUL-1998; Project End 31-DEC-2007 Summary: (provided by applicant): The long-range goal of this work is to determine how B cell tolerance to self-antigens in systemic lupus erythematosus (SLE) is lost. The focus will be the response to the nuclear antigen Smith (Sm), which is unique to human and mouse SLE. We have shown that in non-autoimmune mice some anti-Sm B cells are regulated by negative selection (anergy, developmental arrest, central deletion), while others are positively selected into the marginal zone and B-1 subsets and are functional. This coexistence of negatively and positively selected B cells is unusual and suggests a possible model for the anti-Sm response. The hypothesis to be tested is that one or few positively selected anti-Sm B cells are activated initially, and that the antibody they produce activates additional anti-Sm B cells, including those that are negatively selected. In Aim 1 we will determine which mechanism(s) of anti-Sm B cell regulation are defective in autoimmune MRL and lpr mice by generating a series Ig H and L chain transgenic mice regulated by different mechanisms. These mice will be followed for anti-Sm B cell activation to identify the mechanism(s) activated. In Aim 2 we will determine whether the repertoire of anti-Sm B cells involved in the response expands during its course to include a larger repertoire of B cell clones. Whether anti-Sm antibodies generated early in the response can activate other anti-Sm B cells will also be
Studies 23
determined. In Aim 3 we will examine the anti-Sm response in human SLE. We can detect anti-Sm B cells in the peripheral blood of SLE patients and find that they express unusually high CD19 levels, although non-Sm binding naIve cells have unusually low CD19 levels. We will test the hypothesis that the anti-Sm response in human SLE is antigen-driven and that intra-clonal diversity and affinity maturation are additive through successive periods of active disease. In addition, we will test the hypothesis that the unusual pattern of CD 19 expression affects tolerance and activation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: APOPTOTIC CELLS AS IMMUNOGENS IN SLE Principal Investigator & Institution: Elkon, Keith B. Professor of Medicine; Medicine; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2001; Project Start 28-SEP-2001; Project End 31-JUL-2006 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies to nucleoprotein antigens. We have shown that complement and certain acute phase proteins are deposited on the surface of apoptotic cells and facilitate phagocytosis of the dying cell. Based on the clinical observations that patients with deficiencies of the early complement components develop SLE, that mice deficient in Clq or SAP develop lupuslike autoimmunity and that Clq deficient mice have increased numbers of apoptotic cells in their kidneys, we propose that autoantibodies in SLE arise through failure to process and clear dying cells, particularly at sites of inflammation. To test this idea, we will perform the following studies: In Aim 1, we will define how complement is activated on the surface of dying cells and how pentraxins modulate this process. In Aim 2, we will determine which receptors on macrophages are engaged by different opsonins on the dying cells and will determine the consequences of receptor engagement in terms of anti- or pro-inflammatory cytokine production. In Aim 3, uptake and processing of dying cells will be examined in vivo using two different experimental systems under baseline and inflammatory conditions. The responses will be compared between wild type and mice deficient in opsonins (Aim 1) or receptors (Aim 2) implicated in phagocytosis of dying cells. These studies should elucidate whether the hypothesis proposed is correct. If correct, it will provide the solid scientific background from which to define the molecular basis of human SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: AUTOIMMUNE REGULATION AND TCR REPERTOIRE IN SLE ANTI CD40L Principal Investigator & Institution: Chess, Leonard; Professor; Columbia University Health Sciences New York, NY 10032 Timing: Fiscal Year 2001 Summary: We hypothesize that there are four principal events in the development of SLE: (1) genes predisposing to SLE establish a T-cell repertoire capable of recognizing self peptides intrinsic to the autoimmune process of SLE: (2) previously tolerant autoreactive CD4 T cells: (3) regulatory mechanisms including the activation of TH1 and TH2 CD4+ T cell subsets as well as those involving CD8 T-cells fail, through processes such as clonal deletion or changes in the cytokine milieu and (4) injurious IgG autoantibodies develop through cognitive T-cell B-cell interactions and in concern with potentially self reactive T-cells induce tissue damage and disease. The overall aim of this grant is to study patients at different stages of SLE activity and to use the opportunity
24 Lupus
presented by an ongoing clinical trial of a humanized monoclonal antibody (moAb) to CD40L in systemic lupus erythematosis (SLE) to study the immunopathogenesis of SLE and the basic mechanisms of the therapeutic intervention in this disorder. In principle, interruption of the CD40 ligand-dependent pathway could down-modulate SLE activity by acting at the distal level of the cognitive T-B interaction involved in IgG autoantibody production or at the induction and regulation of autoreactive T-cell clones. These more proximal mechanisms for anti-CD40L treatment would diminish the number of autoreactive cells in the T cell repertoire. We propose to test hypotheses relating to both the immunopathogenesis of SLE and the basic mechanism of the therapeutic intervention using anti-CD40L treatment. Specifically our aims are: (1) To identify by PCR based spectratyping techniques and T cell receptor (TCR) sequencing, oligoclonal and putatively autoantigen-driven expansions of the CD4 alphabeta TCR repertoire in SLE patients compared with those treated with moAb to CD40L: (2) Identify changes in the T cell functional response to autoantigens including Ro, La. These functional studies will include assay of T-B interactions in T cell activation, tolerance and help assays and (3) directly study the regulatory interactions of TH1, TH2 cells as well as CD8+T cells in controlling the TCR repertoire in SLE during anti-CD40L treatment. In select patients we will directly study the function and repertoire of T cells at the site of inflammation (kidney) using HVS immortalization techniques. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AUTOIMMUNE TOXICITY OF CHLORINATED COMPOUNDS Principal Investigator & Institution: Schiffenbauer, Joel; Associate Professor; University of Florida Gainesville, FL 32611 Timing: Fiscal Year 2001 Summary: It is estimated that upwards of 10 million Americans have some form of autoimmune disorder. It is clear that for most of these disorders both genetic and environmental factors contribute to the development and progression of disease. In specific disorders such as systemic lupus erythematosis, women of childbearing age are affected at a rate 9-10 times that of men, and data both from human and animal studies suggest that estrogens can have an adverse impact on the course of the autoimmune process. Recently considerable interest has focused on possible environmental factors that may contribute to the development of autoimmune disorders in general, and lupus in particular. Several chemicals have been shown to have estrogen-like effects, and one mechanism by which environmental toxicants might influence the appearance of severity of autoimmune diseases is by mimicking the effects of estrogen. Preliminary studies in our laboratory have found that three chlorinated pesticides (o,p'-DDT, chlordecone, and methoxychlor) previously shown to have estrogenic effects in vivo significantly accelerate the development of autoimmune disease in a lupus model, (NZB x NZW)F1 (or BW1) mice. A proposed series of experiments 3will extend these observations by establishing dose-response relationships for this effect for each of the three toxicants and determining on-effect levels. To facilitate extrapolation to other species including humans, body burdens in key tissues corresponding to these dosages will be determined. A related objective will be to determine whether autoimmune effects can be elicited by these agents following fetal and neonatal exposure or in a mouse strain that does not normally develop spontaneous lupus. The hypothesis that effects are due to estrogenic activity will be tested in mice administered an estrogen antagonist, and a potential mechanism will be examined. Using o,p'-DDT, methoxychlor, and chlordecone as prototype environment estrogenic autoimmune
Studies 25
disease, as well as provide important information regarding mechanisms through which immune function is altered by these agents. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: B CELL AUTOREACTIVITY IN ESTROGEN-INDUCED LUPUS Principal Investigator & Institution: Grimaldi, Christine M. Microbiology and Immunology; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): The research plan outlined in this proposal will provide the opportunity to incorporate years of postdoctoral training in B cell immunology and predoctoral training in cellular, molecular and biological chemistry and to explore challenging new areas of B cell biology and autoinimunity. Interactions with the many, accomplished scientists at the Albert Einstein College of Medicine have provided a foundation to investigate cellular and molecular aspects of B cell regulation and tolerance induction. In conjunction with the dynamic research environment at AECOM, training will be enhanced by departmental and institutional seminars and courses. The long-term goal is to continue a research career as an immunologist and as an independent investigator in a medical research institution. Plans are described to perform research related to the understanding of the regulator, mechanisms associated with B cell development, B cell activation and autommunity using model systems of systemic lupus erythematosus (SLE). The effects of estrogen (E2) in the immune system and its role in SLE are poorly understood. Since there has long been suggestive evidence for a role of E2 in SLE, they have been studying the impact of E2 treatment on B cell tolerance. Examination of non autoimmunity mice that are transgenic for the heavy chain of a pathogenic anti DNA antibody revealed that a sustained increase in E2 disrupts normal B cell tolerance of anti DNA B cells and leads to an increase in anti DNA antibody titers, an expansion of anti DNA B cells and glomerular immunoglobulin deposition. The autoimmune phenotype observed in E2 treated mice is characterized by the altered distribution of splenic B cell subsets, with a diminished immature transitional population and an increase in marginal zone B cells. This shift in B cell development correlates with the increased number of in vivo activated marginal zone B cells that secrete anti DNA antibody. Since little is known about the role of the T cell independent marginal zone B cell immune response in autoimmunity, the E2 induced mouse model of lupus provides a system to explore the contributions of this B cell subset in B cell mediated autoimmune disorders. In an effort to delineate pathways that regulate the threshold for tolerization and activation, they will explore the hypothesis that E2 induced expression of CD22 and SHP 1 contributes to the escape of autoreactive B cells in this model of lupus. Results from these studies are intended to provide insight into the mechanisms of B cell tolerance, and, importantly, may help identify key regulatory pathways in patients in which disease is hormonally regulated, as well as in patients in which disease is not hormonally regulated. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: B CELL FUNCTIONAL AND SIGNALING ABNORMALITIES IN SLE Principal Investigator & Institution: Anolik, Jennifer H. Medicine; University of Rochester Orpa - Rc Box 270140 Rochester, NY 14627 Timing: Fiscal Year 2002; Project Start 26-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): It is thought that defects in the process of B lymphocyte tolerance are central to the pathogenesis of systemic lupus erythernatosus
26 Lupus
(SLE). Moreover, the importance of B cell receptor (BCR) signal strength for the regulation of tolerance is evident from murine models of autoimmunity which suggest that specific defects in lymphocyte signaling can lead to SLE-like phenotypes. In order to define tolerance mechanisms and test the above hypotheses in humans, they have developed a novel system to track self-reactive B cells. This system utilizes a specific antibody variable region gene segment, VH4.34, as a surrogate marker of autoreactivity. Preliminary results support the hypothesis that B lymphocyte tolerance is defective in SLE and is in particular perturbed during the naive to memory B cell transition. Exactly where and how tolerance is abrogated during this transition remains to be defined. The present proposal will focus on further refining the distribution of autoreactive B cells into distinct subsets by flow cytometry and immunohistochemistry along the naive to memory transition in peripheral blood and lymphoid tissue from lupus patients in order to determine specifically where tolerance is abrogated. Aim 2 will ask what the mechanism of tolerance escape is by characterizing defects in signaling and function in lupus naive B cells compared to normal controls and correlating this with loss of tolerance. Signal transduction will be measured by calcium fluxes, tyrosine phosphorylation, and recruitment of downstream signaling pathways. Functional analysis will include determination of proliferative responses and expression of activation markers. Aim 3 will define what happens to the above abnormalities after treatment with a specific immunotherapy that targets B cells in a systematic fashion. By enumerating B cell subsets and characterizing defects in signal transduction and function in lupus B cells before and after B cell depletion, they will further elucidate the critical role of B cells in the immunopathogenesis of SLE. Given the long-term goal of understanding the basic mechanisms underlying the loss of immune tolerance, as an academic rheumatologist, this project and career award combined with the interactive research environment and the mentoring available at the University of Rochester is intended to foster the candidate's development as a basic science immunologist and independent investigator. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: B CELL TOLERANCE TO NUCLEAR ANTIGEN LA Principal Investigator & Institution: Farris, a Darise. Assistant Member; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2002; Project Start 05-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): A primary knowledge of normal mechanisms of immune tolerance to self constituents is required before the immunological events leading to autoimmunity can be understood. Much has been learned about T and B cell tolerance to membrane and secreted proteins in recent years, largely through the use of transgenic technology, but there is a gap in knowledge regarding normal mechanisms of immune tolerance to protein antigens of the nucleus. Yet the majority of autoantigens, like La/SS-B, targeted in a number of systemic autoimmune diseases including Sjogren's syndrome and systemic lupus erythematosus are composed of protein and localize to the nucleus. The highly conserved nature of these house-keeping proteins (and DNA) has hampered their study in relation to immune tolerance. Moreover, the pathways through which nuclear proteins become visible to the immune system may be fundamentally different from those of previously studied membrane and secreted self antigens. In order for tolerance and autoimmunity to nuclear antigens to be comprehended, a requirement for unraveling the etiology(ies) of systemic autoimmune disease(s), it is therefore imperative that nuclear proteins be studied as a special class of self antigen. To address these issues, B cell tolerance to the La nuclear antigen will be
Studies 27
investigated as a paradigm for protein antigens of the nucleus. These studies will utilize mice transgenic for the human La (hLa) gene in its natural form to allow the study of Laspecific B cells in environments where self antigen is either present or absent. By focusing on B cells that recognize human specific epitopes of the hLa neoself antigen, the existence and extent of tolerance to the hLa antigen in the B cell compartment will be determined. Both transgenic and homologous recombination technology will be used to clarify the nature of B cell tolerance to La and determine the mechanisms of its occurrence. These studies will advance our collective understanding of anti-nuclear autoimmunity underlying a constellation of rheumatic disorders. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BONE MINERAL DENSITY IN SYSTEMIC LUPUS ERTHEMATOSUS IN CHILDREN Principal Investigator & Institution: Von Scheven, Emily; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001 Summary: The increased survival of children with Systemic Lupus Erythematosus (SLE) into adulthood has resulted in novel morbidities such as osteopenia and the associated increased risk of spontaneous vertebral compression fractures and fractures secondary to trauma related to sports and other normal childhood activities. Like other children with severe illness, these children are particularly susceptible to the development of osteopenia due to the limited window of time during childhood dedicated to achieving peak bone mass, and the critical role of peak bone mass in the future development of osteopenia. Additionally, individuals with SLE may be at particularly high risk for the development of osteoporosis due to their use of glucocorticoids, sunshine avoidance with possible vitamin D deficiency, renal disease with potential effect on vitamin D hydroxylation, and decreased activity due to severe illness and arthritis. Very few studies have been performed to evaluate bone mineral density (BMD) in children with SLE and thus neither the incidence nor the appropriate treatment is known. The proposed study will characterize the incidence and severity of osteopenia in pediatric SLE with particular attention to the bone age and sexual development of the child. Additionally, the study will begin the evaluate the clinical risk factors associated with osteopenia in this population. Identification of risk factors will ultimately contribute to the development of preventive and therapeutic interventions. The evaluation of BMD in the context of Tanner stage and bone age in this project may result in the development of bone age-specific treatment protocols for pediatric SLE. Furthermore, information gained from this research may have application to the study of osteopenia in other autoimmune disorders, such as rheumatoid arthritis; and to other patients receiving corticosteroids such as those with nephrotic syndrome and inflammatory bowel disease. The utilization of two densitometric techniques, Dual photon x-ray absorptiometry (DXA) and Single-energy quantitative computerized tomography (SEQCT) will permit us to address the current question of the most appropriate methodology for measuring bone mineral density in children. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CD1 ERYTHEMATOSUS
RESTRICTED
T
CELLS
IN
SYSTEMIC
LUPUS
Principal Investigator & Institution: Sieling, Peter A. Associate Professor; Microbiology; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024
28 Lupus
Timing: Fiscal Year 2001; Project Start 01-DEC-1998; Project End 30-NOV-2003 Summary: The goal of this proposal is to investigate the role of CD1-restricted T cells in autoimmune disease using systemic lupus erythematosus (SLE) is a model. Pathogenic autoantibody production against non peptides in SLE is dependent upon T cell help, thus SLE represents an immunological enigma according to the existing paradigm of T cell recognition of peptide-only antigens. CD1 antigen presentation of non-peptide molecules in SLE produce IL-4 and recognize autoantigen presented by B cells in the context of CD1. We hypothesize that CD1-restricted T cells in SLE recognize non peptide antigens and provide help for B cells to produce autoantibodies against the same antigen, thereby contributing to the pathogenesis of autoimmune disease. To test this hypothesis we first propose to ascertain the restricting element and cytokine patter of CD1- restricted autoreactive T cells in SLE by deriving T cell lines and clones from the cutaneous lesions and blood of patients and compare them to T cell lines derived from healthy donors. Second we will investigate whether the cognate interaction of CD1crestricted autoreactive T cells and CD1c+ B cell deletion or activation. We will also investigate the role of CD1+ antigen presenting cells (APCs) in directing CD1 peptide antigens which stimulate CD1-restricted autoreactive T-cell responses. This final goal will be achieved by purifying the CD1c ligand from a soluble endogenously loaded CD1c and use the ligand to stimulate CD1-restricted autoreactive T-cells. The studies within this proposal should provide a comprehensive understanding of the interaction between T-B cells in the generation of immunoglobulin to non-peptide antigen in SLE. Furthermore, the studies should also provide a more complete definition of CD1c+B cells, a population of B cells that arises early in the development of the immune system, but whose function remains unknown. Finally, we anticipate that our findings will be useful in establishing new mains of treating autoimmune disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CD19 TYROSINE MEDIATED SIGNAL TRANSDUCTION IN VIVO Principal Investigator & Institution: Carter, Robert H. Associate Professor; Medicine; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2002; Project Start 01-JAN-1998; Project End 31-JAN-2007 Summary: (provided by applicant): B cells are involved in many autoimmune diseases. CD19 regulates B cell functions in vivo that are important in autoimmunity. Extensive studies in vitro have identified multiple signaling pathways, linked to specific CD19 cytoplasmic tyrosines, that may mediate CD19 function but their relative importance in vivo is unknown. We have developed a system for studying CD19 tyrosine-based signaling in vivo. Mice that are deficient in CD19 lack peritoneal B 1 and marginal zone B cells and respond poorly to T-independent antigens. We reconstituted these functions with an unmutated CD19 transgene, but a construct with mutation of all cytoplasmic tyrosines completely failed to do so, demonstrating the dependence of these B cell functions on CD19 tyrosine signaling. To determine the relevance in vivo of the multiple pathways that have been linked to particular tyrosines and the molecules that bind them in vitro, we produced four transgenes each containing mutations of distinct pairs of homologous CD19 tyrosines. Founder lines for each have been crossed onto the CD19-/background. We initially studied the role of Y482 and Y513, which bind Lyn and phosphatidylinositol 3-kinase (PI3K), and found an absolute requirement for these in all in vivo CD19 functions tested. The likely mechanism is the abrogation of phosphorylation of all CD19 tyrosines by these mutations. This finding establishes that targeting particular CD19 tyrosines can modulate B cell responses in vivo. We propose to: 1) Identify the CD19 structures that control phosphorylation of the molecule, as these
Studies 29
would be targets for global control of CD19 function, 2) Determine the role of binding of cytoplasmic molecules to particular CD19 tyrosines, as these mediate CD19 function, 3) Determine which downstream pathways are controlled by particular CD19 tyrosines in vivo, as these would be identified as regulating important B cell functions, and 4) Determine the role of CD19 tyrosine-based signaling in a mouse model of lupus, to test the hypothesis that targeting specific CD19 tyrosines could be used for therapeutic manipulation of B cells in autoimmune disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CD4(-) AND CD8(-) T CELLS IN AUTOIMMUNE & NORMAL MICE Principal Investigator & Institution: Strober, Samuel; Professor of Medicine; Medicine; Stanford University Stanford, CA 94305 Timing: Fiscal Year 2003; Project Start 01-MAY-1997; Project End 31-MAR-2007 Summary: (provided by applicant): The object of the proposed studies is to test the hypothesis that the interaction between anti-CD1 reactive NK T cells and marginal zone B cells (IgM(hi) IgD(lo) CD21(hi) CD1(hi)) via CD1 is a critical step in the pathogenesis of lupus in two mouse models; hereditary lupus in NZB/NZW female mice and lupus induced in adoptive BALB/c hosts after the injection of anti-CD1 TCRalpha/beta transgenic T cells. In both models, this interaction is theorized to help activate the marginal zone B cells to secrete autoantibodies, such as anti-dsDNA antibodies, of the pathogenic IgG2a isotype. In vivo and in vitro experiments focus on purifying NK T cells, and on purifying the marginal zone B cells with appropriate mAbs by flow cytometry. We have recently shown that the marginal zone B cells from NZB/NZW mice are the only subset that spontaneously secretes IgM autoantibodies, and is also expressing the IgG2a constant region gene. The sorted NK T cells and non-NK T cells and a variety of B cells subsets from NZB/NZW and transgenic BALB/c mice will be incubated in vitro or transferred to adoptive hosts to determine whether pathogenic autoantibodies are produced resulting in clinical lupus (proteinuria, anti-dsDNA antibodies, increased mortality, etc.) in the adoptive hosts. We will perform immunohistopathological studies to search for the interactions between the critical T and B cells in the marginal zone of the spleen of mice with lupus. In addition, we will attempt to determine patterns of autoantibodies identified in the serum of BALB/c mice injected with anti-CD1 transgenic T cells and compare to NZB/NZW mice. The latter studies will use immunoprecipitation, Western blots, and protein chip arrays. We will assess the impact of anti-CD1 mAb treatment in mice that develop lupus by monitoring disease parameters as well as the spectrum of autoantibodies secreted. Finally, we will attempt to establish a line of NZB mice expressing the green fluorescence protein (GFP) transgene such that transgenic NZB/NZW mice can be used as donors of labeled B cell subsets. The latter cells will be transferred to non-transgenic NZB/NZW hosts to determine their contribution to IgG autoantibody secretion. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CD40 LIGAND IN SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Crow, Mary K. Professor; Hospital for Special Surgery 535 E 70Th St New York, NY 10021 Timing: Fiscal Year 2001; Project Start 01-JAN-1998; Project End 31-DEC-2002 Summary: Systemic lupus erythematosus (SLE) represents the prototype systemic autoimmune disease, in which the human immune system fails to regulate immune reactivity to chromatin and other well-characterized self antigens. The T helper (Th) cell,
30 Lupus
the central immunoregulatory cell in the normal immune system, mediates the hypergammaglobulinemia and autoantibody formation that result in tissue damage in SLE. CD40 ligand (C40L), a member of the TNF gene family, mediates the Th cell signals that drive B cell activation and differentiation. Several new observations regarding the regulation of CD40L expression on lymphocytes from patients with SLE represent the important preliminary data for the proposed studies: 1) Baseline expression of CD40L is increased in patients with active SLE; 2) Expression of CD40L is prolonged following treatment of SLE T lymphocytes with PMA and ionomycin, a stimulus that bypasses TCR signaling events; 3) Soluble CD40L circulates in SLE and is readily detectable in serum samples from patients. The proposed experiments will investigate potential mechanisms and functional consequences of altered regulation of CD40L expression in SLE. The hypothesis to be pursued is that in SLE, impaired regulation of T cell activation results in excessive Th cell function, autoantibody formation, inflammation, and disease. CD40L is both a marker and mediator of this abnormal T cell help. The specific aims of the project are: I. To Study the Regulation of CD40L Expression in Human T Cells. We will investigate the T cell stimuli required for induction of CD40L mRNA and protein expression, the biochemical pathways that mediate induction of CD40L, the regulation of soluble CD40L production, and the effect of cytokines on expression of cell surface and soluble CD40L. II. To Study the Regulation of CD40L Expression in SLE. We will characterize the baseline activation status of SLE T cells, study the effects of costimulatory molecules and cytokines on induction of CD40L expression in SLE, study transcription and post-transcriptional regulation of CD40L mRNA in SLE, and assess cleavage and clearance of cell surface CD40L in SLE. III. To Study the Functional Properties of Cell Surface and Soluble CD40L in SLE. We will characterize the functional properties of T cell subpopulations expressing CD40L in SLE, and study the functional effects of soluble CD40L on target cell populations. These studies should elucidate disease pathogenesis and identify new targets for therapy in SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CELL MEDIATED RENAL INJURY IN LUPUS Principal Investigator & Institution: Kelley, Vicki R. Associate Professor; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 01-FEB-1985; Project End 30-NOV-2002 Summary: The broad objective of this proposal is to test the hypothesis that increased intrarenal macrophage colony stimulating factor (CSF-1) expression is central to the pathogenesis of autoimmune renal disease in MRL-lpr/lpr mice. Using the MRL-lpr/lpr mouse with rapid, uniform, severe and predictable renal disease regulated by the lpr gene we will investigate the importance of CSF-1 in the pathogenesis of lupus nephritis. We propose to test whether the increase in circulating CSF-1 detected in neonatal MRLlpr/lpr mice is contributed by the kidney alone or if other tissues are responsible for elevating serum levels. We will establish whether a molecule(s) in the circulation of MRL-lpr/lpr mice induces intrarenal CSF-1. We will determine whether increased renal expression of CSF-1 recruits macrophages. We will then investigate whether an increased expression of CSF-1 can induce renal disease in mice with normal kidneys including another strain with the lpr gene (C3H- lpr/lpr) and C3H-++ mice or accelerate an indolent, mild nephritis in congenic MRL-++, lacking the lpr gene. We will eliminate CSF-1 by creating a cytokine deficient MRL-lpr/lpr mouse and evaluate the impact on the development of lupus nephritis. In the event that the CSF-1 deficient MRL-lpr/lpr strain does not develop lupus nephritis we will determine if the inability of renal cells to express CSF-1 is responsible for preventing kidney disease. Through the advent of
Studies 31
cellular and molecular techniques we now have the capacity to transfer a cytokine gene using a retroviral vector and establish tubular epithelial (TEC) and mesangial cell lines which can constitutively secrete high levels of a stable cytokine. By implanting these cells under the renal capsule we have created a system to introduce the continuing presence of CSF-1 (or other cytokines) into the kidney. We can then establish if CSF-1 recruits macrophages and determine whether CSF-1 will induce or accelerate renal injury in the MRL-++, C3H-lpr/lpr strains. To definitely establish whether CSF-1 or other cytokines have an enhanced glomerular expression prior to the influx of macrophages, we will isolate and pool individual glomeruli (glom) from MRL-lpr/lpr, congenic, and normal mice at varying ages and quantitate the level of cytokine and macrophages specific marker mRNA using the competitive template polymerase chain reaction. Finally, we will cross the MRL-++ or the C3H- lpr/lpr mice with CSF-1 transgenic mice and select for hybrids with these backgrounds overexpressing macrophage growth factors. In addition, we will eliminate CSF-1 from MRL-lpr/lpr mice by crossing them with the op/+ strain and select for a strain with op/op (producing a non-functional CSF-1) and lpr genes. By increasing or eliminating CSF-1, we will test the impact of this cytokine in promoting renal disease. In addition, we will use the approach of transplanting a kidney into a bilaterally nephrectomized recipient to determine when the MRL-lpr/lpr kidney is responsible for increasing serum CSF-1 and establish if this production is constitutive or is dependent on a stimulus. In addition, we will determine whether a circulating stimulant in the serum of MRL-lpr/lpr mice induces intrarenal CSF-1 and at what age this begins. Finally, we will test whether a kidney unable to express CSF-1 transplanted in the MRL-lpr/lpr mice develops renal injury. Taken together, using several novel approaches we will be able to clarify the importance of CSF-1 in the pathogenesis of lupus nephritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHANGES ERYTHEMATOSUS
IN
EBV
INFECTION
IN
SYSTEMIC
LUPUS
Principal Investigator & Institution: Gross, Andrew J. Microbiology and Immunology; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 30-APR-2008 Summary: (provided by applicant): We will use the human herpesvirus, Epstein-Barr virus (EBV), to probe immune dysfunction in systemic lupus erythematosus (SLE) by studying changes in EBV latent infection in these patients. EBV establishes lifelong infection in >90% of humans. In healthy individuals this is a tightly regulated process, which is evident in the blood, where: the virus is only carried by memory B cells; the viral load is stable over long periods of time; and there is little or no viral gene expression. We have preliminary data that indicates EBV latent infection is disrupted in patients with SLE. First, there are very high viral loads in the blood of these patients. Previous work by our laboratory showed that treating organ transplant recipients with immunosuppressive medications leads to similarly elevated viral loads. However, analysis of the preliminary data indicates that these high loads of EBV in the blood of patients with SLE cannot be accounted for by treatment with immunosuppressive agents. Furthermore, the increase in the viral load is related to the presence of flares of SLE; when patients are symptomatic and immune deregulation is at its maximum, the viral load increases. Second, a latency gene of the virus, LMP1, is atypically expressed in the blood of these patients. We hypothesize that there are two aspects of the immune dysfunction in SLE that could disrupt EBV infection. First, defects in cytotoxic lymphocyte (CTL) function can result in increases in viral load, but not affect other
32 Lupus
aspects of the infection. Second, defects in the cells that harbor the virus, memory B cells, could lead to the appearance of atypical states of viral infection in the blood and/or result in an increase in viral loads in the blood. In this proposal we will assess the relative contributions of these two mechanisms. We will evaluate 1) whether there are defects in CTL function in patients with SLE; 2) whether there are alterations in EBV infection in the blood of patients with SLE, such as infection of other B cell types and expression of EBV genes; and 3) changes in CTL function and EBV infection are affected by SLE disease activity. These studies will provide new insight into the immune dysfunction in SLE and shed light on the cause of this disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHARACTERIZATION OF SLE SUSCEPTIBILITY LOCI ON MOUSE CHROMOSOME 4 Principal Investigator & Institution: Morel, Laurence; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001 Summary: Sle2 on mouse chromosome 4 is a strong recessive locus associated with lupus nephritis in the NZM2410 model. Other groups have identify other SLEassociated loci in the centromeric half of this chromosome. Congenic analysis has showed that Sle2 is associated with B cell hyperactivity resulting in producing of polyclonal IgM antibodies, in vivo and in vitro hyper-responsiveness, increased B7.2 expression, and enlargement of the Bl1 population. Characterization of polycongenic strains combining Sle1, -2. and -3 has shown that Sle2 is necessary for full disease expression, and that, in combination of Sle3, Sle2 results in highly penetrant nonpathogenic hyaline and mesangial renal lesions that might constitute an accelerating factor for lupus nephritis. Using the congenic dissection approach, and following the steps that we are following in the functional and genetic dissection of the role of telomeric chromosome 4 in SLE pathogenesis. To achieve this goal, we have produced a series of 10 sub-congenic strains covering the area. We will use these strains in two specific aims: 1) We will assess whether the various phenotypes associated with Sle2 result from a single or several loci and generate a high resolution genetic map of these loci. The immunological defects and gene expression profile associated with each of these loci will be established. 2) We will determine the contribution of these loci to SLE pathogenesis by combining the corresponding sub-intervals to either Sle3 or the Sle1/Sle3 combination to reconstitute the Sle2/Sle3 or Sle1/Sle2/Sle3 immunopathology, respectively. Preliminary results indicate that the elevated B7.2 expression, but not increased Bl1 compartment, is associated with increased pathogenicity. These experiments are a necessary step towards the identification of the SLE-susceptibility genes on mouse chromosome 4. A high resolution genetic map that leads to the physical map and ultimate cloning of the gene cannot be constructed without a solid evaluation of the number of loci and their associated defects. Finally, the understanding of their relative contribution to the disease process will establish priorities for gene identification. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CHROMOSOME 1 REGIONS LINKED TO SLE IN MULTIPLEX FAMILIES Principal Investigator & Institution: Tsao, Betty P. Professor; Medicine; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024
Studies 33
Timing: Fiscal Year 2002; Project Start 30-SEP-1996; Project End 31-MAR-2007 Summary: (provided by the applicant): Systemic lupus erythematosus (SLE) is a hetereogeneous autoimmune disease characterized by a plethora of immunopathologic manifestations including the production of autoantibodies to various nuclear components. Genetic predisposition clearly plays an important role in risk for developing SLE, however, many remain elusive. Genome scans have mapped many SLE susceptibility loci in both murine and human SLE. One genomic region on the distal end of mouse chromosome 1 and its syntenic human counterpart 1q23-44 has shown strong evidence to harbor SLE susceptibility genes in multiple independent genome scans of both mice and humans. We have observed evidence for linkage to SLE at 1q23 and 1q4142 in our cohort of affected sibpair families. In this application, we propose to identify genetic polymorphisms within 1q21-44 that are associated with SLE or SLE subsets in our cohort, and to explore if the same or functionally related genes contribute to syntenic conservation in susceptibility intervals between mice and humans. Our aims are as follows: 1) To continue family ascertainment for an additional 150 SLE multiplex families and an additional 500 simplex families and to assess familiality of SLE manifestations. The enlarged multiplex families will allow more samples in each stratified subset (by ethnicity, SLE-manifestations, or age of disease-onset), hence greater statistical power to localize the linked interval. The enlarged simplex families will allow greater statistical power for association studies in each ethnic group. Familial SLE manifestations will be used in stratification of our family collection to reduce genetic heterogeneity in subsequent linkage and association studies. 2) To perform a targeted genome scan of chromosome 1q21-44 and to narrow the intervals linked to SLE susceptibility. 3) To perform linkage disequilibrium mapping of the narrowed genomic intervals to eventually localize genetic polymorphisms associated with SLE susceptibility. 4) To assess human homologues of candidate murine SLE susceptibility genes (Sle1d, Nba2, and Lbw7) for evidence of association in our cohort. The identification of SLE susceptibility genes can reveal underlying mechanisms in the pathogenesis of autoimmunity and provide potential targets for disease management. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHROMOSOME 6P AND DEVELOPMENTAL DEFECTS Principal Investigator & Institution: Rosen, Fred S. Professor of Pediatrics; Cbr Institute for Biomedical Research 800 Huntington Ave Boston, MA 02115 Timing: Fiscal Year 2001; Project Start 01-JUL-1999; Project End 30-JUN-2004 Summary: Systemic lupus erythematosus (SLE) is an autoimmune disorder which affects over 200,000 women in the USA and it is characterized by anti-nuclear antibodies and a high incidence of glomerulonephritis. A major risk factor for SLE is deficiency in early classical pathway complement components C1, C2 or C4. This association presents a paradox because it is not expected that an immune deficiency would result in an autoimmune disease. One explanation is that early complement is involved in maintenance of B cell tolerance and in its absence, self-reactive B cells accumulate in the periphery where they potentially may be activated. The goal of this proposal is to test this hypothesis and it is divided into 3 specific aims: (i) Test the hypothesis that early classical pathway complement components C1, C4 and C3 are directly involved in negative selection of self-reactive B lymphocytes. The approach used in this aim is to breed mice deficient in C1, C4, or C3 with two well established immunoglobulin transgenic models (anti-HEL and anti-dsDNA) and determine if complement is essential in B cell anergy. (ii) The second aim will test the hypothesis that deficiency in classical pathway complement results in increased severity of disease in a well defined mouse
34 Lupus
model of lupus, i.e. lpr strain. The advantage of this aim is that it will examine the importance of early complement in the autoimmune response to natural lupus antigens such as dsDNA and nuclear proteins. (iii) The third aim will test the hypothesis that impaired self-tolerance in C4null mice can be rescued by protein replacement or gene therapy and if so compare C4A and C4B isotypes. It will also examine the mechanism of C4 in B cell tolerance using a fusion protein of C4d linked to sHEL antigen to uncouple solubilization of immune complexes from targeting of antigen to the lymphoid compartment via C4d. This aim is important as it will establish the feasibility of protein or gene therapy in lupus and clarify our understanding of B cell tolerance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: COMPLEMENT C4 AND HLA CLASS III GENES IN HUMAN SLE Principal Investigator & Institution: Yu, Chack Y. Associate Professor; Ohio State University 1800 Cannon Dr, Rm 1210 Columbus, OH 43210 Timing: Fiscal Year 2001; Project Start 01-JUN-2001; Project End 31-MAR-2006 Summary: Complement proteins are essential in the dissolution of immune complexes (IC). The circulation of pathogenic IC is an etiologic factor for kidney and autoimmune disease. Systemic lupus erythematosus (SLE) is multi-factorial disease with complex genetic trait and diverse clinical manifestations. Lupus nephritis (SLE-N) is a severe form of the disease. Specifically, this proposal seeks to determine the roles of complement component C4, which has an amazing degree of variations in the quantities and qualities of the genes and proteins present in each individual, in the disease etiology of SLE and SLE-N. The quantitative variations include the number of the C4A and C4B genes present that may determine the levels of proteins expressed in patients and in normals. We hypothesize that over-expression of C4 (C4A, C4B or both), underexpression of C4 (C4A or C4B or both), and malfunction of C4A or C4B contribute to the etiological processes of SLE and SLE-N. In other words, the pathogenesis of SLE may be related to the C4 gene dosage (number of C4 genes), C4 gene types (long and short C4 genes), and C4 protein functions (C4A and C4B isotypes and allotypes). Over-expression of C4 may aggravate the disease by directly promoting the local activation of the complement system that causes tissue injuries. Under-expression of malfunction of the CR would lead to impairment in the dissolution of IC. Specific techniques to determine the qualitative and quantitative variations of the RCCX constituents including complement C4A and C4B have been established by our laboratory. Many novel genes have been discovered in the MHC class III region. These breakthroughs allow the following Specific Aims to be addressed: I) To determine the RCCX modular variations in SLE, SLE-N and normals; II) To determine the molecular bases of C4A and C4B deficiencies in SLE-SLE-N and normals; IV) To investigate the molecular genetics of the MHC class III genes in SLE, SLE-N and normals. The study population include 250 individuals with SLE-N, and equal numbers of SLE patients with non- renal involvement, affected family based controls and case matched controls. The results of this proposal will directly influence the philosophy on the treatment (and possibly cure) of SLE and SLE-N. It may have enormous impact on the medicare of the SLE and other rheumatic disease patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: COMPLEMENT, CARDIOVASCULAR DISEASE, AND SLE Principal Investigator & Institution: Ahearn, Joseph M. Associate Professor; Medicine; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260
Studies 35
Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-MAY-2007 Summary: (provided by applicant): Inflammation is now recognized as a critical process in the development and rupture of atherosclerotic plaques, and in the morbidity and mortality that result from cardiovascular disease. Recent studies have also suggested that this process may be accelerated and exaggerated in patients with systemic lupus erythematosus. We have recently established a research program focused on vascular biology and pathology, with a focus on lupus as a model of accelerated atherosclerosis. The stimulus for these investigations was a recent surprising observation by Manzi and colleagues who demonstrated a strong linear association between elevated serum levels of C3 and C4 and aortic stiffness in premenopausal women with SLE. Whereas decreased serum levels of C3 and C4 have traditionally been used to monitor disease activity in patients with SLE, association of elevated serum levels of serum complement components with any disease process is unprecedented. This observation led us to investigate the potential role(s) for complement C3 and C4 in the immunopathogenesis of cardiovascular disease in SLE. Vascular imaging studies led to several intriguing and unexpected observations that will be further explored here. First, we discovered that complement components C3 and C4 are present in several distinct patterns within the arterial walls of both humans and mice. Specifically, proteolytic fragments of C3 and C4 co-localize with, and may be covalently bound to, elastin within the arterial wall. This entirely unexpected observation suggested that complement deposition within the arterial wall may increase vascular stiffness, an early event in atherosclerosis, through direct interference with elastic fiber flexibility. Second, we observed aggregates of complement deposition within the vessel wall, the site at which plaque formation is now known to initiate. Third, we demonstrated that complement components are specifically present within the vasculature of mice with lupus-like syndromes as compared with controls. These observations, together with those of Manzi and colleagues, have led to the following specific aims that are based on our central hypothesis that the complement system may influence vascular stiffness and contribute significantly to the atherosclerotic process by directly reducing vascular elasticity within the arterial wall. The long-term goal of this proposal is to perform an initial characterization of the role of the complement system in atherosclerosis, using normal and abnormal human and mouse vascular systems. Specific Aim 1 is to characterize the spatial and temporal localization of complement proteins C3 and C4 within the arterial wall. Specific Aim 2 is to characterize the distribution of complement C3 and C4 within the arterial tree. Specific Aim 3 is to determine the capacity of complement C3 and C4 within the arterial wall to increase arterial stiffness. These studies will represent the first rigorous investigation of the role of the complement system in atherosclerosis, using SLE as a model of accelerated coronary vascular disease. Ultimately, the data generated by the proposed studies should identify therapeutic targets in SLE and in atherosclerosis in general. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CORE--CELL SCIENCES AND IMMUNOCHEMISTRY Principal Investigator & Institution: Gaskin, Felicia; Charlottesville Box 400195 Charlottesville, VA 22904
University
of
Virginia
Timing: Fiscal Year 2001 Summary: The Cell Sciences and Immunochemistry Core (CIC) is an integral part of the SLE SCOR Center, providing services to the four principal investigators on projects that rely on the following laboratory support. First, routine histology and electron microscopy studies and their interpretations. Second, tissue and cellular localization of
36 Lupus
antigenic molecules by immunohistology, and messenger RNA (mRNA) by in situ hybridization. Third, acquisition and management of human and murine recombinant proteins and peptide libraries for the "lupus antigens" including Ro60, Ro52, La, Sm, calreticulin, U1-70 RNAP, dsDNA and phospholipids. Fourth acquisition and management of a library of essential monoclonal Ab and cDNA probes for Ag localization and flow cytometry, as well as B cell hybridomas generating sufficient monoclonal Ab for in vivo experiments. Fifth, quantitation and isotype determination of human and murine autoAb by immunoprecipitation and ELISA. Sixth, quantitation of murine cytokines by ELISA. The CIC will function, in collaboration with the project investigators, in experiments that: 1) analyze autoAb responses to the "lupus Ag" and their overlapping peptides, 2) require peptides for mapping their T and B cell epitopes, 3) evaluate the immunopathologic end points including the detection and semiquantitation glomerulonephritis, sialoadenitis and diseases affecting other organs that result from autoimmune response to the lupus Ag, 4) evaluate the quality of the autoimmune response with respect to the specificity and Ig isotype of the autoAb and the nature of the cytokines produces by peptide specific T cells. In summary, the CIC will support the research project of the SLE SCOR by providing common reagents and by conducting several essential and common techniques. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CORE--METHODOLOGY AND DATA MANAGEMENT Principal Investigator & Institution: Chang, Roland W.; Northwestern University Office of Sponsored Programs Chicago, IL 60611 Timing: Fiscal Year 2001 Summary: The Methodology and Data Management Core is critically important to the success of this MAMDC and its EEHSR Component in particular. The centralized availability of expertise in database and study form construction; data entry, monitoring, and retrieval, and the various analytic techniques used to test hypotheses and control for potential confounders are essential resources for all investigators. The Core has assisted educators, epidemiologists, and health services researchers from several divisions and departments in studying a wide variety of disease and demographic groups including systemic lupus erythematosus, osteoarthritis, juvenile dermatomyositis (JDMS), rheumatoid arthritis, and the elderly. Core resources have been used efficiently because of the economies of scale in addition to excellent coordination with the EEHSR component. Recognizing that newer analytic techniques have become available and more accepted and that this proposal represents an expansion of our work in clinical epidemiology and health services research, this proposed Core is both larger to support a greater volume of work and broader t utilize these new analytic techniques. As a result of the Executive Committee's decision to emphasize longitudinal and cost-effectiveness research, experts in advanced statistical techniques (generalized estimating equation (GEE), classification and regression trees (CART), econometric approaches to controlling selection bias, meta-analysis), economics, and decision analysis (stochastic tree modeling, continuous- risk utility assessment) have been recruited as Core co-investigators. Clinical Epidemiology has also been formally included within the Core structure. The Core will support the four EEHSR proposals in this grant application and the funded activities of the JDMS registry and Children's Memorial Hospital. It will continue to contribute to the MAMDC research environment by providing data management and methodologic assistance to investigators who engage in arthritis related research. The institution of a EEHSR/Core research conference and enhanced viability and support for health services research on
Studies 37
Northwestern University's Chicago Campus will further heighten the Core's influence on the environment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CORE--MOUSE GENETIC Principal Investigator & Institution: Brown, Michael D. Assistant Professor; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2002; Project Start 27-SEP-2002; Project End 30-JUN-2003 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a systemic autoimmune disease process contributed by multiple genes in humans and mouse models. The usefulness of the mouse as a model organism for studies of factors that contribute to pathogenesis has been well accepted. To date, as many as 12 distinct loci have been implicated in mouse lupus, but strong genetic linkages have been assigned to chromosomes 1, 4, 7 and 17. Newly available genetic tools are transforming studies of autoimmune disease in mouse models and have the potential to support rational exploration of complex physiological pathways and genetic traits. Congenic mice are especially useful in this regard since they are amenable to the study of the effects of single genes or multiple tightly linked genes in genetically controlled experiments, thus enabling directed and rational approaches toward the study of multigenic traits. Moreover, congenic mice and recombinant congenic mice derived from them, provide the foundation for genetic mapping and positional gene cloning strategies. Ultimately, information and resources gained from investigation of mouse models may be exploited to identify and characterize human genes that contribute to autoimmune disease. In order to make these genetic tools available to the members of the SCOR, we have established the Mouse Genetics Core (MGC). The primary role of the Mouse Genetics Core is to maintain extant mouse models and to develop and maintain novel mouse models for studies of autoimmune disease in well characterized and appropriate genetic backgrounds as needed by SCOR Principal Investigators serving all four SCOR projects. Novel congenic, recombinant congenic and gene targeted mice will be generated using a "speed congenic" strategy utilizing genome-wide microsatellite screening sets that others and we have previously characterized. In particular, the MGC will serve as a breeding unit and genetic analysis facility for efficient and controlled propagation and selection of specified genetic traits or mutant genes in mice. Genetic characterization will include genotyping, microsatellite linkage, DNA sequencing, and gene expression analyses. In addition, the MGC will serve as an educational resource, providing information and training for all aspects of mouse breeding and handling and genetic characterization for SCOR Investigators and their laboratory personnel. Hence, the MGC will service the needs of the SCOR Investigators by providing an operational and centralized mouse core facility to maintain and develop the necessary mouse models for continued SLE investigation and will encourage expansion of biochemical and physiological studies from cell culture systems into whole animal models, facilitating linkage of basic research with preclinical studies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: COSTIMULARTORY ERYTHEMATOSUS
ANTAGONISTS
/SYSTEMIC
LUPUS
Principal Investigator & Institution: Davidson, Anne; Associate Professor; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2007
38 Lupus
Summary: Although, the lifespan with SLE has improved considerably over the last several decades, safer and more effective therapies as needed. The discovery of costimulatory molecules has led to the consideration of new therapies for SLE aimed at decreasing activation thresholds of both B and T cells. Use of costimulatory blocking agents in appropriate combinations may allow us to treat SLE flares with short term regimens, so as to avoid the morbidity associated with long term immunosuppressive therapy. In Aim 1 of this proposal we will develop modulators of two newly discovered members of the B7/CD28 and TNF/TNFR families and test them in prevention and remission induction studies in SLE prone mice in combination with a knock effective agent, CTLA4Ig. The CD28 like molecule ICOS that costimulates activated T cells and secondary B cell responses will be blocked with a soluble murine ICOS-IgG2a fusion. The CD40L like molecule BAFF, that costimulates both naive B cell development and germinal center formation, will be blocked with either a dimeric or pentameric fusion protein of its receptor TACI. In Aim 2 we propose to understand at which stage of C cell development these new agents are effective by using novel methods of isolating populations of naive and antigen activated autoreactive B cells that can be studied using a variety of immunochemical and molecular techniques. In Aim 3 we will determine the effect of CTLA4Ig with and without addition of ICOS or BAFF blockade on two populations of T cells that may influence disease progression- memory and NK T cells. The mechanistic studies proposed in this application will have direct clinical relevance because they permit us to understand the effect of showing signs of disease activity. As immune modulating agents begin to enter early clinical trials in humans, understanding their short and long term effects on the B cell repertoire and B cell activation in the autoimmune host is critically important. Analysis of the pathways that are inactivated by these new reagents will pave the way towards development of more specific targeted therapy for SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CR2 AS A MURINE LUPUS SUSCEPTIBILITY GENE Principal Investigator & Institution: Boackle, Susan A. Assistant Professor; Medicine; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, CO 800450508 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 31-DEC-2007 Summary: (provided by applicant): The major murine systemic lupus erythematosus (SLE) susceptibility locus, Sle1, corresponds to 3 loci independently affecting loss of tolerance to chromatin in the NZM2410 mouse. The congenic interval corresponding to Sle1c, derived from NZW, contains Cr2, which encodes complement receptors I and 2 (CR1/CR2, CD35/CD21). CR1/CR2 deficiency has been associated with autoimmune disease in both humans and in animal models. A structural difference in a critical ligand-binding domain has recently been identified in Sle1c CR1/CR2 which results in significant impairment in receptor function. These results strongly support the role of Cr2 as a disease susceptibility gene in the Sle1c interval. The project outlined in this proposal will be directed towards characterizing the role of NZW CR2 in the NZM2410 mouse model for lupus. The specific aims are to prove that CR2 is the lupus susceptibility gene in the NZM2410 Sle1c interval, to identify the structural domains in NZW CR2 that are critical in loss of tolerance, and to determine the mechanisms by which NZW CR2 results in loss of tolerance. Proof that CR2 is the lupus susceptibility gene in the Sle1c locus will be provided by demonstrating that the Sle1c phenotypes resolve in the presence of normal gene products. Recombinant strains that contain narrowed intervals containing Cr2 will be assessed to ensure that CR2 dysfunction
Studies 39
continues to track with autoimmune disease, The critical receptor domains that result in the autoimmune phenotypes will be determined, using both CR2-deficient cell lines transfected with recombinant proteins as well as B cells from BAC transgenic mice that express various forms of the polymorphic NZW CR2. Finally, the mechanisms by which the altered NZW CR2 allele results in loss of B cell tolerance will be characterized using the 3-83 and HEL models for B cell tolerance. These studies will clarify the specific functions of CR2, impaired in the NZM2410 mouse model, that may impact on the development of autoimmune disease and thus be important targets for therapeutic interventions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CYCLOOXYGENASE/NUCLEOBINDIN INTERACTION Principal Investigator & Institution: Simmons, Daniel L. Chemistry and Biochemistry; Brigham Young University A-261 Asb Provo, UT 846021231 Timing: Fiscal Year 2001; Project Start 01-SEP-1999; Project End 30-JUN-2003 Summary: Cyclooxygenases (COXs) catalyze the rate-limiting step in the synthesis of prostaglandins, prostacyclin, and thromboxanes. Derived from arachidonate, these biomediators act as local hormones affecting inflammation, pain, and a host of other physiological processes in the cell and organism. There are presently two known COXs. COX-1 is typically expressed constitutively like a housekeeping protein. COX-2, in contrast, is highly inducible by growth factors and hormones. Many cells contain both COX-1 and COX-2. In these cells arachidonate is delivered selectively by an unknown mechanism to COX-2 following mitogen stimulation. Also, COX-1 and COX-2 can have very different sensitivities to NSAIDs in vivo than in their purified states in vitro. These data suggest that, in spite of the colocalization of COX-1 and COX-2 to the lumen of the endoplasmic reticulum and nuclear envelope, unknown factors confer unique, isoenzyme-specific properties on these structurally and enzymatically similar proteins. While in search of proteins which might confer these properties, our laboratory determined that the calcium binding protein, nucleobindin (Nuc), associates with cyclooxygenases in the yeast 2-hybrid system and in other in vitro and in vivo assays. Extracellular release of Nuc into the blood stream has been associated with systemic lupus erythematosus-like symptoms in mice. Indeed, Nuc was first isolated as a protein responsible for the generation of anti-DNA antibodies in a genetic mouse model for lupus. Moreover, injection of purified Nuc into normal mice elicits some of the symptoms of lupus. Similar to COXs, Nuc is widely expressed in tissues; however, its function in these tissues is unknown. Our hypothesis is that Nuc functions in COX/Nuc complexes to regulate prostaglandin synthesis and that COXs may function in the release of Nuc in autoimmune disorders. In these proposed studies, Nuc and COX isoenzymes will be co-expressed at high levels in insect cells and the enzymatic and physical properties of COX/Nuc complexes relative to unbound COX- 1 and COX-2 will be studied. Pharmacologically important aspects to be analyzed include the effect of Nuc on the rate of prostaglandin synthesis and on the inhibitory action of non-steroidal antiinflammatory drugs (NSAIDs). Additionally, a new variant of Nuc expressed in fibroblasts that lacks 33 amino acids in its COX binding domain will be examined. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CYTOKINES AS PREDICTORS OF FLARE IN SLE Principal Investigator & Institution: Rus, Violeta; Medicine; University of Maryland Balt Prof School Baltimore, MD 21201
40 Lupus
Timing: Fiscal Year 2001; Project Start 15-SEP-2000; Project End 31-AUG-2005 Summary: The candidate, Violeta Rus, is a rheumatology fellow and future Clinical Instructor at the University of Maryland Medical School, where she is developing a career in clinical research in lupus. The proposed work draws on her bench-research experience on the role of cytokines in a murine models of lupus, and requests support for a Mentored Patient-Oriented Career Development Award to acquire new expertise in the science of clinical investigation in order to be able to translate new biomedical advances to the bed-side. The candidate will work under the mentorship of Dr. Charles S. Via and Marc C. Hochberg in the superb research environment, resources and opportunities for career development at the University of Maryland Medical School. The candidate's immediate goals are to obtain the training necessary to become a successful, independent clinical investigator able to pursue high quality hypothesis-driven disease oriented research. The proposal is an outgrowth of the PIs previous work in a murine model of lupus where she identified the role of several cytokines important in driving autoantibody production. The present study will define the cytokine pattern in active and inactive lupus patients and determine whether changes in cytokine levels will predict future flares. Hypothesis: High levels of B cell stimulatory cytokines IL-10 and IL-6 and low or variable levels of IFN-g and TNF-a will correlate with disease activity in lupus patients and precede disease flare. Conversely, reciprocal changes of these cytokines will accompany or precede disease remission. Specific Aim 1) Determine in a cross sectional study whether disease activity is correlated with altered serum or mRNA levels of IL-10, IL-6, IFN-g and TNF-a. Cytokines will be measured in serum specimens by ELISA and in peripheral blood mononuclear cells mRNA by semiquantitative RTPCR. Specific Aim 2) Determine in a longitudinal study whether alterations in these cytokines precede or accompany disease flare and post-flare improvement. Samples of serum and mRNA from 50 patients followed longitudinally will be tested every 3 month in stable patients, biweekly during flares and monthly until remission. For both aims, flares will be defined using SLEDAI and Physician Global Assessment scores. Statistical analysis using exploratory analyses and regression models will be used. The proposed research will have high relevance to our understanding of the role of cytokines in inducing flares and remissions in SLE, assess their predictive value for future flares, and develop a rationale for future cytokine-based therapies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DELINEATION PATHOGENESIS
OF
GENETIC
PATHWAYS
TO
LUPUS
Principal Investigator & Institution: Liu, Kui; Microbiology; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001; Project Start 01-SEP-2001 Summary: (provided by the applicant): Systemic lupus erythematosus (SLE) is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. Development of congenic mouse models carrying lupus susceptibility gene intervals has provided powerful tools for studying the mechanism of lupus pathogenesis. Sle1 mediates the loss of tolerance to nuclear antigens and the initiation of autoimmunity. Our recent study demonstrated that Sle1 mediates the abnormal expression of several genes, including the c-myc protooncogene, that control B-cell activation and proliferation. We hypothesize that autoreactive B-cells are generated from B-cell populations that have aberrant c-myc expression. We propose to identify the mechanisms leading to the aberrant c-myc expression and to characterize the B-cell populations that have aberrant c-myc expression in B6.Sle1 mice. We also
Studies 41
propose to use powerful, microarray-based approaches to identify the molecular mechanisms by which Sle1 and S1e3 interact to cause lupus. Furthermore, we propose to identify lupus susceptibility gene(s) in the S1e3 interval using fine mapping in combination with functional genomics. By identifying the lupus susceptibility genes and the molecules involved in the pathogenesis, we can select candidate therapeutic targets for curing lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DYSREGULATION OF THE IMMUNE SYSTEM IN AUTOIMMUNITY Principal Investigator & Institution: Flavell, Richard A. Professor and Chairman; Immunobiology; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2001; Project Start 01-SEP-1994; Project End 29-SEP-2002 Summary: The goal of this program is to understand the regulation and dysregulation of the immune system in autoimmunity. The program involves collaborative interaction between members of three Departments, and is organized into four projects supported by three Core facilities. Expertise in the field of immunology, molecular biology, and biochemistry will focus on the vents that initiate and sustain autoimmune responses, and the regulatory processes. which contain autoimmunity. We will address the following questions. What are the requirements to initiate autoimmune responses? Are autoimmune responses regulated, and if so, by what mechanisms? Does immune regulation contain autoimmune responses under normal circumstances? Finally, do sustained autoimmune responses remain chronic because they diversity from a single initiating response to responses to other autoantigens from the same tissue? These questions will be addressed by collaborative interactions between the Principal Investigators of these projects, which are as follows: (1)R.A. Flavell- Using transgenic mice expressing a T cell receptor specific for myelin basic protein (MPB) and gene targeted mice lacing L- selectin or E- and P-selectin, the role of selectins in the development of EAE will be determined. The requirement of selectins for the development of disease, as well as the mechanisms which underlie this requirement will be determined, focusing on the cell types which must express L-selectin, the role of selectins in the entry of leukocytes into the CNS and the potential role of selectins within the CNS. (2) C.A. Janeway Jr.- This project will investigate four aspects of the regulation of experimental allergic encephalomyelitis (EAE): Why are mice lacking B cells unable to fully resolve their disease; why does the inability to form cells with other receptors lead to spontaneous disease in mice transgenic for a TCR that recognizes myelin; why do mice cells with other receptors lead to spontaneous disease in mice transgenic for a TCR that recognizes myelin; why do mice with the same receptor who are heterozygous for gld get spontaneous disease; and what is the role of L- selectin in EAE, in collaboration with project 1. (3) M.J. Shlomchik- Transgenic mouse models will be used to study the regulation of B cells expressing a disease-related autoantibody, rheumatoid factor (RF), in normal and autoimmune mice. In contrast to some other autoantibody models, RF B cells from these transgenics are competent to initiate an immune response. Thus, studies will focus on how RF B cells are regulated after Ag stimulation in normal mice and propagated in autoimmune mice, and what prevents chronic autoimmunity in RF transgenic mice. (4) M.J. Mamula, PI- This project will examine the role of self-peptides in the initiation and perpetuation of both Band T cell autoimmunity in models of systemic lupus erythematosus (SLE) and multiple sclerosis (EAE). The role of B cells as autoantigen in models of systemic lupus erythematosus (SLE) and multiple sclerosis (EAE). The role of B cells as autoantigen presenting cells will be examined with relevance to mechanisms that lead to epitope spreading in autoimmunity. Finally, this
42 Lupus
work will study a novel post-translational peptide modification that arises naturally in cells and confers immunity to self peptides. These four projects will be supported by an administrative core to coordinate the project as a whole, a genetically modified mouse core to provide gene targeted and transgenic rodents essential to most of these studies, and a FACS core, to allow us to separate cells for analysis and to analyze cells in all of these projects. The program is coordinated by frequent meetings of the program faculty bringing together these diverse approaches to address a common goal. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ECTOPIC GERMINAL CENTER REACTION IN SYSTEMIC LUPUS Principal Investigator & Institution: Pascual, Maria V. Pediatrics; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001; Project Start 15-SEP-2000; Project End 31-AUG-2005 Summary: (Adapted from the Investigator's abstract): Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ involvement and widespread immunologic abnormalities, the most relevant of which are hypergammaglobulinemia, immune complex formation, and complement system activation. Highly specific autoantigen-driven responses, particularly those directed at protein and nucleic acid components of intracytoplasmic and intranuclear particles, are characteristic of SLE patients. At the peak of disease activity and antibody secretion, however, SLE patients are known to display peripheral blood lymphopenia. The investigator's laboratory has previously described methods to isolate human peripheral B cell subpopulations. Now they show that the peripheral blood B cells of children with SLE differ from healthy adults and children. In particular, the conventional recirculating naive B cell pool is decreased in children with SLE, while B cells with pre-germinal center phenotype are expanded. Consistent with this information, genes restricted to the germinal center (GC) reaction can be amplified from SLE peripheral B cells. Additionally, a cell population that co-purifies with B cells but lacks the pan-B cell markers CD19 and CD20 is expanded in these patients. This population is composed of a CD79a+ B cell subset, and a CD79a- (dendritic cell?) subset. These data lead the investigators to propose that an ectopic and accelerated GC reaction takes place in SLE. The experiments described below are designed to test their hypothesis and to gain understanding of the nature of B cell alteration(s) in SLE. The aims of the current application are: 1. To further demonstrate that the IgD+ CD38+ population + _ expanded in the blood of SLE patients corresponds to pre-GC cells. 2. To + _ characterize the phenotype and function of the recirculating B cell subpopulation(s) expressing surface CD40-L in SLE blood. 3. To identify the + _ nature and function of the SLE-restricted CD19- CD20- cell populations that co-purify with B cells. 4. To determine the clinical relevance of the + _ accumulation f IgD+ CD38+ B cells and/or CD19- CD20cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ELICITATION OF HIV SPECIFIC CATALYSTIC IMMUNITY Principal Investigator & Institution: Paul, Sudhir; Professor; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2001 Summary: In view of the enhanced antigen neutralizing capability imparted by the catalytic function, we propose to isolate antibodies (Abs) for passive immunotherapy of HIV-1. This goal has become feasible because of the recent acquisition of the following
Studies 43
information: a subset of Abs expresses peptidase activity; synthesis of peptidase Abs, including gp120 cleaving Abs incresed in lupus mice; the peptidase activity is encoded by a germline gene encoding the variable (V) region of the light chain subunit; and, the catalytic site is structurally similar to the active sites found in non-Ab serine proteases. In Aim 1, the specificity of the gp120 cleaving Abs in lupus mice will be enhanced by immunization with gp120, a B cell epitope derived from the CD4 binding site of gp120, and if needed, intact HIV-1 particles. Recombinant Fv constructs with peptidase activity will be selected from phage display libraries (constructed by Core B) using gp120 analogs designed to bind covalently to the serine protease-like site found in certain Ab light chains (covalently reactive analogs, CRAs). The CRAs to be employed include derivatives of the whole gp120 molecule and the gp120 epitope. The essential features of the CRAs include: the presence of an electrophilic ester group reactive with nucelophilic Ser residues, a phosphonate structure mimicking the tetrahedral transition structure, a basic flanking residue reactive with the germline peptidase sites, and additional flanking residues derived from gp120 to permit high affinity gp120 recognition by the Abs. In Aim 2, the CRAs will be employed as immunogens to permit the selective recruitment and somatic maturation of the catalyst gene(s) for synthesis of gp120cleaving Abs. Improvements in the catalytic turnover are predicted over the course of clonal selection because superior CRA binders are likely to be superior transition state stabilizers. This will be evident as increased binding of the Abs to the CRAs relative to the unmodified gp120. As in aim 1, the best catalysts will be isolated from Fv phage display libraries using CRA selections. Cleavage of the gp120 B cell epitope, monomer gp120 in solution, and native gp120 expressed on the HIV surface will be studied by electrophoresis, HPLC and radioassay methods. The catalytic Ab responses in autoimmune and non-autoimmune mice will be compared to determine whether the regulatory factors limiting catalytic Ab synthesis by the healthy immune system are surmounted by the CRA immunizations. In vitro HIV-1 infectivity studies will be carrie dout by Core C to compare the HIV neutralizing activity of catalytic and noncatalytic Abs. The desired result from these studies is that we will have in hand catalytically efficient Fv constructs derived from autoimmune mice with the capability of specifically cleaving gp120 and potently neutralizing the infectivity of HIV-1. In addition, if the gp120 CRAs provoke a catalytic Ab response in non-autoimmune mice, these reagents can be considered as prototypes for a prophylactic vaccine capable of eliciting catalytic immunity to HIV. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EPIDEMIOLOGY OF CARDIOVASCULAR DISEASE IN LUPUS WOMEN Principal Investigator & Institution: Manzi, Susan M. Associate Professor of Medicine and Epid; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2001 Summary: Cardiovascular event rates in women with lupus represent a 5 to 10 fold increase as compared to population-based studies of normal women. The pathogenesis for premature atherosclerosis in women with lupus is likely multifactorial. The first aim of this study is to estimate the prevalence of clinical and sub-clinical atherosclerosis in women with lupus. The second aim is to determine the association between atherosclerosis and markers of inflammation in women with lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
44 Lupus
•
Project Title: EPIDEMIOLOGY OF OSTEOPOROSIS IN WOMEN WITH LUPUS MAMDC PROJECT Principal Investigator & Institution: Ramsey-Goldman, Rosalind; Northwestern University Office of Sponsored Programs Chicago, IL 60611
Professor;
Timing: Fiscal Year 2001 Summary: The specific aims to be addressed in this study are: 1) To estimate the differences in bone mineral density (BMD) at the hip and lumbar spine between 128 Caucasian and 128 African-American women with lupus and a comparable control group matched by age, race, and menopause status; 2) To determine the association of lupus risk factors with low BMD in women with lupus, after controlling for traditional risk factors for low BMD; 3) To follow the subjects entered in the cross-sectional study over a two year period in order to estimate the difference in the rate of bone loss at the hip and lumbar spine between women with lupus and a comparable control group matched by age, race, and menopause status; and 4) To determine the association of lupus risk factors with increased rates of bone loss in women with lupus, after controlling for traditional risk factors for low BMD. The hypotheses to be examined in this study are: 1a) Women with lupus have lower BMD at the hip and spine than matched controls; 1b) the negative effect of lupus on bone mineral density at the hip and spine is greater in Caucasian than in African-American women; 2a) Traditional risk factors for low BMD (nulliparty and menopause status, irregular menstrual cycles or premature menopause, avoidance of oral contraceptives and/or hormone replacement therapy, lower physical activity level, and decreased vitamin D levels) are associated with lower BMD at the hip or lumbar spine in women with lupus; 2b) lupus risk factors (greater disease activity, greater disease severity, higher corticosteroid burden, use of anticonvulsant drugs, and the presence of renal disease) are associated with lower BMD at the hip or lumbar spine in women with lupus, after controlling for traditional risk factors forlow BMD; 3a) Women with lupus have accelerated bone loss at the hip and spine during two additional years of lupus disease compared with matched controls followed for two years; 3b) the effect of lupus on the rate of bone loss at the hip and spine is greater in Caucasian than in African-American women; 4a) Traditional osteoporosis risk factors (mentioned in 2a above) are associated with accelerated bone loss in women with lupus; and 4b) lupus risk factors (mentioned in 2b above) are associated with accelerated bone loss at the hip or lumbar spine in women with lupus, after controlling for traditional risk factors for low BMD. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ESTROGEN AND GENDER BIASED AUTOIMMUNITY Principal Investigator & Institution: Rider, Virginia C. Associate Professor; Biology; Pittsburg State University Pittsburg, KS 66762 Timing: Fiscal Year 2001; Project Start 01-MAY-2001; Project End 30-APR-2004 Summary: (Scanned from the applicant's abstract) Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women during their childbearing years. The effects of SLE are physically and emotionally debilitating and can be life threatening due to the involvement of a variety of organs including the renal and central nervous systems. Estrogen is a female sex hormone that acts on target cells though specific receptor proteins. Estrogen receptors (ER) are ligand activated transcription factors that bind to unique DNA sequences of target genes and alter the rates of transcription. In lupus T cells, estrogen increases the expression of calcineurin, a protein phosphatase involved in T cell activation. The proposed research will investigate the
Studies 45
molecular mechanisms by which estrogen, acting through the ER, significantly increases calcineurin, activates nuclear factor of activated T cells (NFAT), and augments CD4O ligand (CD4OL) expression in the T cells from female lupus patients but not in T cells from healthy individuals. The molecular mechanisms responsible for increased calcineurin expression in lupus T cells will be investigated using assays that distinguish transcriptional and posttranscriptional gene regulation. To determine if the differential control of calcineurin in lupus T cells is due to differences in the ratio of ER subtypes (ERa versus ERB), reverse transcription and quantitative polymerase chain amplification will be used to determine the amount of ER subtype transcripts. Altered ER binding to DNA regulatory elements will be assessed by electrophoretic mobility shift assays (EMSA). To investigate if the estrogen-dependent increase in calcineurin hyperactivates the transcription factor NFAT, the duration of NFAT dephosphorylation will be analyzed and the ratio of phosphorylated and dephosphorylated NFAT will be compared between T cells treated without and with estradiol. Biological activity over the same time points will be measured by comparing the amount of NEAT binding to consensus DNA sequences of the CD4OL promoter and by measuring changes in CD4OL expression in response to estradiol. The knowledge gained by completion of this study will identify potential sites in human lupus T cells at which to block signal transduction and decrease the abnormal synthesis of cytokines that promote inflammation and B cell activation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EUBACTERIA ERYTHEMATOSUS
IN
PATHOGENESIS
OF
SYSTEMIC
LUPUS
Principal Investigator & Institution: Kovacs, Shirley A.; California State University Fresno Fresno, Ca 93740-9999 Fresno, CA 93740 Timing: Fiscal Year 2001 Summary: Systemic Lupus Erythematosus (SLE) is an autoimmune arthritic disease of unknown etiology but behaves as an antigen-driven response to the nuclear component, U1 small nuclear ribonucleoprotein particle (U1snRNP). U1nsRNPs are highly conserved among eukaryotes but not thought to exist in prokaryotic organisms. However our work suggests that ribonucleoprotein particles displaying high homology to U1snRNPs do exist among eubacteria. As bacterial snRNPs could provide an antigenic stimulus which might lead to autoimmunity, a small group of SLE-susceptible MRL-lpr/lpr mice were injected with extracts from one of the "U1snRNP"-positive bacteria. The mice injected with bacterial extracts displayed early expression of certain clinical and immunological symptoms of SLE, commencing about two weeks after inoculation (mice were one month old at inoculation). This proposal intends to examine the details of this immune response through variations in immunization dose and time relevant to "natural" disease course and to include more thorough clinical, histopathological and immunological assessments of the outcomes. Clinical criteria will principally include assessments of arthritis, lymph node hyperplasia and alopecia; histopathological criteria will principally monitor tissue abnormalities in joints, kidneys and lymph nodes; and immunologic criteria will use ELISA reactivity to human nuclear extract and to bacterial extract for titers and isotypic profiles, direct immunofluorescence to detect glomerulonephritis, indirect immunofluorescence to detect reactivity to specific nuclear antigens, and western blotting to determine reactivity to U1snRNP-specific proteins MRL-lpr/lpr mice immunized with physiological saline and random genetic mice (Swiss-Webster) injected either the bacterial extract or saline will serve as experimental controls. Other candidate "U1snRNP"- containing bacterial organisms will
46 Lupus
be identified and/or evaluated using computer-based genomics, the polymerase chain reaction (PCR), northern hybridization and RNase protection assays, and mRNA splicing- complementation assays. Finally, the ability of these candidate prokaryotic cell extracts to stimulate SLE will be assessed by immunization of MRL-lpr/lpr mice and assessment of clinical, histopathological and immunological symptoms. The outcome of these experiments should provide a cleared indication of whether: inoculation with prokaryotic cells; and these latter pro-karyotic cells can also stimulate SLE symptoms in MRL-lpr/lpr model mice. The possibility that bacteria are etiological agents of SLE unveils a wealth of antibiotic therapeutic opportunities for treatment of SLE or other autoimmune, rheumatological disorders. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: EXPERIMENTAL INDUCTION OF SLE BY ALTERED IA Principal Investigator & Institution: Eisenberg, Robert A. Professor; Medicine; University of Pennsylvania 3451 Walnut Street Philadelphia, PA 19104 Timing: Fiscal Year 2001; Project Start 01-JUL-1984; Project End 30-JUN-2003 Summary: (Adapted from the applicant's abstract) - The chronic GVH reaction is induced in inbred strains of mice by a transfer of spleen and lymph node T cells that recognize a foreign MHC class II determinant in the recipient. This syndrome is characterized by a spectrum of autoantibodies and immunopathological changes that closely parallels those found in human systemic lupus erythematosus. Previous work by the principal investigator's laboratory and others have shown that this disease is induced by the donor alloreactive T cells that recognize foreign MHC class II on B cells of the recipient that are in turn, induced to produce autoantibodies. In the current application, the principal investigator proposes to study the specific role of the donor and recipient T cells as well as the recipient B cells in chronic GVH. His specific aims are (1) To learn how B cell tolerance is lost in the chronic GVH reaction; (2) to understand what the role is of endogenous T cells in the chronic GVH response; and, (3) to determine what the role is of donor T cells in the chronic GVH response. These studies, the applicants expect, will provide important insight into the mechanism of autoantibody production and loss of tolerance in the chronic GVH response. This, they believe, will help in understanding the underlying mechanisms that produce the loss of tolerance characteristic of spontaneous systemic lupus erythematosus (SLE), both in mice and in humans. Such understanding will, eventually, lead to more rational therapy for this disease and other autoimmune diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: FC RECEPTOR FUNCTION IN NORMALS AND SLE Principal Investigator & Institution: Salmon, Jane E. Professor; Hospital for Special Surgery 535 E 70Th St New York, NY 10021 Timing: Fiscal Year 2001; Project Start 01-DEC-1992; Project End 31-AUG-2003 Summary: (Adapted from Investigator's abstract): Human Fc receptors (FcgR) consist of three families with extensive diversity of structure and function. Recent advances bring into focus four observations pertinent to SLE: 1) FcgRIIa is a crucial receptor mediating phagocytic function; 2) FcgRIIa is unique among FcgR in that it is targeted for oxidant and protease-induced amplification of effector function as well as avidity modulation, independent of receptor number; 3) the H131 allele of FcgRIIa is the only human FcgR which recognizes IgG2 efficiently; 4) the distribution of FcgRIIa alleles is skewed in SLE patients compared to normals, with a highly significant decrease in FcgRIIa-H131 in
Studies 47
lupus nephritis. In SLE, FcgR-specific immune complex removal by the mononuclear phagocytes system is impaired. This defect is related to renal disease, emphasizing the possible role of FcgR dysfunction in immune complex deposition and the pathogenesis of SLE. Despite the decrease in FcgR function in vivo , there is a paradoxical increase in FcgR binding in vitro. Preliminary data indicate that FcgRIIa is a compelling candidate for the FcgR dysfunction in SLE. Monocytes in SLE patients have increased FcgRIIamediated binding, but markedly decreased FcgRIIa phagocytosis, indicating dissociation of receptor-effector coupling. Disease-induced dysfunction superimposed upon inherited polymorphisms of FcgRIIa with decreased functional capacity may provide the milieu for the development of immune complex deposition and nephritis. Recent evidence for a role of IgG2 autoantibodies in nephritis underscores the importance of FcgRIIa in disease phenotype. Based on these observations, the investigators hypothesize that 1) abnormal FcgRIIa function provides a basis for diseaserelated defects in SLE, and 2) that alleles of FcgRIIa which affect ligand binding are important heritable disease susceptibility factors. Therefore, the specific aims of this application: 1. to define the mechanism of activation of FcgRIIa; 2. to define the basis for the defect in phagocytosis by FcgRIIa in SLE; 3. to define the role of FcgRIIa alleles as risk factors for lupus nephritis: (a) to establish genetic linkage of lupus and nephritis to FcgRIIa and (b) to define the relative importance of FcgRIIa alleles among different ethnic groups; and 4. to define subclasses of IgG deposited within glomeruli in lupus nephritis and their relationships to FcgRIIa alleles, autoantibodies, and induction of glomerular injury. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: FREQUENCY OF MIF GENE POLYMORPHISM AND CUTANEOUS EXPRESSION Principal Investigator & Institution: Kang, Insoo; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2004 Summary: This is a new application for a YSDRCC pilot/feasibility grant from a clinically trained rheumatologist without NIH fundings who now seeks a financial support to initiate his research in determining Mif gene polymorphisms and cutaneous expression of MIF in patients with systemic lupus erythematosus (SLE). The mucocutaneous system is affected in 85% of patients with SLE. An important concept in the pathogenesis of SLE is that there is an intrinsically heightened state of T lymphocyte responsiveness that contributes to sustained T cell activation and autoantibody production. These events lead to recruitment and activation of inflammatory cells, such as macrophages, and subsequent tissue destruction in inflammatory sites. Several studies showed the requirement of macrophages in the development of murine lupus nephritis, suggesting an important role of macrophages as a pro-inflammatory migration inhibitory factor (MIF) is a pro-inflammatory cytokine secreted from monocytes, macrophages and T cells and has a pivotal, upstream role in activation of macrophages and T cells. Recently, a study identified promoter polymorphisms of the Mif gene that comprises the tetranuclotide repeat sequence (CATT)5-8. In rheumatoid arthritis (RA), a systemic autoimmune disease like SLE, a study showed that patients with RA had a decreased frequency of a single 5-CATT allele (lowest Mif expression), which was even lower in RA patients with mild disease. This suggests a potential role of Mif in the pathogenesis of T cell- and marcrophage-mediated autoimmune inflammatory diseases such as SLE and RA. Of interest, in psoriasis, an increased level of MIF was found in the skin and serum, suggesting a role of MIF in inflammatory skin
48 Lupus
diseases. Furthermore, a study showed induction of MIF in the skin by UVB, which is a well-known environmental factor for SLE. Based on these observations, a hypothesize that patients with SLE have increased expression of MIF, as a result of genetic predisposition, that promotes macrophasge-mediated inflammation and possibly T cell activation will be tested. To investigate this hypothesis, the following will be done. First, define the frequency of low- and high-expression Mif alleles in patients with SLE and correlate them with plasma MIF levels and disease activity. Second, determine the expression of MIF in skin lesions from patients with SLE and discoid lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: FUNCTIONAL EXPRESSION OF SLE3/5 ON NON-B CELL APC'S Principal Investigator & Institution: Sobel, Eric S. Associate Professor; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001 Summary: This proposal is part of a systematic effort to functionally dissect the complex genetic leading to systemic lupus erythematosis (SLE or lupus). Essentially, our approach has been to use genome wide scanning to identity susceptibility loci contributing to the development of SLE, to transfer these loci onto the non-autoimmune C57BL/6 (B6) background, to determine a reproducible phenotype conferred by the interval, and to determine the lineage(s) in which the susceptibility locus is expressed. We identified 3 genomic intervals from NZM2410 that contributed to the development of glomerulonephritis. One of these, B6.NZMc7 [containing Sle3/5 on chromosome 7] develop low titers of IgG anti-nuclear antibodies, elevated CD4:CD8 ratios and mild-tomoderate immune complex glomerulonephritis (GN). We have recently completed a set of bone marrow adoptive transfer studies with B6.NZMc7 congenic mice. The NZMc7 locus was functionally expressed on bone-marrow-derived cells but not on radioresistant host cells. Moreover, the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated DD4:CD8 ratio was not an intrinsic property of the T cells, and that a trend toward an elevated ratio could also been seen within the single-positive thymocytes. Preliminary data also indicated that the low-penetrant autoantibodies were not due to expression of the NZMc7 locus in B cells. Taken together, these data suggests that the effect is mediated by a bone-marrow-derived, non-B-cell antigen presenting cell (APC) present both in the thymus and secondary lymphoid organs. We propose that these APCs are dendritic cells, and may be the recently described lymphoid organs. We propose that these APCs are dendritic cells, and may be the recently described lymphoid subset, which are thought to be regulatory in nature. To test these hypotheses, we propose three Specific Aims: 1) to test the prediction that B6.NMc7 non-B-cell APCs are responsible for the elevated CD4:CD8 ratio; 2) to test the prediction that B6:NZMc7 dendritic cells have an altered threshold for inducing negative selection; and 3) to test the prediction that there is a difference in the kinetics of proliferation and apoptosis induced by BB6.NZMc7 APCs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENDER SPECIFIC T CELL HOMING AND AUTOIMMUNITY Principal Investigator & Institution: Adams, Matthew D. Internal Medicine; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 01-JUL-2001
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Summary: Women are more susceptible to autoimmune diseases, and the reason is unknown. Female sex hormones appear to play a role in this predisposition to autoimmunity, but extensive analysis of the effects of the female sex steroids on immune responses in vitro have failed to identify the mechanism(s). The Richardson lab has used a new model of drug-induced lupus to identify novel gender- specific immune mechanisms. In this model, D10 cells, a cloned Th2 line, are made autoreactive by treatment with DNA methylation inhibitors, then injected into syngeneic mice. The autoreactive cells cause a more severe autoimmune disease in females than in males, and disease severity is diminished by oophorectomy. Significantly more of the cells, treated or untreated, are retained in the female spleens, and this selective retention also decreases following oophorectomy. Finally, splenectomy prevents the development of autoimmunity. These results demonstrate that T cell splenic homing differs between males and females, and that the spleen is essential for the development of disease. These results suggest that the greater disease severity in females is due to more autoreactive cells accumulating in the female spleens. The reversal by oophorectomy implicates female sex hormones in these differences. We hypothesize that gender-specific differences in T cell homing, due to effects of female sex hormones on adhesion molecule expression, contribute to increased severity of autoimmune diseases in females by modifying lymphocyte trafficking patterns. Gender-specific trafficking differences could be important both in the induction of disease as well as later in the disease process. Our model system provides a unique opportunity to directly test the role of sex hormones in modulating endothelial cell adhesion molecule expression and lymphocyte homing, and to relate these findings to the development and severity of autoimmunity. The specific aims are to: 1) Characterize the effects of sex hormones on T cell homing in vivo, 2) Define the effects of sex hormones and other signals on T cell and endothelial cell adhesion molecule expression and function in vitro, and 3) Define the role of those adhesion molecules whose expression is modified by sex hormones in splenic homing and disease severity. These studies may identify novel and important mechanisms contributing to the increased incidence and severity of autoimmune disease in women. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENE MAPPING IN WOMEN W/ SYSTEMIC LUPUS Principal Investigator & Institution: Messner, Ronald; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, MN 554552070 Timing: Fiscal Year 2001 Summary: Systemic lupus erthematosus is an autoimmune disease of unknown etiology. There is convincing epidemiological evidence that SLE clusters in families, suggesting a genetic basis for the disease. Our hypothesis, based on a large body of evidence, is that SLE is an oligogenic disease, with the inheritance of a few genes other than MHC and TCR contributing to susceptibility. We propose to use gene mapping with highly informative, short tandem repeat polymorphism's (STRPs) in sibling pairs and multiplex families with SLE. In the course of the study we will establish a detailed database of clinical and family history data, determine Class II genotypes, and establish a repository of sera, DNA and immortalized cell lines for each subject. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENES AND CHEMICAL EXPOSURE ASSOCIATED WITH SLE RISK Principal Investigator & Institution: Fraser, Patricia A. Associate Professor; Cbr Institute for Biomedical Research 800 Huntington Ave Boston, MA 02115
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Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 29-SEP-2003 Summary: Systemic lupus erythematosus (SLE) is an autoimmune, disabling, disfiguring systemic rheumatic disease that preferentially afflicts women and AfricanAmericans. The excess risk of SLE in African-Americans is not entirely explained by the genetic markers of susceptibility that have been identified to date. Sex hormones are immunomodulatory. During the interval between menarche and menopause women are exposed to significantly higher estrogen levels when compared to men of similar age. This gender difference in estrogen exposure may explain the gender imbalance in SLE risk. Similarly, African-Americans have higher levels of sex hormones than Caucasians. Genetic determinants of the observed ethnic differences in sex hormone levels may contribute to the ethnic differences in predisposition to SLE. Several polymorphic cytochrome P-450 genes encode enzymes in critical pathways of estrogen and androgen synthesis and degradation. Inter-relationships among these genes may be important genetic determinants of hormone levels that may also influence the hormonal effects on lupus susceptibility. Gene-hormone interactions affect hormone homeostasis of function and these, we hypothesize, can be affected by environmental agents. Through a variety of mechanisms, organochlorines in the environment such as 2,2-bis(rho-chlorophenyl)1,1,1- trichlorethane (DDT), and its long-lasting metabolite DDE may affect sex hormone homeostasis. We hypothesize that interactions of genes that affect sex hormone homeostasis and function (androgen receptor (AR) and estrogen receptors (ERs) and cytochrome P450 genes) with endogenous and/or exogenous estrogens and also with organochlorine exposures explain the gender and ethnic differences observed in SLE. The specific aims of this proposal are to: 1. Determine AR, ERs and cytochrome P450 genotypes in SLE subjects and controls by PCR based methodologies in a large SLE case/control study; 2. Determine the relative importance of genetic markers in Aim number 1 with endogenous and exogenous estrogens and with exposure to organochlorines in predicting risk of SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC ANALYSIS OF ANTIPHOSPHOLIPID ANTIBODY SYNDROMES Principal Investigator & Institution: Ortel, Thomas L. Associate Professor; Duke University Durham, NC 27706 Timing: Fiscal Year 2001 Summary: Purpose: The purpose of this study is to understand the molecular basis of inherited antiphospholipid antibody syndromes as well as define the molecular mechanisms underlying the diverse pattern of clinical phenotypes and laboratory findings that have been observed in patients with antiphospholipid antibodies. Methods: We have established a registry of all individuals identified with a positive lupus anticoagulant and/or anticardiolipin antibody at Duke during the last five years. To date, we have identified over 570 patients with antiphospholipid antibodies. These patients are then recruited to participate in the studies described below. In addition, we have also enrolled family members from 39 of these patients, obtaining genomic DNA, plasma, and serum for characterization. Results: [1] To investigate the genetics of primary antiphospholipid antibody syndromes, we have identified 7 multiplex families that met stringent clinical and laboratory criteria for the diagnosis of a familial antiphospholipid antibody syndrome (APS). In these families, 23 out of 94 family members (24.5%) met criteria by either serologic or coagulation defects, or a combination of clinical, serologic and coagulation defects. Segregation studies rejected environmental and autosomal recessive inheritance patterns, and suggested that a
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dominant or co-dominant model would best fit the observed data in these families. Linkage studies showed independent segregation of APS and markers for several candidate genes. [2] To investigate the molecular basis of the observed clinical heterogeneity in these patients, we are screening patients for additional primary hypercoagulable states and correlating the presence of a second disorder with the occurrence of a thrombotic event. To date, we have screened 99 patients for the presence of three commonly encountered thrombophilic polymorphisms by restriction enzyme analysis of PCR-amplified genomic DNA: factor VR506Q; the 3'-untranslated prothrombin gene (PTG) polymorphism; and the thermolabile variant of methylene tetrahydrofolate reductase (MTHFR). Fifty-eight patients had primary antiphospholipid antibody syndrome (PAPS), 25 had systemic lupus erythematosus (SLE), 6 had other connective tissue disorders (CTD), and 7 were asymptomatic. Seventy-seven patients sustained one or more TE: 53 had venous TE, and 36 had arterial TE (12 had both venous and arterial TE). Fifteen of 58 female patients (25.9%) sustained one or more miscarriages. Eighteen patients had one or more thrombophilic polymorphisms: 8 were heterozygous for factor VR506Q, 3 were heterozygous for the 3' PTG polymorphism, and 9 were homozygous for the MTHFR polymorphism (including 2 who were also heterozygous for factor VR506Q). Eleven of 18 had PAPS (19%), 6 had SLE (24%), and 1 had CTD (16.7%). Sixteen of the 18 patients had sustained a venous and/or arterial TE (p=0.347). Two individuals homozygous for the MTHFR polymorphism had not sustained a TE. The presence of factor VR506Q or the 3' PTG polymorphism were associated with a significantly increased risk for a venous TE (10 of 11 patients; p=0.0095). In contrast, only 5 patients with the MTHFR polymorphism sustained a venous TE, including the 2 who were also heterozygous for factor VR506Q. None of the thrombophilic genotypes, alone or combined, were associated with an increased risk for arterial TE or miscarriage. Significance: The significance of these studies is that we will understand the genetics of an inherited autoimmune disorder as well as the molecular basis of the phenotypic heterogeneity observed in these patients. This will enable us to better identify patients at risk for developing a thromboembolic complication due to the presence of these antibodies. By defining which patients are at risk, we will be able to develop better therapeutic agents to prevent recurrent complications in these patients and also develop new clinical laboratory assays in order to better monitor their therapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC ANALYSIS OF EPITOPE SPREADING Principal Investigator & Institution: James, Judith A. Associate Professor; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2001; Project Start 30-SEP-1998; Project End 31-AUG-2005 Summary: Systemic lupus erythematosus (SLE) is a serious autoimmune disease of which the etiology and mechanisms of pathogenesis are incompletely understood. It is clear that there is an important genetic component to lupus. High titers of autoantibodies, which may include anti-Sm and anti-RNP are characteristic of lupus. Recent work shows that the natural history of these autoimmune responses is to increase in complexity by involving additional structures of the autoantigen in the autoimmune response. This process is termed epitope spreading detected in this proposal as added antigenic spine specificity through time. A similar phenomenon in T cell epitopes is very important in other models of disease, such as experimental autoimmune encephalitis. The applicants suspect that this process is also very important in lupus pathogenesis. Recently a new model of lupus autoimmunity was discovered by this group induced by immunization with a short sequence from Sm B/B'. This new
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model of induced SLE presents opportunities to explore the genes involved in B cell epitope spreading as well as the autoimmunity of lupus. Work here with the AKXL recombinant inbred set of mouse strains has preliminarily established linkage on chromosome 4 at B cell marker 72. They propose to apply the impressive tools of mouse genetics to identify the genomic region and perhaps the specific genes associated with anti-Sm B cell epitope spreading. They will subsequently explore the syntenic regions and homologous genes in human lupus. The goals of this proposal are to analyze genetic contributions to epitope spreading and recombinant inbred strains of mice and to confirm the findings by classical genetic approaches. It will seek to confirm and narrow the region of chromosome 4 by classical back cross experiments. Simultaneously the investigators will evaluate the candidate gene Cd72 for its potential role in the observed linkage. If Cd72 does not explain linkage in this recombinant inbred set, then confirmation of this region, and a search for different candidate genes will then pursue. They will then seek linkage in other recombinant inbred strains of mice and work to identify the responsible genes. Finally, linked regions and identified genes will be tested for their potential contribution to human lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC ANALYSIS OF T CELLS IN LUPUS Principal Investigator & Institution: Craft, Joseph E. Professor; Internal Medicine; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2001; Project Start 01-JUL-1996; Project End 30-JUN-2006 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is characterized by IgG autoantibodies to certain intracellular components, including chromatin and ribonucleoproteins. Several inbred mouse strains also develop spontaneous lupus, with the same spectrum of autoantibodies. Certain of these specificities are pathogenic, including those against chromatin that induce immunecomplex glomerulonephritis. Autoantibodies in lupus arise as a consequence of autoantigen-specific alpha/beta CD4+ T cell help, including T cells specific for peptides of chromatin-associated proteins. Such autoreactive T cells bypass normal tolerance mechanisms in the periphery; however, the mechanism of activation of T cells responsive to self peptides in lupus is unknown, as are the tissue source(s) of such peptides and the events leading to autoreactive CD4+ T cell-B cell collaboration with resultant pathogenic autoantibody production. In this proposal, an in vivo approach will be used to dissect the mechanisms that lead to peripheral T cell tolerance abrogation and T cell help for autoantibody production in lupus. It is hypothesized that these events arise in two stages: first, that lupus T cells have intrinsic (genetic) defects that render them susceptible to activation after contact with the ubiquitous self peptide-class II MI-IC complexes that are sufficient for CD4+ T cell survival in normal animals; second, that such activation, initiating tolerance loss with polyclonal expansion of autoreactive T cells, leads to oligoclonal T-B cell collaboration in the setting of specific autoantigen presentation by autoreactive B cells. The hypothesis will be addressed in two aims. First, it will be determined if normally displayed (ubiquitous) MHC-self peptide complexes can activate autoreactive T cells from Fas-intact mice MRL/+Fas-lpr mice, in comparison to non-autoimmune control T cells. Second, it will be determined if T cells from MRL/+Fas-lpr mice can provide B cell help in the setting of autoantigen presentation by autoreactive B cells, an event that leads to antigen-specific expansion of cells from both lineages. These objectives fit well within the overall context of this IRPO proposal centered around developing a better understanding of T cell-B cell interactions during the development and maintenance of systemic autoimmunity.
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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC CONTROL OF AUTOIMMUNITY IN LUPUS PRONE MICE Principal Investigator & Institution: Mcduffie, Marcia J. Associate Professor; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2001 Summary: We have identified three chromosomal segments, apart from the MHC complex, which contain genes that independently regulate progression of the pancreatic autoimmunity and beta-cell loss in non-obese diabetic (NOD) mice. Two of these genes, found on chromosomes 4 and 11, co-localize with chromosomal segments associated with one or more aspects of lupus-like disease in the NZB/NZW F1 model. This observation led to our first hypothesis: that genes located on chromosomes 4 and 11, which control- diabetes-susceptibility in NOD mice, promote the development of lupuslike disease in mice. We propose to define the location of lupus-susceptibility genes on chromosomes 4 and 11 in a recombinant-inbred mouse model of SLE, NZM2328. This will be accomplished by inhibiting disease expression with defined intervals of chromosome 4 and 11 taken from the non-autoimmune C57L/J strain and correspond to the locations of the chromosome 4 and 11 genes in autoimmune diabetes (Specific Aim 1). Using targeted breeding, we will then determine directly whether homologous NOD interval can reestablish disease expression after replacing C57L/J segments in this model (Specific Aim 2). Taken together, the results of the proposed experiments in Aims 1 and 2 will test the hypothesis that the lupus- susceptibility genes identified on chromosomes 4 and 1 represent "autoimmunity genes" common to at least two distinct models of spontaneous autoimmunity in mice. In addition to protection from insulitis, we show that introduction of C57L/J derived alleles into a defined segment of chromosome 11 supports normalization of defective immune responses and protection from invasive insulitis in NOD mice. These observations suggest the second hypothesis: that this region of chromosome 11 plays a fundamental role in the regulation of specific immune reactivity, with particular relevance to autoimmunity. In order to test this hypothesis, we will determine the mechanism of action of the chromosome 11 gene(s) in an induced model of autoimmunity resulting from immunization with Ro60 antigen (Specific Aim 3). These studies will allow us to define the genetic relationship between pathogenic immune responses and the regulation of inflammatory responses by genes on chromosome 11. The results of these experiment will provide insight in disease pathogenesis in lupus-like autoimmunity in mice, particularly in dependence regulation of immune responses and inflammation. In addition, they will determine whether common "autoimmunity genes" control the development of disease in two autoimmune disease with different manifestations. We postulate that genes of this type are very likely to have homologues in human autoimmune disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC EXPRESSION IN SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Aune, Thomas M. President; Vanderbilt University 3319 West End Ave. Nashville, TN 372036917 Timing: Fiscal Year 2001; Project Start 01-MAY-1994; Project End 30-APR-2004 Summary: Systemic lupus erythematosus is a multisystem autoimmune disorder of unknown etiology and poorly understood pathogenesis. The heterogeneity of lupus makes it especially difficult to characterize and quantitate in either routine clinical care or in the setting of controlled clinical trials. These problems limit clinical studies of new
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therapeutic approaches. We propose to apply methods to analyze gene expression using microarrays to characterize patients with lupus with the following specific aims: 1. To compare 7 lupus patients and 3 control subjects for differences in gene expression on gene filter microarrays analyzing 20,000-30,000 gene sequences. 2. To further evaluate groups of related genes suggested by Aim #1 to be of importance in lupus using either selected microarays or RNA (Northern) blot analyses. 3. To examine the gene expression findings for correlations with clinical features, activity and severity of lupus. Studies of gene expression in subjects with lupus offer several advantages over existing approaches. In addition to providing a noninvasive, easily repeatable measure of immune system activation, the results can be quantified and compared for many subjects. More importantly, there is the real possibility of identifying new pathways of immune activation at the molecular level which may in turn suggest approaches to the development of novel therapeutic agents. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC FINE MAPPING IN SLE PAIR FAMILIES Principal Investigator & Institution: Behrens, Timothy W.; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2001 Summary: The familial clustering of systemic lupus erythematosus (SLE) and the high rate of concordance in monozygotic twins suggests a strong genetic component for susceptibility to SLE. As one approach to the identification of lupus genes, our laboratory is currently performing a genome-wide marker screen in an effort to localized the chromosomal regions that harbor lupus genes. Over 180 families with at least two affected SLE relatives (mostly sib pair families) are enrolled in this study. In preliminary studies, three distinct regions of human Chromosome 1 show evidence of possible linkage in an initial cohort of 105 SLE sib pair families. As the genome screen proceeds it is likely that other candidate intervals will emerge on other chromosomes. The primary goal of the SCOR project is to accelerate the gene search in the MN SLE family collection. This will be accomplished by sharing ongoing genotyping data with our colleagues at OMRF (project #2) to compare results in the two SLE family collections. Preliminary mapping results at MN will also be shared with our colleagues at UAB (project #3) to assist in their screening of Chromosome 1q candidate genes. The MN family collection will then be genotyped with markers from the OMRF study, so that linkage results can be directly compared. Interesting chromosomal regions ill then be prioritized for high density marker screens and fine mapping. Trio families (one SLE patient with both parents) collected as a component of this SCOR application will be used in disequilibrium mapping in the targeted areas. A variety of strategies will then be employed to identify the SLE gene(s) in these regions. The ultimate isolation and cloning of SLE genes will provide the first opportunity to understand at the genetic level the defects that lead to the clinical immune dysregulation characteristic of patients with lupus. These insights should suggest new avenues of treatments for this patients, including the potential for somatic gene therapy. Interestingly, lupus- prone families also have an increased incidence of other autoimmune diseases including rheumatoid arthritis, thyroid disease, and diabetes. Thus, the identification of genes that cause human SLE is likely to improve our understanding of the genetics and pathophysiology of organ- specific autoimmunity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC LINKAGE IN LUPUS Principal Investigator & Institution: Harley, John B. Professor; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2001; Project Start 01-JUL-1987; Project End 31-JAN-2005 Summary: The genetic basis of systemic lupus erythematosis has been pursued using the last five years of funding from this grant (AR24717-07 to -11) to help perform and evaluate a genome scan in lupus. These results considered with those of our closest competitor show 26 possible genetic linkages with 11 of these having some support for linkage from both studies. In addition, we confirm evidence supporting the presence of linkage at D1s229. Other work has advanced Epstein-Barr virus as a possible etiologic agent in lupus. Data show association, are consistent with Epstein-Barr virus infection preceding lupus onset, and advance a plausible mechanism for lupus autoimmunity in some patients. Virus exposure data and differences between the anti-viral immune responses of lupus patients and normal are amenable to genetic analysis in our pedigrees. In aggregate, we have 2109 pedigrees multiplex for lupus containing 1227 subjects (275 affecteds & 752 unaffecteds). We hope to continue our work by pursuing the following specific aims: 1. Enlarge the pedigree collection; 2. Establish linkage using: A. Lupus (by revised ACR criteria), B. An environmental factor and intermediate phenotypes: i. Lupus and Epstein-Barr virus infection, ii. Anti-peptide antibodies against Epstein-Barr virus (& against lupus autoantigens), iii. Anti-Ro and anti-nRNP autoantibody responses, and C. Multi-locus effects, and 3. Reduce linkage intervals and evaluate candidate genes. Hopefully, results from this resubmitted project will help elucidate the complex genetics of lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC MOUSE MODELS FOR CHRONIC INFLAMMATORY DISEASE Principal Investigator & Institution: Bullard, Daniel C. Assistant Professor; Genetics; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2007 Summary: Systemic lupus erythematosus, Wegener's granulomatosis, and polyarteritis nodosa are chronic inflammatory diseases that have vasculitis as a major component. The pathogenesis of vasculitis in these and other disorders involves a complex interaction among inflammatory, genetic, and environmental factors. Evidence from both patient studies and in vitro models supports a central role for leukocyte/endothelial cell adhesion molecules, such as ICAM-1 and its beta2 integrin counterreceptors, in the development of vasculitis. However, the specific mechanisms by which ICAM-1 mediates vasculititic lesion formation are not clear. This project will use a straightforward genetic approach using the MRL/MpJ-Faslpr mouse model to define the ICAM-1-dependent pathways responsible for mediating vasculitis. MRL/MpJ-Faslpr mice containing mutations in ICAM-1, LFA-l, Mac-1 and P150/95 have now been generated and will be used to investigate the roles of leukocyte/endothelial interactions in lesion formation in vivo. The specific aims are to: (i) define, by comprehensive qualitative and quantitative analysis, the effects of ICAM-1 deficiency on development and progression, organ distribution, and inflammatory characteristics of lesions of vasculitis in MRL/MpJ-Faslpr mice; (ii) define the relative contributions of ICAM-1 in mediating neutrophil, lymphocyte, and monocyte adhesion to MRL/MpJ-Faslpr endothelial cells; (iii) determine the roles of ICAM-1 in neutrophilmediated damage to MRL/MpJ-Faslpr endothelial cells; and (iv) determine whether
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MRL/MpJ-Faslpr mice with null mutations in the ICAM-1 ligands CD11a (LFA-1), CD11b (Mac-1), or CD11c (p150/95) will alter development and progression, organ distribution, or inflammatory characteristics of lesions of vasculitis. Upon successful completion of these aims, detailed mechanistic information regarding the roles of ICAM-1 in mediating leukocyte/endothelial adhesion and damage during the development of vasculitis will be obtained. In addition, new insights will be gained towards the general understanding of the pathogenesis of vasculitis as well as other leukocyte-mediated vascular injuries such as transplantation arteriosclerosis, and other reperfusion injuries. There have been many requests for these mutant mice or materials derived from these models; therefore, these models are contributing significantly to ongoing research that is relevant to several NIH institutes, including NIAMS, NHLBI, and NIAID. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC PATHWAYS CONTRIBUTING TO SLE PATHOGENESIS Principal Investigator & Institution: Wakeland, Edward K. Professor; Microbiology; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2003; Project Start 30-SEP-1998; Project End 29-FEB-2008 Summary: (provided by applicant): This research program is focused on characterizing the genetic interactions that mediate fatal disease in the lupus-prone NZM2410 mouse model of SLE. During the previous grant period, we identified four suppressive modifiers and two epistatic susceptibility loci that impacted disease development in our B6-congenic model of lupus autoimmunity. We subsequently produced a collection of B6-congenic strains carrying these disease-enhancing or disease-suppressing genes and initiated a detailed characterization of their component phenotypes and epistatic interactions. These experiments have led to the development of a model proposing that severe disease is mediated by epistatic interactions among genes in three separate pathways, each impacting a distinct element of disease pathogenesis {Wakeland, Liu, et al. 2001 2262/id}. In this application, we are proposing to identify three of the genes that were detected in our genetic dissection of this epistatic disease model. The identification of these genes will provide insights into genetic mechanisms that can suppress the breach in tolerance mediated by Sle1, and will identify genes that exacerbate the severity of glomerulonephritis as a consequence of immune complex deposition. We have three specific aims: 1). To fine map and identify kidney-targeting genes in the Sle1 gene cluster. We have developed a nephrotoxic antisera assay that allows the rapid detection of genes that exacerbate the development of glomerulonephritis (GN) as a consequence of immune complex deposition in the kidney. We propose to utilize this assay to complete the fine mapping and identification of these two genes via positional cloning strategies. 2). To fine map and identify Sles1. Sles1 was the strongest suppressive locus detected in our linkage analysis of disease modifiers in the NZW genome. This gene specifically suppresses the breach in immune tolerance mediated by the Sle1 gene cluster. We have produced a series of congenic recombinants with truncated intervals that will allow the localization of Sles1 into a ~950 Kb genomic interval. We will complete this fine mapping analysis and identify Sles1 3). Characterize the component phenotypes and genetic interactions of Sles2, Sles3, Sles4, and Sle6. We propose to create a series of bi- and tri-congenic strains to assess their impact on disease pathogenesis. The long-term goal of these studies is to characterize the genetic interactions that enhance and suppress lupus pathogenesis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC RISK FACTORS FOR PNEUMONIA IN SLE PATIENTS Principal Investigator & Institution: Freemer, Michelle M. Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2003; Project Start 15-JUL-2003 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) can be a severely disabling disease and may affect any portion of the respiratory tract. The most frequent lung disease in SLE patients is pneumonia. It constitutes a major source of morbidity as well as mortality in these patients. While many of the clinical factors predisposing SLE patients to infections have been investigated, the genetic risk factors for pneumonia have not been thoroughly examined. The University of California, San Francisco (UCSF) Lupus Genetics Project, represents a well-characterized, ethnically diverse cohort of patients. Furthermore, the availability of genotype analysis for these patients provides an ideal opportunity to determine the impact of genetic polymorphisms on SLE patients' risk of developing pneumonia. Interestingly, prior investigators have identified genetic risk factors for infections in healthy populations that correspond to some of the polymorphisms associated with the development of SLE Evaluation of the relationship between pneumonia and these polymorphisms in an SLE population will yield insight into whether the genetic risk factors identified in healthy hosts also account for the high infection rate observed in SLE patients. This information has significant clinical implications regarding SLE patients' need for immunization and antibiotic prophylaxis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC RISK FACTORS FOR SUSCEPTIBILITY TO INFECTION IN SLE Principal Investigator & Institution: Fessler, Barri J.; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2001 Summary: Although mortality in patients with systemic lupus erythematosus (SLE) has decreased over the past three decades, infectious complications remain a significant cause of morbidity and mortality, accounting for approximately 20- 50% of all deaths. The profound influence of genetic background on susceptibility to infections has been well established. Recently, specific variants in the tumor necrosis factor alpha (TNFalpha) promoter and the genes encoding Fcgamma receptors and mannose binding lectin have been associated with an increased susceptibility to infections in certain populations. Polymorphisms affecting these same genes have also been associated with an increased risk of developing SLE or expression of specific lupus manifestations. However, the link between these genetic polymorphisms and risk of infections in SLE patients has never been examined. The aims of this study are: a) to determine the incidence of specific polymorphisms in the TNFalpha promoter and the genes encoding Fcgamma receptors and mannose binding lectin in SLE patients b) characterize the frequency, type and severity of infectious complications in a group of SLE patients as compared with controls c) determine whether the presence of one or more polymorphisms in the genes of interest is associated with an increased risk of infectious complications in SLE. One hundred and five SLE patients and controls matched for age, sex and ethnicity will be assessed over an 18-month period to determine the incidence and severity of infectious complications. The incidence of genetic polymorphisms affecting these candidate genes and the frequency of infections will be compared between the two groups to determine the level of risk. The identification of genetic
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markers that would distinguish SLE patients at increased risk for infection would make a profound impact on the outcome of this disease by allowing clinicians to institute appropriate prophylactic therapies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETIC RISK PROFILE IN LONGITUDINAL SLE COHORTS Principal Investigator & Institution: Petri, Michelle; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: There is a genetic predisposition to the disease systemic lupus erythematosus. Recent research has concentrated on several genes on chromosome 1. Several lupus centers are cooperating in the further study of these genes, especially whether they predict which organs lupus affects in individual patients. SLE patients and their parents are being invited to participate in this study. Patients and parents agreeing to participate will have blood drawn at the time they enter the study (approximately 50 cc or a little more than 3 tablespoons), which will be sent to the University of Alabama-Birmingham for genetic studies. Patients alone will have blood drawn (approximately 30 cc or 2 tablespoons) to determine autoantibodies. Socioeconomic and demographic data (i.e., age; gender; race; occupation; income; marital status; housing; health habits; and health insurance) will be obtained, because past studies have shown these factors are important in the outcome of lupus. Information on the past course of SLE will be obtained from medical records, and disease activity will be assessed (by Dr. Petri) during regular appointments, on a yearly basis. There will be updates by telephone (about every 6 months). Genetic material will be stored to help in the future search of genes that contribute to lupus. Confidentiality will be respected by coding the genetic samples. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETIC RISK PROFILE IN LONGITUDINAL SLE COHORTS Principal Investigator & Institution: Alarcon, Graciela S. Professor; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2001 Summary: Both genetic and non-genetic factors contribute to the clinical presentations, clinical course, and outcome in SLE. To develop the theme of this SCOR, the genetics of SLE, and to translate the findings to the bedside by characterizing the clinical manifestations associated with the identified genetic markers, we propose to gather a population of well characterized SLE patients by joining efforts with investigators who already have ESTABLISHED longitudinal cohorts and the mechanisms to enroll NEW patients (Drs. Petri, John Hopkins University, MD; Reveille, University of Texas Health Sciences Center, Houston, TX; Ramsey-Goldman, Northwestern University, Chicago, IL) which will constitute the largest single available cohort of SLE patients for such studies (The PROFILE cohort)> With this cohort we will determine the extent to which a given genotype (PROFILE) determines the clinical phenotype. We will also contribute to the constitution of TRIO families for TDT analysis in order to confirm and narrow regions of genetic interest. The specific aims of the study are to: 1) Establish a multi-center common core database of SLE patients from multiple ethnicities comprised of patients who are already in local cohorts (ESTABLISHED) as well as NEW patients to be recruited; 2) Assess the ability of chromosome 1 genes (q21-32, see projects #1-3) to predict disease phenotype [renal, cardiovascular, pulmonary, and central nervous
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system, (CNS) involvement] in this PROFILE COHORT of SLE patients; 3) Establish a core set of TRIO families and work in conjunction with projects #1-3 and the Genetic Epidemiology and Biostatistics Core to use transmission disequilibrium to confirm the identification of candidate regions and genes in SLE. We have based our power and sample size calculations on the frequency of FcgammaRII H131/H131 in SLE patients with and without renal disease (8% versus 20%) and the frequency of renal disease among lupus patients from one of the center's cohorts. With total number of SLE patients of 600-700, we will have adequate power to demonstrate the skewing of this and other genes in the phenotype of the disease. Descriptive and analytical methods will be used where the phenotype will be the dependent variable and genetic (and other factors) the independent variables. The ability to predict the expression of the disease may have important implications for optimizing therapeutic strategies or developing new therapies for lupus patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GENETICS OF AUTOIMMUNITY Principal Investigator & Institution: Kotzin, Brian L. Professor; Medicine; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, CO 800450508 Timing: Fiscal Year 2001; Project Start 01-APR-1986; Project End 31-MAR-2004 Summary: (Adapted from Investigator's abstract): The major goal of the studies proposed in this competitive renewal is to define further the genetic contributions to autoimmune disease in New Zealand hybrid mice that is a model of systemic lupus erythematosus (SLE). In previous studies, the investigator showed that the major dominant NZW contribution to disease was linked to H2, the murine major histocompatibility complex. Additional studies have mapped NZB susceptibility loci to chromosomes 1, 4, 7, 13, and 17 at H2. Congenic mice carrying these loci have been generated in the investigator's laboratory with the hypothesis being that each locus underlies an intermediate phenotype that in isolation reflects a single immunopathologic component of overall disease process. In Specific Aim 1 the investigator proposes to find out whether Ez and Az interact to control disease or if other H2 loci contribute to the MHC affect. Previously, the investigator identified a locus on chromosome 4 in NZB mice (Nba1) that he has introgressed onto NZW. This locus results in increased nephritis and mortality. In Specific Aim 2, the investigator proposes to identify the intermediate phenotype associated with this locus and to refine the support interval and number of candidate genes via congenic mapping. In Specific Aim 3, similar studies will be carried out using C57BL/6 and SM/J congenic mice carrying Nba2NZB, an NZB susceptibility locus on chromosome 1. The investigator purports to have identified a lupus-susceptibility locus that maps to chromosome 13 in C57BL/10 mice but is absent in C57BL/6 mice. Therefore, in Specific Aim 4 he proposes to take advantage of what he believes is a unique opportunity to identify this lupusenhancing gene. In Specific Aim 5 the investigator proposes to continue to derive and characterize SM/J mice congenic for NZB lupus-susceptibility loci on chromosomes 7 and 13. Taken together, the investigator suggests that the results of the studies proposed in this application will provide new insight into the genes that allow for autoimmune disease in SLE and the intermediate phenotypes that they control. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETICS OF CHILDHOOD ONSET SLE Principal Investigator & Institution: Jacob, Chaim O. Associate Professor of Medicine and Immu; Medicine; University of Southern California 2250 Alcazar Street, Csc-219 Los Angeles, CA 90033 Timing: Fiscal Year 2001; Project Start 20-SEP-1999; Project End 30-JUN-2004 Summary: (Adapted from Investigator's Abstract) The etiology of systemic lupus erythematosus (SLE) includes poorly understood genetic, environmental and sexhormone factors acting on the immune system. The long term goal of this application is to identify genes involved in the etiopathogenesis of human SLE and to characterize the mechanisms by which these genes influence disease development. This application will also serve as a follow-up study of the initial linkage analyses performed in mouse models and in multiplex families. For this stage of mapping, the investigators propose to rely on association (linkage disequilibrium) studies in nuclear families as they will be able to collect a much larger study population than if they relied on the affected sib-pair pedigree approach. Childhood-onset SLE represents a potentially unique subgroup of patients because its early disease onset may be an indicator of increased genetic predisposition and penetrance, and because childhood-onset disease is more severe than adult-onset involving many organs and carrying a worse prognosis. The investigators therefore propose to study nuclear families of childhood-onset SLE subjects. Given that probands will be children, they anticipate that their parents and siblings will be available and strongly motivated to participate. The specific aims are as follow. 1) Recruitment and blood and DNA collection from 850 nuclear families containing at least one subject with childhood-onset SLE. They will classify all subjects for clinical and laboratory evidence of SLE, its organ involvement, severity of disease and complications. This will be accomplished using four recruitment study sites, which will provide large pediatric lupus populations. 2) Testing for association with specific candidate genes suggested by linkage, synteny and functional relevance using the family-controlled generalized transmission disequilibrium test (TDT) approach. 3) Exploring candidate regions of about 5 cM suggested by linkage and synteny for patterns of linkage disequilibrium by testing for marker associations and haplotype sharing. Initially, they will use markers spaced roughly 0.5 cM apart in each region, with promising leads followed up at a 0.05 cM marker spacing. The investigators point out that this application integrates the talent of a multidisciplinary team, combining clinical expertise with highly qualified basic scientists and with genetic, epidemiological, molecular biological and genetic analytic expertise. They further state that this is a multi-center application from some of the largest pediatric lupus clinics in the country and, therefore, represents a major and unique resource for the study of the genetics of childhood-onset SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: GENETICS OF LUPUS-RELATED AUTOIMMUNITY IN HUMANS Principal Investigator & Institution: Moser, Kathy L. Assistant Professor; Medicine; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, MN 554552070 Timing: Fiscal Year 2001; Project Start 20-SEP-1999; Project End 31-AUG-2003 Summary: The possibility that different autoimmune phenotypes might share particular genetic linkages has been bolstered by a meta-analysis of the available data (Becker, et al, PNAS 95:9979-9984, 1998). In addition, our collection of pedigrees multiplex for systemic lupus erythematosus (SLE), as a clinical disease, contain many members who do not have lupus, but who do have autoimmune findings. Examples include lupus-
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related positive serology and other disorders thought to be autoimmune in origin such as myasthenia gravis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, psoriasis, and diabetes. Recently, we published the results of a genome scan using clinical lupus as a phenotype (Moser, et al. PNAS 95:14869-14874, 1998). In 94 pedigrees studied, there are 223 confirmed SLE affecteds and 594 family members, 17 percent of who report the presence of another autoimmune disorder and over 30 percent with positive autoimmune serology. In classic work, Bias and coworkers (Am J Hum Genet 39:584602, 1986) have shown that pedigrees ascertained on lupus and evaluated using humoral autoimmunity as an intermediate phenotype segregate this trait in an autosomal dominant pattern. We propose to use our now larger collection of pedigrees multiplex for SLE as a basis from which to seek evidence for the predicted autosomal dominant linkage as well as for other genetic effects. We will use our currently available collection of 173 pedigrees containing 1300 individuals to: 1) evaluate for a LupusRelated Autoimmune (LRA) phenotype, 2) seek linkage, and 3) perform fine mapping in regions providing evidence for linkage. Identification of genes that govern the propensity to develop autoimmunity has potential to provide important insight into mechanisms of etiology and pathogenesis that are common among multiple autoimmune diseases. Understanding these underlying pathological events will lead towards new opportunities for development of more effective mechanism-based therapeutic strategies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GL701 ERYTHEMATOSUS
IN
FEMALES
WITH
ACTIVE
SYSTEMIC
LUPUS
Principal Investigator & Institution: Petri, Michele; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: Current medications for systemic lupus erythematosus do not completely control symptoms of the disease and/or can have side-effects. GL701 is a form of DHEA (dehydroepiandrosterone), an investigational drug, that has shown benefit in studies of lupus patients. This multi-center study will determine if DHEA (200 mg) works better than a placebo for SLE in 300 women who have active lupus, but are taking prednisone doses of 10 mg or less. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: GUIDELINES FOR STEROIDS IN CHILDREN WITH LUPUS Principal Investigator & Institution: Brunner, Hermine I. Assistant Professor; Children's Hospital Med Ctr (Cincinnati) 3333 Burnet Ave Cincinnati, OH 45229 Timing: Fiscal Year 2003; Project Start 08-MAY-2003; Project End 30-APR-2006 Summary: (provided by applicant): BACKGROUND: This is a pilot study to evaluate the use of steroids and other medications in children diagnosed with SLE (cSLE). After having revolutionized the prognosis of lupus in the 1950s, steroids remain the mainstay of therapy of cSLE. Recent studies suggest that, despite their proven benefits, steroids contribute to the development of permanent disease damage in both adult and pediatric SLE patients. 10-year patient survival is only at 85%. Preliminary data support that there is a considerable degree of practice variation among pediatric rheumatologists treating cSLE and that these differences in treatment approach may have an impact on patient outcomes. There are no published guidelines of how to best treat cSLE, especially how to use steroids for its the treatment and when to introduce other steroid-sparing
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medications. GOALS: 1) To document treatment patterns of pediatric rheumatologists for patients with cSLE in order a) To identify key factor that prompt physicians to choose a certain steroid dose and document the factors that make physicians change a given dose of steroids; b) To identify the key variables that prompt physicians to introduce of immunosuppressive therapies for patients diagnosed with cSLE. 2) To measure quality of life and specific outcomes (damage, costs) associated with the treatment of children and adolescents diagnosed with cSLE. STUDY DESIGN: A cohort of consecutively sampled patients treated for cSLE (n=70) at 4 pediatric US and Canadian Rheumatology Centers (Chicago, Cincinnati, Minneapolis, Toronto) will be assessed in at least tri-monthly intervals over an 18-month period regarding their disease course (disease activity, number of flares, infection and hospitalization), treatments, and outcomes (damage, quality of life). Key determinants that prompt the physicians to use certain therapies will be recorded. Relevant retrospective patient information will be obtained by chart review. Patient quality of life and treatment costs will be measured. Correlation, regression, multivariable modeling including repeated measure analysis will be used to analyze the relationship of cSLE therapies to outcomes (damage, quality of life, costs) and to key determinants of treatment decisions in cSLE. SIGNIFICANCE: The proposed pilot study will provide information regarding physician treatment patterns, cost of cSLE and patient quality of life. Data will be collected to support that there are important differences in the approach to cSLE therapy that have a significant impact on patient outcomes. Results of the study will be used to generate hypotheses towards improved treatment approaches for cSLE. The proposed study constitutes a first step towards the development of evidence-based guidelines. Information on HRQL and costs of patients diagnosed with cSLE is required for future cost-effectiveness analyses of treatments for cSLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFICATION OF SLE1B AND IT'S ROLE IN AUTOIMMUNITY Principal Investigator & Institution: Wandstrat, Amy E. Center for Immunology; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001; Project Start 01-SEP-2001 Summary: Systemic lupus erythematosus (SLE) is a complex genetic disorder and occurs 8-9 times more frequently in women than men with variable penetrance. In the lupus-prone mouse strain, NZM 2410, four regions have been identified on chromosomes 1, 4, 7, and 17. We are interested in identifying the gene(s) known to be responsible for the autoimmune response seen in SLE. Isolated in congenic mouse strains against a C57BL/6 background, the chromosome 1 region, Sle1, has been associated with either loss of tolerance to chromatin or increased immune response leading to splenomegaly and production of autoantibodies. The region of murine chromosome 1 that Sle1 maps to is syntenic to human chromosome 1 where a locus for lupus susceptibility has been linked (29- 33). Therefore, identification of the mouse gene may also help in identifying the human gene. We have been able to further narrow the Sle1 region using congenic meiotic recombinants and have found that there are three genes in the Sle1 region, Sle1a, Sle1b, and Sle1c that confer autoantibody production. Our studies have revealed that Sle1b is the strongest of the three loci regarding antichromatin IgG production. We have a sequence-ready BAC contig of the region and will use both BAC sequencing and cDNA direct selection to identify candidate genes for Sle1b. Candidate genes will then be analyzed for proper expression and the ability to reproduce the phenotype in a B6 mouse knockout. As Sle1b may be important in
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focusing the autoimmune response to selected targets, identification of the gene will be important in understanding how the autoimmune cascade is initiated. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFICATION OF THE EARLY ONSET SLE GENE ON 17P13 Principal Investigator & Institution: Sestak, Andrea L.; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2003; Project Start 01-SEP-2003; Project End 31-AUG-2006 Summary: (provided by applicant): Ongoing studies at the OMRF have recruited and genotyped over 160 families multiplex for systemic lupus erythernatosus (SLE). Subgroup analysis of those families containing at least one SLE patient with age of onset less than 16 has revealed a putative susceptibility gene for early onset lupus with a lod score of 3.0 at 17p13. Independent analysis of affected relative pairs in the 160 family collection, using a principal component approach, confirms that age of onset is a major covariate, producing a lod of 4.4 at the same site, D17s1298. We have designated this putative susceptibility gene SLE pediatric 1 (SLEP1), and the goal of this application is to find and characterize this gene. First, we expect that an additional 30-45 families containing at least one member with pediatric onset SLE will become available from ongoing efforts to recruit multiplex pedigrees at OMRF, and we will seek to confirm the effect at D17s1298 in a second cohort. If successful, we will use fine mapping techniques, first with additional microsatellite markers and later with SNPs, to narrow the region of interest to 2-3 cM. Next, we will evaluate potential candidate genes in the narrowed susceptibility region by genotyping at SNP markers in known genes in the region and analyzing these by linkage disequilibrium methods. At this stage, priority will be given to any genes in the region with a plausible role in autoimmune pathogenesis. Finally, if the SNP selected to identify the gene through linkage disequilibrium to SLE does not prove to be the causative mutation, we will sequence the putative susceptibility gene and attempt to discover the functional mutations leading to SLEP1. This project is proposed in support of a K08 Mentored Clinical Scientist Development Award. It is relevant to the career goals of the principal investigator, both as a pediatric rheurnatologist and as a new scientific investigator. This project has the potential to increase our understanding of the genetic factors contributing to the pathogenesis of early onset SLE, as well as lupus in general. It will allow the principal investigator to interact with a number of collaborators and to become more familiar with the state of the art genotyping and biostatistical analysis techniques currently used in the OMRF lupus genetics project. In addition, this project has the potential to generate related projects in molecular biology that would allow the principal investigator to become fully independent. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: IDENTIFYING GENES FOR NEUROPSYCHIATRIC LUPUS Principal Investigator & Institution: Mishra, Nilamadhab; Internal Medicine; Wake Forest University Health Sciences Winston-Salem, NC 27157 Timing: Fiscal Year 2002; Project Start 11-SEP-2002; Project End 31-JUL-2005 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a chronic, idiopathic autoimmune disease characterized by episodic flares and progression of disease, substantial morbidity and mortality(l, 2). It is a multisystern rheumatic disease with a wide variety of associated clinical neurological and psychiatric syndromes including cognitive, behavioral, affective, and/or motor manifestations that may effect
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up to 75 percent of SLE patients(3). Both morbidity and mortality remain high because of lack of understanding of the underlying mechanisms related to abnormal central nervous system (CNS) function. In addition, progress has been hampered by the lack of specific diagnostic methods and therapeutic regimens. A long-standing challenge has been to discover drugs that can halt the progression of disease by inhibiting the ongoing pathologic immune responses while maintaining physiologic immune surveillance. An ideal therapeutic approach would be to modify the expression of the genes that contribute to immunopathogenesis of neuropsychiatric lupus (NPLE). Although the genes responsible for neurological disturbances in SLE is not finely dissected out, preliminary studies in mouse models of lupus suggests aberrant cytokine genes expression in hippocampus and cerebellum are responsible for the neurological deficit(3-5). Our laboratory has recently demonstrated that the histone deacetylase (HDI) inhibitor Trichostatin A (TSA) reverses the skewed expression of several genes implicated in the immunopathogenesis of SLE(6). TSA significantly down-regulated CD154 (CD40-ligand) and IL-10 mRNA and protein, while simultaneously upregulating IFN-g message and protein levels in human SLE PBMC/T cells. Furthermore, our preliminary data in MRlJIpr mouse model of lupus demonstrates that this inhibitor down-regulates IL-10, IL-6, IL-12p30, IL-12p40 and IFN-g mRNA and protein secretion in MRL/Ipr splenocytes. Since IL-6, IL-10 and IFN-g genes are over expressed in cerebellum and hippocampus in MR/Ipr lupus, we propose the concept that HDIs may be useful for the prevention or treatment of neuropshychiatric lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IDENTIFYING THE VITILIGO RELATED SLE GENE ON 17P13 Principal Investigator & Institution: Nath, Swapan K.; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2002; Project Start 05-AUG-2002; Project End 31-JUL-2005 Summary: (provided by applicant): Recently, we published tantalizing evidence locating a gene affecting susceptibility to systemic lupus erythematosus (SLE), and possibly vitiligo, in vitiligo related lupus families on 17p13 (1). Our goal for this proposal is to narrow the susceptibility region and to identify the susceptibility gene. We will achieve this by iterative reduction in the size of the chromosomal region. To improve the power of the study, specific aim 1 is to first augment our current data with new data, identified from our ongoing SLE genetic linkage projects. We will then narrow our previously identified susceptibility region in two steps. First, in specific aim 2, we will choose microsatellite markers to form a 1-2 cM map across the current susceptibility region and analyse these data using genetic linkage methods. Second, in specific aim 3, we will choose single nucleotide polymorphism (SNP) markers to form a 0.5 cM map across the reduced region from specific aim 2 and analyse these data using linkage disequilibrium methods. In the final step, specific aim 4, we will search the public databases for SNPs in genes known to be located in the narrowed susceptibility region established by specific aim 3 and to analyse these using linkage disequilibrium methods. Specific aim 5 is to sequence the gene to find the causal mutations. We ascertained families with European American ancestry, multiplex for SLE and each family has at least one member afflicted with vitiligo. Since autoimmune diseases are thought to share some of their genetic origins, decreasing sample heterogeneity would increase the power to identify the susceptibility gene(s). As the presence of vitiligo in the family was used as a pedigree ascertainment criterion, and there was significantly higher risk associated with developing vitiligo among the family members affected with SLE compared to non-SLE family members, we postulated the following hypotheses: SLE and vitiligo may share
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common autoimmune genetic determinant(s) for their pathophysiology. Alternatively, we may assert that there are genes that lead primarily to develop SLE, which may also modify the risk of developing vitiligo, at least among the ascertained families. Our preliminary results support the hypothesis that SLE and vitiligo may share common genetic determinant(s) (1). This project is directly relevant to the goals of NIAMS SMALL GRANT PROGRAM FOR NEW INVESTIGATORS and has the potential to reveal important, previously unappreciated, susceptibility genes, which contribute toward understanding the etiology of SLE and vitiligo. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IG CLASS SWITCHING IN LUPUS B CELLS Principal Investigator & Institution: Casali, Paolo; Professor; Pathology and Laboratory Medicine; Weill Medical College of Cornell Univ New York, NY 10021 Timing: Fiscal Year 2001; Project Start 01-MAY-2000; Project End 30-APR-2005 Summary: The long term goal of this proposal is to gain insight into the mechanisms underlying the generation of autoantibodies in systemic lupus erythematosis (SLE), a major human autoimmune disease. The predominant and pathogenic anti-self response in these autoimmune patients consists of autoantibodies to nuclear components, including DNA. The origin of these autoantibodies remains enigmatic, but antibodies with similar binding activity are expressed by the normal B cell repertoire (natural autoantibodies). Compared to naturally occurring anti-DNA autoantibodies, lupus autoantibodies of similar specificity are "affinity mature", i.e., they are somatically mutated and antigen-selected. In addition, while the former are IgM, the latter are in general IgG, suggesting that class switching, a crucial mechanism in the maturation of any antibody response, is also important in the generation of autoantibodies in SLE. In class switching to IgG, the constant (C) region of the mu H chain is replaced by Cgamma region, resulting in the acquisition of novel biological activities, including the ability to pass into the extravascular space, and, therefore, in the case of autoantibodies to produce tissue damage. IgG-switched B cells are numerous in the circulation of SLE patients, and IgG accounts for the majority of the pathogenic autoantibodies in these patients. We formally argue here that class switching to IgG occurs more frequently and more effectively in lupus mu+ B cells than in normal mu" B cells. We further argue that this enhanced IgM->IgG switching results from a higher expression of CD40L by lupus T and B cells, as well as from a higher switching propensity of the B cells of these patients, due to a polymorphism of the Cgamma gene promoter or switch regions, and/or to dysregulation of the CD30-dependent mechanism that, as we have recently shown, physiologically dampens IgG-inducing stimuli. To test our hypothesis, we propose: (i) to study CD40L expression in SLE B cells, and their capacity to promote switching to IgG hypothesis, we propose: (i) to study CD40L expression in SLE B cells, and their capacity to promote switching to IgG using our unique in vitro human monoclonal (CL-01) IgM+ IgD+ B cell system; (ii) to analyze the in vitro spontaneous and CD40:CD40-induced IgG class switching in SLE IgM+ IgD+ B cells; (iii) to analyze the regulatory regions upstream of the Cgamma1, Cgamma2, Cgamma3, and Cgamma4 genes in SLE patients, their family members, and for comparison healthy subjects; and finally, (iv) to analyze the down-regulation of the CD40-mediated Ig class switching by CD30, another cell surface molecule of the TNFR family, and the interference with this mechanism by soluble CD30. The proposed experiments should further our understanding of the means that lead to Ig class switching and generation of IgG autoantibodies in lupus, and may help design specific means of therapeutic intervention.
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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNE ABLATION AND HEMATOPOIETIC STEM CELL SUPPORT IN PATIENTS WITH SLE Principal Investigator & Institution: Burt, Richard; Northwestern University Office of Sponsored Programs Chicago, IL 60611 Timing: Fiscal Year 2001 Summary: Systemic lupus erythematosus pursues an aggressive course in a minority of patients, marked by Class III and Class IV nephritis, catastrophic anti-phospholipid syndrome, severe hematologic aberrations, or vasculitis, manifested by recurrent neurologic deficits, pulmonary parenchymal disease, or myocarditis. This subset of patients currently is treated with repeated courses of high dose glucocorticoids or cyclophosphamide. It is the nature of the persistent loss of self-tolerance that characterizes SLE that anti-self antibodies recur after a course of immunosuppression. Anecdotal evidence, derived from the responses of individuals with immunologic diseases who have received bone marrow transplantation for malignancy, suggests that the immune reconsistitution that occurs after transplantation can occur with acquisition of new tolerance to self-antigens. To allow the immune system to reconstitute itself from the stem cell progenitor after immune ablation, in the absence of any previously inciting antigenic stimulus, may allow extended remission from immunologic disease. The prior success of cyclophosphamide therapy, given in more moderate dosage, in curtailing organ-threatening and life-threatening manifestations of lupus, makes it a logical choice for immune ablation therapy. The use of anti-thymocite globulin in conjunction with cyclophosphamide therapy should prolong the inversion of CD4/CD8 cells, which normally characterizes immune reconstitution for a year after marrow ablation. It is hoped that a prolonged tolerance of self-antigens will be facilitated by this condition. By serologic as well as clinical parameters of disease activity, it should be possible to monitor the durability of this tolerance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: IMMUNE MECHANISMS IN PRISTANE INDUCED LUPUS NEPHRITIS Principal Investigator & Institution: Reeves, Westley H. Professor; Medicine; University of Florida Gainesville, FL 32611 Timing: Fiscal Year 2001; Project Start 01-JAN-1999; Project End 31-DEC-2003 Summary: Intraperitoneal injection of pristane (2,6, 10, 14- tetramethylpentadecane) induces a lupus-like syndrome in nearly all "normal" strains of inbred mice. This syndrome is characterized by disease-specific autoantibody production (anti-Sm, RNP, Su, ribosomal P, double stranded DNA), hypergammaglobulinemia, and severe immune complex-mediated glomerulonephritis closely resembling lupus nephritis. In preliminary studies, it was shown that the disease develops in two phases, each with characteristic types of autoantibodies. cytokines, and renal involvement. Microbial stimulation was found to be an important co-factor in progression to the second. more severe, phase. This project will examine the hypothesis that immune complex deposition is necessary, but not sufficient, for the development of nephritis in pristane-induced lupus. Further, it is hypothesized that a systemic abnormality in macrophage or monocyte phenotype resulting from pristane and/or microbial stimulation leads to the production of proinflammatory cytokines and disease progression. The goal of this project is to define pathways leading to glomerulonephritis in pristane-treated mice and
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ultimately to relate them to human lupus nephritis. Three specific aims are proposed. The pathology of the renal lesions will be defined in Aim 1. Mesangial and mesangiocapillary lesions will be studied by immunohistochemical techniques to determine whether hypercellularity reflects proliferation of endogenous (mesangial or endothelial) cells vs. influx of exogenous macrophages, lymphocytes or neutrophils. In addition, mesangial matrix deposition will be evaluated, and the time course of the renal changes will be studied. The roles of pro-vs. anti- inflammatory cytokines will be evaluated in Aim 2. Cytokine production in the glomerulus will be compared with that by phagocytes in the peritoneal exudate, spleen and liver to see if systemic abnormalities are present. Expression of cytokine-inducible markers will be studied as a means to evaluate whether the effects of pro-or anti-inflammatory cytokines predominate. The contribution of microbial stimulation to the development of nephritis in pristane-induced lupus will be examined in Aim 3. It is hypothesized that enhanced intestinal permeability resulting from pristane injection increases the translocation of microbial products, such as lipopolysaccharide, into the bloodstream. This may cause systemic activation of monocytes and macrophages, which then are recruited to the glomerulus in response to immune complex deposition, causing progression instead of resolution of the renal lesion. In view of the widespread susceptibility among "normal" mice to pristane-induced lupus, it seems likely that pristane causes lupus- like disease by its effects on a common, distal, part of a lupus pathway, largely bypassing the genetic abnormalities that predispose to spontaneous forms of the disease. The mechanisms involved in this new inducible model of SLE may, therefore, be common to other forms of lupus, including human SLE. Future studies will address the question of whether renal abnormalities similar to those induced by pristane are involved in the pathogenesis of human lupus nephritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNOLOGIC MECHANISM IN LUPUS NEPHRITIS Principal Investigator & Institution: Madaio, Michael P. Professor of Medicine; Medicine; University of Pennsylvania 3451 Walnut Street Philadelphia, PA 19104 Timing: Fiscal Year 2001; Project Start 01-JAN-1985; Project End 30-JUN-2003 Summary: (Adapted from Investigator's Abstract): The overall aim of this project is to develop a better understanding of the immunologic events leading to glomerular immune deposit formation in individuals with Systemic Lupus Erythematosus. In previous studies, murine and human monoclonal anti-DNA antibodies (Ab) were identified that produced glomerulonephritis following transfer to normal mice. Of particular relevance, the location of immune deposit formation and disease phenotype varied with the mAb. Furthermore, these individual pathogenic Ab bound directly to glomerular cell surface antigens, however each monoclonal anti-DNA Ab recognized a different cell surface proteins. Based on these observations, it was postulated that different autoantibody-glomerular antigen interactions, in vivo, contributes to the phenotypic diversity observed both among the monoclonal Ab and among individuals with lupus. A primary goal of this project is to fully identify the glomerular cell surface antigens for three nephritogenic lupus autoantibodies: anti-DNA MES and anti-DNA SE, derived from MRL-lpr/lpr mice; and RH-14, a human anti-DNA Ab. Anti-DNA MES produces mesangial deposits and binds to mesangial cells, whereas anti-DNA SE produces subendothelial deposits and binds to glomerular endothelial cells. RH14 produces massive subendothelial deposits on transfer to SCID mice, and it binds to glomerular endothelial cells. Candidate cell surface protein antigens were isolated for the autoantibodies. Peptides derived from the isolated proteins will be sequenced and
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then used to generate both degenerate oligonucleotides and anti-peptide antibodies to screen cDNA libraries, in order to define the full sequence and identity of the immunoreactive proteins. Another primary goal of the project is to further determine the pathogenic relevance of these autoantibody-glomerular cell interactions by examining: i) the immune response to the purified cell surface proteins, ii) other spontaneously produced autoantibodies with anti-cell surface protein activity; and iii) the cellular and functional consequences of Ab ligation of the cell surface proteins. Studies will be performed to begin to determine the overall relevance of direct binding of human lupus autoantibodies to glomerular antigens, in general, using: human lupus sera from the Lupus Collaborative Study and controls, the purified cell surface antigens, and individual glomerular cells. Collectively, the results should identify diseaserelevant glomerular antigens for pathogenic lupus autoantibodies and provide insights into the overall pathogenic relevance of autoantibody-glomerular cell surface antigen interactions in lupus nephritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMMUNOREGULATORY ERYTHEMATOSUS
NK
T
CELL
/SYSTEMIC
LUPUS
Principal Investigator & Institution: Porcelli, Steven A. Associate Professor; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2007 Summary: A population of T lymphocytes known as NK T cells recognizes specific lipid ligands in the context of MHC class I-like CD1d molecules and has recently been found to contribute to the regulation of immune responses and the maintenance of immunological tolerance to self antigens. Numerous published reports have linked the loss of NK T cells and changes in their function to the progression of autoimmune diseases, including systemic lupus erythematosus (SLE) and mouse models of this disease. The central hypothesis of the current proposal is that NK T cells constitute an important regulatory arm of the immune system that normally assists in preventing the development of aggressive autoimmunity such as that which occurs in full-blown SLE. The proposed studies will evaluate changes in NK T cells during the progression of spontaneous lupus-like disease in NZB/W F1 mice, and will determine the influence of NK T cells on SLE-associated autoantibody production in murine models of spontaneous and induced SLE-like disease. These studies will build on preliminary findings that strongly implicate NK T cells as a major factor in the regulation of marginal zone B cells, a distinct B cell subset with inherent autoreactivity that has recently become a significant focus for research into the origins of autoantibody production in SLE. Methods that should allow the direct stimulation of NK cells in vivo will be investigated as potential approaches to therapy of SLE that could take advantage of the natural functions of these regulatory T cells. The program project format provides an optimal environment for these studies by providing numerous collaborative interactions with the other members of the program who bring it a wealth of expertise and resources for the analysis of murine models of SLE that are highly relevant to this proposal. These studies will also benefit from extensive usage of all of the Program's proposed core facilities. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMPACT OF HRES-1 ENDOGENOUS RETROVIRUS IN SLE Principal Investigator & Institution: Perl, Andras; Professor; Medicine; Upstate Medical University Research Administration Syracuse, NY 13210
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Timing: Fiscal Year 2001; Project Start 01-JUL-2000; Project End 30-JUN-2005 Summary: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by circulating antinuclear autoantibodies and dysfunction of T and B lymphocytes. Both genetic and environmental factors are believed to influence development of the disease. Common lupus autoantigens show potent immunological cross-reactivities with proteins of viruses and endogenous retroviral elements. Autoantibodies to HRES-1/p28, a 28 kD nuclear protein encoded by the HRES-1 human endogenous retrovirus, were found by several laboratories in up to half of the patients with SLE and overlap syndromes (OLS). We documented molecular mimicry between HRES-1, another nuclear autoantigen, the 70K component of U1 snRNP, and infectious viral core proteins. Analysis of molecular mimicries may provide clues to the identity of viral antigens responsible for triggering cross- reactive immune responses. We detected and cloned the HRES-1 human endogenous retrovirus and mapped it to chromosome 1 at q42. We identified polymorphic genotypes in the long terminal repeat (LTR)/promoter region of the HRES-1 genomic locus and revealed their association with SLE. HRES-1 is centrally located at 1q42 with respect to microsatellite markers associated with disease susceptibility. Thus, HRES-1 or a gene in linkage disequilibrium with this genomic locus may influence autoimmunity in SLE. Genetic variations of the HRES-1 LTR may be linked to a high degree of spontaneous and 5-azacytidine-inducible fragility of the 1q42 chromosomal region. 5-azacytidine, a demethylating agent, capable of triggering autoreactivity of T cells, may influence structure and activity of the HRES-1 LTR. The specific aims will test the hypotheses that (i) identification of HRES-1 autoepitopes with regions of homology to viral proteins and other autoantigens may pinpoint pathogens responsible for initiating autoreactivities, (ii) genetic composition of the HRES-1 LTR directly or indirectly influences development of SLE, and (iii) genotypes of the LTR region determine promoter activity and expression of HRES1/p28. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: IMPACT OF LUPUS SUSCEPTIBILITY LOCI ON B CELL TOLERANCE Principal Investigator & Institution: Mohan, Chandra; Associate Professor; Internal Medicine; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001; Project Start 15-JAN-1998; Project End 31-DEC-2002 Summary: Problem: The function of the immune system is to distinguish self from nonself. When self-tolerance is lost, auto-immunity ensues. Systemic lupus erythematosus is a prototypic auto-immune disease, where loss of tolerance to nuclear antigens leads to a plethora of autoimmune pathology. Yet, little is known about which tolerance check points or mechanisms are actually breached in this disease. Parallel advances in the fields of B- cell tolerances, and genetics of autoimmunity, now allow us to investigate this in a systematic manner. Research Design: The use of immunoglobulin transgenics allows us to assess the integrity of B-cell tolerance at successive check points: deletion, receptor-edition, follicular exclusion, and anergy. Also, the availability of B6 congenic strains bearing individual lupus susceptibility intervals, allows us to evaluate the impact of each of these loci on B-cell tolerance, in a systematic way. This proposal will focus on two such potent loci, originally derived from the NZM2410 lupus strain: Sle1, which is responsible for triggering a strong anti-nuclear humoral response, and Sl32, which leads to spontaneous B-cell hyperactivity. By crossing DNA-specific, or lysozyme-specific immunoglobulin transgenes into these congenic backgrounds, we plan to study how Sle1 and Sle2 impact B-cell tolerance, to these two antigens.
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Significance: These experiments will tell us if the different lupus susceptibility loci breach B-cell tolerance in a global, or (nuclear) antigen-specific manner. Appreciating how the lupus genes tip the delicate balance from tolerance, towards flagrant autoaggression, together with their actual identification, will enrich our understanding of lupus and humor autoimmunity, and pave the way towards more rational and effective therapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: INHIBITION OF LUPUS NEPHRITIS IN IRF-1 DEFICIENT MICE Principal Investigator & Institution: Reilly, Christopher; Biomedical Scis/Pathobiology; Virginia Polytechnic Inst and St Univ 460 Turner Street, Suite 306 Blacksburg, VA 24060 Timing: Fiscal Year 2002; Project Start 23-SEP-2002; Project End 31-MAY-2004 Summary: (provided by applicant): MRL/Ipr mice spontaneously develop immune complex glomerulonephritis similar to human lupus. Prior to overt disease manifestations, MRL/lpr mice overproduce nitric oxide (NO) secondary to increased gene expression of inducible nitric oxide synthase (iNOS). Blockade of iNOS pharmacologically reduces disease expression in MRL/lpr mice. Both macrophages and mesangial cells respond to IFN-gamma with increased production of iNOS and this response can be potentiated further by the addition of TNF-a. IFN-gamma constitutes one of the most potent macrophage activating factors. Either IFN-7 or IFN-y receptor gene deletion modulates disease activity in lupus mice although other adverse effects in these genetic knockouts were noted. Mesangial cells are the principal immunoregulatory cells in the glomerulus possessing both macrophage and smooth muscle cell characteristics. In addition to expressing Fc receptors, mesangial cells contain receptors that bind cytokines and chemokines including receptors for IFNgamma and TNF-oc. Binding of IFN-gamma to its receptor induces transcription of various genes including IFN-gamma regulatory factor 1 (IRF-1). Many of the inflammatory effects of IFN-gamma in macrophages and mesangial cells are mediated through IRF-1 including up-regulation of IL-12, vascular cell adhesion molecule 1, interferon-p, major histocompatability complex I, and iNOS. The molecular events triggered by IRF-1 activation leading to iNOS expression are not completely elucidated. We hypothesize that IRF-1 plays a key role in the initiation and propagation of the inflammatory response in the murine lupus kidney. Targeting IRF-1 may thus serve as a novel mechanism for blocking inflammation in the lupus kidney. The specific aims described below investigate the role of IFN-y signaling and define the role of IRF-1 in lupus nephritis in MRL/lpr mice. Specific Aim 1: Determine the relationship between IFN-gamma, IRF-1 and NFKB activation on inflammatory mediator production including iNOS, IL-12, COX2, and TNF-c in mesangial cells. We will also determine the effects of specific immune modulators on IFN-y signaling and IRF-1 expression in macrophages and mesangial cells from MRL/lpr IRF-1 (-/-, +/-, +/+), B6 IRF-1 (-/-) and control, wiId type mice stimulated with LPS/TNF-oc. Specific Aim 2: Study the in vivo effect of gene deletion of IRF-1 on MRL/lpr mice by backcrossing C57BL6 (IRF-1- /-) mice onto the MRL/lpr background. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: INVESTIGATION INTO THE FUNCTION OF FIG1 Principal Investigator & Institution: Chu, Charles C.; North Shore University Hospital 300 Community Dr Manhasset, NY 11030 Timing: Fiscal Year 2001; Project Start 01-MAR-1999; Project End 28-FEB-2003
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Summary: This proposal is aimed at understanding the function of a recently discovered interleukin-1 (IL-4) induced gene, Figl. Because its expression is regulated by IL-4 and it is only expressed in lymphoid organs, we are particularly interested in understanding its role in immune function. The Figl protein is most similar to the L-amino acid oxidase (LAO) flavoenzyme found in snake venom. Interestingly, LAO kills cells by apoptosis through the production of hydrogen peroxide. Because Figl contains a hydrophobic leader peptide, we propose that Figl is secreted by cells to induce apoptosis of themselves or adjacent cells and thus regulating the immune response. We plan to test this by isolation nd purification of adjacent cells and thus regulating the immune response. We plan to test this by isolation and purification of Figl protein followed by determination if it has similar biochemical properties as LAO. We also plan to heavily explore the role of Figl in the immune system. Besides determination of the ability of Figl protein to induce apoptosis in immune cells, we plan to examine its ability to induce or modify other IL-4 mediated events, especially changes in cell surface protein expression. In addition, we plan to further characterize the expression pattern of Figl in immune cells and confirm whether or not it is a secreted protein. Figl genetically maps to the same region as Sle3, a systemic lupus erythematosus (SLE) susceptibility locus found in mice. Interestingly, some of the characteristics of Sle3 suggest that it may be Figl. Mice with Sle3 have high levels of polyreactive IgM and IgG and a expansion of CD4 T cells. One possible explanation for this phenotype, based on the predicted features of Figl protein, is that mutant Figl protein may not induce apoptosis in activated B cells or in CD4 T cells, resulting in elevated IgM and IgG and an increase in the CD4 T cells. We plan to determine if Figl and Sle3 are identical by genetic mapping in mice and attempting to recreate the Sle3 phenotype by introducing the affected allele into healthy mice. In addition, we plan to extend these studies to human SLE. If we discover that particular Figl polymorphisms are associated with human SLE, we may be closer to understanding some of the genetic basis for susceptibility to SLE. These proposed studies will not only provide new knowledge into the function of a previously uncharacterized protein, Figl, but may also provide exiting new insights into susceptibility to SLE, basic understanding of the development of autoimmunity, and regulation of the immune system. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: LIF MEDIATES ACTH AXIS RESPONSE TO INFLAMMATION Principal Investigator & Institution: Chesnokova, Vera M.; Cedars-Sinai Medical Center Box 48750, 8700 Beverly Blvd Los Angeles, CA 90048 Timing: Fiscal Year 2001; Project Start 10-FEB-1999; Project End 31-JAN-2004 Summary: The hypothalamo-pituitary-adrenal axis (HPA) plays an important role in the relationship between the immune and neuroendocrine systems. Blunting of the HPA response to stress enhances susceptibility to or severity of inflammatory/autoimmune disease. The cascade of HPA hormones released under stress conditions may exert a dampening role on specific defense mechanisms, especially on most cellular actions of the inflammatory immune response. Cytokines, polypeptide mediators, classically associated with immune system regulation and inflammation, play a significant role in the HPA axis activation during immune disorders and actively mediate signaling between the immune and endocrine systems. Leukemia inhibitory factor (LIF), a pleotropic cytokine with diverse biologic activities, also present in pituitary corticotrophs, stimulates proopiomelanocortin (POMC) gene transcription, and is significantly induced in mouse hypothalamus and pituitary in response to endotoxin. As LIF is an inducible proinflammatory hypothalamo-pituitary cytokine which may
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function as a paracrine regulator of ACTH we hypothesize that LIF is involved in control of the inflammatory/autoimmune process acting through regulation of the HPA axis. Preliminary studies conducted in LIF knockout mice, and shown herein, demonstrate that in the absence of LIF, mice cannot maintain an appropriate level of HPA axis activation in response to stress and exogenous IL 1. In addition, the low HPA response to mycobacterial adjuvant observed in knockout animals compared to normal littermates shows a strong correlation between LIF-regulated activity of the HPA axis and susceptibility to inflammatory process. In this study we will determine the molecular and physiological mechanisms implicating LIF in the neuroendocrine control of inflammation. We will utilize both in vivo whole animal models as well as in vitro molecular techniques to characterize the role of LIF in mediating HPA axis response to pathological stress. The hypothesis that LIF mediates the HPA axis response to acute inflammatory challenges as well as to experimental (experimental allergic encephalomyelitis) and genetically determined (systemic lupus erythematosus) inflammatory/autoimmune disease will be tested. This study will provide insights into the cytokine-mediated neuro-immuno-endocrine interface. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: LUPUS ANTICOAGULANTS AND THE PROTEIN C PATHWAY Principal Investigator & Institution: Esmon, Naomi L.; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, OK 73126 Timing: Fiscal Year 2001 Summary: We have found that the membrane requirements of the APC anti- coagulant complex differ markedly from those of the pro-coagulant complexes, These requirements mimic those of at least a population of autoantibodies found in thrombotic patients, providing both specificity and a link between the APC pathway, lupus anticoagulant/anti- phospholipid antibodies and thrombosis. More recently, we have observed that phospholipid oxidation further enhances APC activity selectively. Oxidation is considered a central feature of many inflammatory diseases including lupus and cardiovascular disease. It is the goal of this application to determine the relationship between the different membrane structural requirements of the prothrombinase and APC complexes with an emphasis on the role of oxidation in these reaction. We will determine whether currently unknown plasma factors are involved in the oxidation, what the active products are and whether any modification to the proteins of the APC complex occur as a result of this oxidation. The lipophilic antioxidant, alpha-tocopherol is regarded as protective against oxidative damage in various diseases, including heart disease. We will determine whether incorporation of tocopherol derivatives in membranes differentially affects the pro- and anti- coagulant reactions which may have direct protective effects against thrombus formation. Patients have been identified whose immunoglobulin inhibits only the oxidation dependent anticoagulant APC activity, while others inhibit the oxidation dependent and independent activity equivalently. It is not known whether these represent different risk groups. Immunoglobulin from thrombotic patients recruited during the first grant period will be screened for the prevalence of anti- APC activity and the binding specificities determined. A chimeric form of protein C whose membrane requirements more closely resemble those of the pro-coagulant complexes will also be used in these studies. We will attempt to correlate APC inhibition and binding patterns with the risk of rethrombosis in the study population to determine whether such classification is useful in the identification of pro-thrombotic antibodies for the diagnosis and/or treatment of thrombosis.
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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: LYMPHOCYTE SIGNALING DEFECTS IN PATIENTS WITH LUPUS Principal Investigator & Institution: Tsokos, George C. Professor of Medicine and Molecular Cell; Henry M. Jackson Fdn for the Adv Mil/Med Rockville, MD 20852 Timing: Fiscal Year 2001; Project Start 15-JUL-1998; Project End 31-MAY-2003 Summary: Systemic lupus erythematosus (SLE) is an idiopathic autoimmune syndrome characterized by disorders of the cellular and humoral immune response that lead to autoantibody production. We have recently demonstrated that lupus lymphocytes display disease-specific, antigen receptor- initiated signaling aberrations and hypothesized that these abnormalities result in altered expression of gene(s) that impair T and B cell immune effector function. The proposed experiments, grouped in 4 specific aims, are based on the finding that lupus T cells display deficient expression of T cell receptor (TCR) zetu chain: 1. We shall test the hypothesis that TCR zetu chain deficiency is disease specific and independent of lupus disease activity. To test this hypothesis we will (a) establish the defective expression of zetu chain in lupus patients and determine its possible association with disease activity, (b) establish whether the defect is limited to lupus, and (c) establish the heritability of the disorder. 2. We shall test the hypothesis that zetu chain is exclusively defective in lupus T cells and that this defect is associated with abnormal phosphorylation of certain cytosolic proteins that are important in T cell signaling. To test this hypothesis we shall determine (a) whether other chains of the CD3 complex and the zetu protein family are deficient in lupus T cells, and (b) which proteins are involved in the increased protein tyrosine phosphorylation that is observed following crosslinking of the CD3 molecule. 3. We shall test the hypothesis that zetu chain deficiency is causally associated with the CD3-initiated hyperphosphorylation of cytosolic proteins in lupus T cells and the increased Ca2+ response. This hypothesis will be tested by (a) establishing an association between the effective expression of the zetu chain and the abnormal CD3- initiated signaling, (b) by transfecting lupus T cells with the zetu chain gene and testing whether successful transfection will reverse the described abnormal cell signaling events, and (c) determining whether the marginally CD3-mediated inositol 1,4,5 trisphosphate (IP3) production increase is due the aberrant phospholipase Cgamma and IP3 receptor phosphorylation. Finally, in the 4th specific aim we will test the hypothesis that zetu chain deficiency is the result of deletion(s) or mutation(s) of the coding or the promoter region of the gene. Experiments will be performed to consider the alternative hypothesis, i.e., zetu chain deficiency in lupus cells is the result of cell activation or other cellular regulatory abnormality. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MECHANISM OF AUTOIMMUNE RESPONSE IN HUMAN LUPUS Principal Investigator & Institution: Datta, Syamal K. Professor; Medicine; Northwestern University Office of Sponsored Programs Chicago, IL 60611 Timing: Fiscal Year 2001; Project Start 01-AUG-1993; Project End 31-JUL-2005 Summary: (Adapted from the Applicant's Abstract): The long-term goal of this project is to define basic mechanisms of autoimmunity in Systemic Lupus Erythematosus (SLE), by focusing on disease-relevant T helper (Th) cells that induce the production of pathogenic anti-DNA autoantibodies in SLE. The full spectrum of major peptide epitopes, including naturally processed peptide epitopes, for the pathogenic autoantibody-inducing Th cells of human lupus that recognize nucleosomes in a promiscuous manner will be defined. Shared epitopes for autoimmune B-cells of lupus
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will also be identified. Immunologic relevance of the epitopes in influencing autoimmune T- and B-cell functions will be studied. T-cell receptor (TCR) and MHCcontact residues in the peptide epitopes will be finely mapped for finding consensus motifs that could lead to autoantigen -specific therapy of lupus in humans using tolerogenic peptides or altered peptide ligands. Altered peptide ligands that are partial agonists or antagonists will be designed and studied for blocking pathogenic anti-DNA autoantibody production in vitro. Nucleosomal peptide-HLA-DR tetramers (or peptideMHC-Ig chimeric dimers) will be made to track autoimmune T-cells in lupus patients and family members for diagnostic and prognostic purposes, and for studying the effects of peptide tolerogens in vitro. The second part of the project will deal with mechanisms of prolonged hyper-expression of CD40 ligand (CD40L) and resistance to anergy induction and maintenance in T-cells of human lupus. Major components of Tcell signal transduction pathways involved in T-cell activation, and anergy, particularly in the context of CD40L hyper-expression will be defined. The role of differential MAPK activity in CD40L hyper-expression and stability of CD40L mRNA in lupus T-cells will be studied. Possible anomalies in B7-CD28, and CTLA-4 expression and function in lupus T-cells that could lead to the above mentioned defects being analyzed. Identification of critical peptide epitopes for the autoimmune T helper cells of lupus and studies on regulatory defects in expression of co-stimulatory signaling molecules are leading towards an understanding of basic mechanisms of the disease and development of specific immunotherapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MECHANISM OF GP120 CLEAVING ANTIBODIES IN LUPUS Principal Investigator & Institution: Tramontano, Alfonso; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, TX 77225 Timing: Fiscal Year 2001 Summary: Antibodies (Abs) with innate proteolytic activity are detected in the mouse and human autoimmune repertoires. Antibodies in human SLE and in a murine lupus model (MRL/lpr) have also been shown to bind HIV-1 antigens with high affinity. The central hypothesis of this proposal is that specific catalytic Abs against HIV-1 gp120 could be expressed in subjects with autoimmune disease. Such antibodies may have therapeutic potential for inactivation of HIV-1 infectivity through specific proteolysis at viral capsid sites. Molecular studies suggest that a serine protease-like active site at or near the antigen binding site of peptidase Abs is encoded by a germline VL gene. Our proposal will explore the occurrence, molecular specificity and biological activity of HIV- 1 gp120-specific Ab proteases that arise in autoimmunity. A variable domain (Fv)phage library, constructed from antibody genes found in human SLE is expected to comprise both catalytic and gp120-specific antibodies. Proteolytic Ab fragments from the phage display libraries will be obtained by chemical selection, using active sitemodifying reagents for specific covalent labeling and affinity capture of Abs with serine protease-like reactivity. A series of reagents having a common reactive group and differing degrees of analogy with the target substrate (gp120) will be compared for their relative efficacy in capturing specific and catalytically efficient Abs from the library. These studies will also examine if proteolytic degradation at specific gp120 sites, including a neutralizing epitope for conventional antibodies leads to more effective inactivation of the virus. The range of binding specificities in the lupus repertoire suggests that broadly inactivating catalytic Abs are likely to be identified. Insights from the mechanistic studies shall be integrated into the studies of the comparison proposals of the program project application. The overall Program Project has significant
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implications for development of a new therapeutic modality for treatment or prevention of HIV infection. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MECHANISMS OF CD40 SIGNALING IN B LYMPOCYTES IN VIVO Principal Investigator & Institution: Noelle, Randolph J. Associate Professor; Microbiology and Immunology; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, NH 03755 Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2007 Summary: (provided by applicant): Humoral immune and autoimmune responses are governed by the interactions of CD154 expressed on helper T cells and CD40 on B cells. Extensive efforts have been made to unravel how CD40 engagement transduces the diversity of signals to trigger B cell expansion and differentiation. Like many of the other TNF Receptor family members, it is believed that the recruitment of TNF Receptor Associate Factors (TRAFs) to the cytoplasmic domain of CD40 plays a critical role in regulating CD40 biology. This proposal focuses on the role that TRAFs, and other signaling elements play in regulating B cell function in vivo. We present a unique cohort of Tg/knock-out mice that express mutations in the cytoplasmic tail of CD40 which disrupt the interaction of the cytoplasmic domain with specific TRAFs. B cells from these mice will be studied in vitro (Specific Aim #1) and in vivo (Specific Aim #2) to address the causal relationships between signaling domains of the cytoplasmic tail of CD40 and the induction of in vitro and in vivo B cell biochemistry and function. Preliminary data shows that the loss in TRAF recruitment in B cells exerts minimal or no impact on early IgM and lgG responses in vivo, however, a major effect of TRAF recruitment is observed on the durability of humoral immune responses in vivo. While loss in TRAF recruitment results in defined lesions in humoral immunity, many aspects of the humoral immune response are intact in the absence of TRAF recruitment. We propose to identify (Specific Aim #3) non-TRAF binding domains in CD40 that are critical for early B cell activation. Genetic and proteonomic approaches are presented that will identify novel, functionally significant sites in the CD40 tail and the factors they bind. Enhanced TRAF recruitment and chronic CD40 signaling in the B cell compartment is believed to contribute to the development of lupus. We propose that the expression of a constitutively active form of CD40 (CD40zip; a leucine-zippered, myristoylated CD40 tail), within the B cell compartment will drive B cell expansion and differentiation in vivo and result in the development of a lupus-like syndrome (Specific Aim#4). Assuming the development of autoimmunity in Tg CD40zip mice, mutations in the TRAF binding sites of CD40zip, will be engineered to evaluate the contribution of specific TRAFs to the development of autoimmunity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MECHANISMS OF DEFICIENT PKAII ACTIVITY IN LUPUS T CELLS Principal Investigator & Institution: Shanks, Ryan A. Internal Medicine; Wake Forest University Health Sciences Winston-Salem, NC 27157 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 30-JUN-2006 Summary: (provided by the applicant): Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disorder of indeterminate etiology characterized by the T cell's inability to carry out programmed physiological immune effector functions, including help and cytotoxicity. Our laboratory identified the first disorder of T cell signaling involving the cAMP/protein kinase A (PKA) pathway. There is a profound deficiency of
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PKA phosphotransferase activity due to diminished type I (PKA-I) and type II (PKA-II) isozyme functions. Deficient PKA-II activity is associated with abnormal nuclear translocation of the beta isoform of the regulatory subunit from the cytosol and its retention in the nucleus. I hypothesize that nuclear RII beta-subunit in SLE T cells is a transcriptional repressor of the c-Fos gene. RII beta-subunit is predicted to bind to the nuclear transcription factor, cAMP response element binding protein (CREB), preventing CREB form binding to the cAMP response element (CRE) of the c-Fos gene and thereby inhibiting the gene's transcriptional activation. Transcriptional repression of c-Fos indirectly diminishes the generation of IL-2. This mechanism may contribute to impaired IL-2 production by SLE T cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MECHANISMS OF DRUG INDUCED LUPUS Principal Investigator & Institution: Yung, Raymond L. Internal Medicine; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 01-JUN-1997; Project End 31-MAY-2004 Summary: The application proposes funding with the specific intent of developing an independent research program by the principal investigator. For the past three and a half years, the applicant has been pursuing his interest in basic science research in the areas of T cell immunology and pathogenesis of lupus. Both the clinical and basic science training have prepared the applicant for an independent career in academic rheumatology/immunology in the near future. The proposal is a natural extension of the applicant~s current research. The applicant has shown that murine Th2 cells overexpress LFA-1 (CD11a/CD18) and become autoreactive following treatment with two distinct DNA hypomethylating agents. Adoptive transfer of these autoreactive cells will also induce a lupus-like disease in syngeneic mice. This proposal will first examine the relationship between different lupus-inducing drugs and T cell DNA hypomethylation. The role of LFA-1 in T cell autoreactivity and in vivo autoimmunity will be determined by overexpressing LFA-1 on T cells directly through transfection of murine LFA-1 constructs, and the use of ICAM-1 deficient mice in the murine system. The role of Th1 and Th2 cytokines in T cell autoreactivity in vitro and autoimmunity in vivo will also be examined. Finally, attempts will be made to knockout the murine T cell DNA methyltransferase gene by homologous recombination to definitively determine the role of the gene in the proposed hypothesis of environmental agents inhibiting T cell DNA methyltransferase , leading to DNA hypomethylation, LFA-1 overexpression, and T cell autoreactivity and in vivo autoimmunity. The murine model is likely to have relevance to human disease, as abnormal T cell DNA methylation, LFA-1 overexpression, and increased production of Th2 cytokines such as IL-6 have all been reported in human lupus patients. The application is now in the position to develop his own independent research program as a junior faculty member in the Department of Internal Medicine. It is expected that the applicant will be promoted to a tenure track position during the time of the award. The sponsor, Dr. Bruce Richardson, who is also head of the division of Rheumatology at the Ann Arbor Veteran Administration Hospital, is commited to contributing protected time and resources to the applicant. The collaborators, Dr. Rossler and Dr. Johnson, as well as the core facilities at the University of Michigan will also provide expert help where it is needed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MECHANISMS OF IMMUNE TOLERANCE INDUCED IN MURINE LUPUS Principal Investigator & Institution: Hahn, Bevra H. Professor; Medicine; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, CA 90024 Timing: Fiscal Year 2001; Project Start 15-FEB-2000; Project End 31-JAN-2004 Summary: This proposal aims to define mechanisms of T cell tolerance induced in vivo in NZB/NZW F1 lupus-like mice lupus by administration of a novel, artificial peptide (consensus peptide, pCONS). Tolerance results in dramatic delay in appearance of high titer IgG anti-dsDNA and nephritis, and prolongs life 7 months or more. PCONS is constructed from an algorithm based on spontaneous proliferation of BWF1 T cells to wild peptides from the VH regions of BWF1 monoclonal antibodies to DNA. The algorithm predicts amino acid sequences likely to stimulate BWF1 T cells. A wild peptide (p33B) sharing 10 of 15 amino acids with pCONS, representing a sequence in the VH region (CDR1/FR2) of a mAb BWF1 IgG2a anti-dsDNA, has similar effects. Treatment with an artificial peptide that violates the algorithm, pNEG, produces no clinical benefits. Mice treated with pCONS or p33B, but not saline or pNEG, have reduced proliferation of T cells to immunization with the tolerizing peptide, and reduced T cell help for production of IgG anti-dsDNA in vitro. They also fail to develop the high plasma levels of IFN (and IL-4 that characterize saline-treated BWF1 females. We hypothesize that the large effects of these tolerogens result from induction of apotosis, cytokine deviation, or both in a large population of helper T cells - possibly a population that recognizes different peptides from multiple autoantigens, either early in life or later, after determinant spreading. These effects may correlate with affinity and stability of peptide/MHC Class II binding. The hypothesis will be tested by determining peptide/Class II binding affinities and stability (including peptide variants), developing surrogate rapid measures of effects on BWF1 T cells that predict clinical efficacy of peptides with different MHC binding characteristics, determining the effect of peptide/MHC engagement of TCR on T cell survival and cytokines, and testing peptide variants in vivo for clinical effects. Differences between the T cell responses to peptide/MHC complexes in BWF1 and normal mice expressing the MHC Class II molecules restricting these peptides, will identify novel characteristics of Th activation that characterize the autoimmune mice. Since patients with SLE have T cells that recognize Ig-derived peptides from human monoclonal antibodies to DNA, the information from these experiments may suggest a novel therapeutic approach to this disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MECHANISMS OF T-CELL INDUCED-APC CYTOTOXICITY IN LUPUS Principal Investigator & Institution: Kaplan, Mariana J. Assistant Professor; Internal Medicine; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2002; Project Start 20-SEP-2002; Project End 30-JUN-2007 Summary: (provided by applicant): The application requests funding with the specific intent of developing an independent research program by the principal investigator. The applicant has been pursuing basic science research in the areas of T cell immunology and pathogenesis of lupus (SLE) for the past four years. The proposal is an extension of the applicant's current research on monocyte/macrophage (M theta) apoptosis. Hypothesis: Apoptosis-inducing molecules mediate the autologous monocyte/M theta
78 Lupus
killing caused by CD4+ lupus T cells. Target cell killing by this mechanism can lead to the generation of autoantibodies. Specific aims: To determine the pathways involved in monocyte/M theta apoptosis induced by lupusCD4+ T cells. The applicant will test whether it's possible to inhibit the development of autoimmunity in an SLE animal model, by blocking the apoptotic pathways involved in monocyte/M theta killing by autoreactive CD4+ T cells. The role of macrophage apoptosis in triggering or augmenting autoimmunity will also be investigated. Methods: a) Measurement of cell surface expression of death-receptor ligands on SLE and control T cells by flow cytometry. b) With cytotoxicity assays, determine whether these apoptotic pathways are functional in SLE monocytes/M theta and whether blocking these molecules can inhibit the autologous monocyte/M theta killing by SLE T cells. c) Given the redundancy of the pathways involved in M theta cytotoxicity, the applicant will test, in vitro, if inhibiting the death signals downstream of the death receptors (FADD, caspases, FLIP) is sufficient to inhibit monocyte/M theta apoptosis induced by these ligands. d) In vivo studies will try to characterize whether the blockade on monocyte/macrophage death-receptor ligands by monoclonal antibodies or fusion proteins, can inhibit the development of murine SLE, and whether the elimination of tissue macrophages; per se (with clodronate liposomes in vivo) is sufficient to induce autoimmunity in an animal model. The results of the studies proposed might identify potential mechanisms involved in the generation of autoantigens in SLE. These could lead into the development of therapeutic interventions designed to reverse these abnormalities and abrogate or block the onset and severity of this disease. The sponsor and the institution are committed to contributing protected time, career development and resources to the applicant. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MIDCAREER INVESTIGATOR AWARD IN PATIENT-ORIENTED RESEAR Principal Investigator & Institution: Brey, Robin L. Professor; Medicine; University of Texas Hlth Sci Ctr San Ant 7703 Floyd Curl Dr San Antonio, TX 78229 Timing: Fiscal Year 2002; Project Start 01-MAY-2002; Project End 30-APR-2007 Summary: (provided by the applicant): Systemic lupus erythematosus (SLE) is a systemic inflammatory autoimmune disease that affects predominantly young, minority, pre-menopausal women. Up to 75% of patients with SLE experience some type of nervous system manifestations during their disease course (termed neuropsychiatric lupus or NPSLE). The most common manifestations include cognitive dysfunction, psychiatric disease and stroke, although the full range of NS involvement has not been well characterized. Two major areas of the candidate's research have been the study of 1) clinical and serologic predictors for the development of NPSLE, and 2) immune-mediated thrombosis related to antiphospholipid antibodies (aPL). The major thrust of this proposal will be to expand a strong clinical research program studying NPSLE in a cohort of predominantly Mexican-American SLE patients by 1) determining the frequency of and risk factors for accelerated atherosclerosis in this well-defined cohort and 2) evaluating the frequency and of microembolic signals (MES) by transcranial doppler (TCD) and the relationship between MES and cerebrovascular ischemia and cognitive dysfunction in 2 cohorts of SLE patients (UTHSCSA and Dusseldorf, Germany) with and without aPL. SLE patients are at increased risk for thrombotic events, including both myocardial infarction and stroke. Several NPSLE manifestations may have cerebral ischemia as a common underlying pathophysiologic mechanism, e.g. cognitive dysfunction and stroke. Potential causes of ischemia in SLE include vasospasm, microvascular disease, thrombosis with or without atherosclerosis,
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and rarely, vasculitis. The frequency of atherosclerosis in SLE, the importance of atherosclerosis in SLE-related stroke risk, or whether there are important genetic or racial influences are unknown. Traditional vascular risk factors, inflammatory, immunological, and treatment-related factors specific to SLE are all likely to be involved. With the support of the Mid-Career Investigator Award, the candidate will work with a multidisciplinary team of established investigators and fellows in the areas of stroke, vascular and brain imaging, immunogenetics, epidemiology, psychiatry and neuropsychology. This collaborative effort will provide very important information regarding prevalence and extent of sub-clinical vascular disease and associated risk factors in SLE. Much will be learned about the relative importance of genetic factors and autoantibodies on vascular disease risk in SLE that is likely to apply to other populations also at increased risk for cerebral ischemia and vascular dementia. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MOLECULAR RECOGNITION OF DNA BY AUTOANTIBODIES Principal Investigator & Institution: Glick, Gary D. Professor; Chemistry; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2002; Project Start 01-AUG-1992; Project End 28-FEB-2006 Summary: (provided by applicant): Autoantibodies that bind DNA (anti-DNA) are a hallmark of the autoimmune disorder systemic lupus erythematosus (SLE). A subset of anti-DNA are pathogenic: they mediate an inflammatory response in kidney tissue resulting in renal damage. During the previous project period, we studied the thermodynamic basis of sequence-specific binding by anti-ssDNA mAb 11F8. These studies were predicated on the observations that 11F8 is pathogenic; injecting hybridoma cells that produce 11F8 into normal mice results in nephritis of a nature and severity similar to that seen in human lupus, and the ability of 11F8 to cause disease is linked to its DNA binding properties. In a second research area, we identified a new benzodiazepine (1) that is pro-apoptotic. This compound is remarkably effective in treating the lupus-like disease in the two most clinically relevant polyclonal animal models of SLE. Significantly, treatment is not accompanied by the broad toxicities and side-effects that plague current therapeutic regimes. Based on these findings, the next phase of this grant proposes a series of basic and translation experiments that could directly impact our understanding of the pathology of lupus and the way in which this disorder is diagnosed and treated: (a) Investigate structural aspects of 11F8 recognition through X-ray crystallography, fluorescence resonance energy transfer experiments, and functional group mutagenesis of the 11F8 consensus sequence (Aims 1-2); (b) Use stopped-flow kinetics to investigate the mechanism by which 11F8 discriminates between specific and non-specific ligands (Aim 3); (c) Revert somatic mutations in 11F8 back to germline residues through site-directed mutagenesis to explore the functional significance of affinity maturation in the evolution of the autoimmune response to DNA (Aim 4); (d) Evaluate the utility of the 11F8 consensus sequence (SEL11F8) as a diagnostic probe for lupus (Aim 5); and (e) Use combinatorial chemistry to identify analogs of 1 possessing increased potency in vitro, and test these molecules in vivo (Aim 6). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: MURINE MODELS OF GENERALIZED AUTOIMMUNITY Principal Investigator & Institution: Theofilopoulos, Argyrios N. Professor; Scripps Research Institute 10550 N Torrey Pines Rd La Jolla, CA 920371000
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Timing: Fiscal Year 2001; Project Start 01-APR-1983; Project End 31-AUG-2002 Summary: Systemic lupus erythematosus is a complex, largely genetically-determined, disease with T and B cell tolerance defects leading to autoantibody production against a wide array of self-molecules. Normal T and B cell tolerance occurs centrally or peripherally either as clonal deletion or anergy. Clonal deletions are mediated by apoptosis, a process controlled by an intricate web of promoting and inhibition genes, while the molecular events underyling anergy are unknown. Whereas immature lymphocytes rapidly undergo apoptosis in response to receptor-mediated signals, death of mature cells requires entry into cell cycle, a process also controlled by multiple promoting and inhibiting genes. The relevance of defective activation-induced apoptosis in systemic autoimmunity has been highlighted by the discovery of mutations in the FAS/FASL system in mice with lymphoaccumulation/lupus syndromes. Based on the documented importance of these processes in the induction of tolerance, the central hypothesis in this proposal is that the lupus-associated tolerance defects must be reflected directly or indirectly in the cell-cycle and apoptosis programs, and that detailed analysis of these systems will be instrumental in understanding the etiology of this disorder. This hypothesis will be addressed in the first specific aim by examining activation-induced cell cycle and apoptosis of T and B cells from unmanipulated lupus mice. Expresison levels of the multiple cell cycle-and apoptosis-controlling genes will be measured and correlated with functional and clinical assessments. Emphasis will be given to validating the hypothesis that accumulation of apoptosis-resistant memory T cells in lupus mice is caused by cell cycle arrest. Associated with increased levels of cyclin kinase inhibitors. In the second specific aim, lg transgenic normal and lupus background mice will be used to define mechanisms of B cell tolerance as reflected in cell cycle and apoptosis genes, and to test the hypothesis that lupus mice differ from normals in B cell responses to antigen and memory B cell generation. In the third specific aim, T cell tolerance will be similarly characterized in T cell receptor transgenic normal and lupus mice under conditions leading to activation, anery or deletion. Additional studies using reciprocal adoptive transfers will test the influence of environment on generation and survival of membory T cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MUTATIONS IN PKA GENE TRANSCRIPTS OF LUPUS T CELLS Principal Investigator & Institution: Laxminarayana, Dama; Internal Medicine; Wake Forest University Health Sciences Winston-Salem, NC 27157 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2008 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disorder of indeterminate etiology with multiple Immune effector dysfunctions, which afflicts females in the child bearing years. Protein kinase A (PKA) plays an important role in regulation of immune effector functions of T cells. Previous research has revealed a disorder of type I protein kinase A (PKA-I) enzyme activity in SLE T cells. Recently the applicant has identified mRNA transcript editing of PKA-I RIalpha-subunit and up-regulation of the transcript editing gene, adesosine deaminases that acts on RNA (ADAR) in SLE T lymphocytes. The RNA editing is the co- or posttranscriptional modification of RNA molecules, which results in the insertion, deletion or substitution of nucleotides, mRNA editing plays an important role in the regulation of gene expression and produces phenotypic variability by diversifying the information encoded within the corresponding genomic sequence. The objective of this proposal is to identify the molecular mechanism(s) leading to mRNA transcript editing in RIalphaand RIbeta-subunits of PKA-I and their role in deficient PKA-I isozyme
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phosphotransferase activity in SLE T lymphocytes. The specific aims of the project are;(1) to quantify mRNA transcript editing in RIa and RIbeta gene transcripts in T cells from patients with SLE and compare this with normal controls as well as patients with rheumatoid arthritis (RA) to characterize its association with SLE pathogenesis;(2) to characterize mutant RIa- and Ribeta-subunit proteins phosphotransferase activity in T cells from SLE patients;(3) to analyze regulation and expression of the ADAR gene in T cells from normal, RA and SLE patients and determine whether there is a selective association with SLE pathogenesis;(4) to quantify ADAR-mediated adenosine to inosine conversion in controls and SLE lymphocyte gene transcripts; and, (5) to quantify ADAR2 transcript editing in T cells of SLE patients and compare this with that in T cells of normals and RA patients to characterize its association with SLE pathogenesis. Therefore, the major goal of this project is to identify RNA editing events in the gene transcripts of SLE T cells and the subsequent dissection of editing mechanisms. The novel data derived from these experiments will prove crucial in addressing questions of functional, biological significance, and regulatory events responsible for deficient PKA-I isozyme phosphotransferase activity in SLE T lymphocytes. Identification of this new mechanism of transcript mutations will provide new insights into the mechanisms of aberrant T cell immune effector functions in SLE and open a new avenue of research to design molecular tools to control aberrant immune functions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NEPHRITIS LINKAGE IN LUPUS Principal Investigator & Institution: Quintero-Del Rio, Ana I.; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2003; Project Start 15-JUL-2003; Project End 30-JUN-2006 Summary: (provided by applicant): Renal disease occurs in up to 75% of the systemic lupus erythematosus (SLE) patients usually appearing shortly after diagnosis. We recently linked a putative susceptibility gene to 10q22.3, which leads to lupus in nephritis related lupus families of European American background (1). Our goal for this proposal is to narrow the susceptibility region and to identify the susceptibility gene. We will achieve this by iterative reduction in the size of the chromosomal region. First, we will choose microsatellite markers to form a 1-2 cM map across the current susceptibility region and analyze these data using genetic linkage methods. Second, we will choose single nucleotide polymorphism (SNP) markers to form a 0.5 cM map across the reduced region and analyze these data using linkage disequilibrium methods. In the final step, we will search the public databases for SNPs in genes known to be located in the narrowed susceptibility region previously established and to analyze these using linkage disequilibrium methods. If this does not locate the functional mutation then the gene will be sequenced to find the causal mutation(s). Since autoimmune diseases are thought to share some of their genetic origins, we ascertained families multiplex for SLE in which at least one affected was diagnosed with nephritis, with the intent of decreasing sample heterogeneity and increasing the power to identify the susceptibility gene(s). Our preliminary results support the hypothesis that SLE and nephritis may share common genetic determinants in families of European American background (1). This project is directly relevant to the goals of NIAMS small grant program for new investigators and has the potential to reveal important, previously unappreciated, susceptibility genes, which contribute toward understanding the etiology of SLE and nephritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: DEPRESSION
NEUROPSYCHOLOGICAL
TESTING
FOR
LUPUS
AND
Principal Investigator & Institution: Kozora, Elizabeth; National Jewish Medical & Res Ctr and Research Center Denver, CO 80206 Timing: Fiscal Year 2001; Project Start 20-JUL-2001; Project End 30-JUN-2003 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which is characterized by multi-system involvement and diverse manifestations. A large fraction of patients with SLE demonstrate organic psychiatric and neurologic disorders indicating central nervous system (CNS) involvement and the prevalence of cognitive abnormalities is approximatley 50 percent. An ad hoc multidisciplinary Committee of 35 members was convened by The American College of Rheumatology Research Committee in April of 1997 for the purpose of developing standard nomenclature for neuropsychiatric SLE and proposed a standard battery of neuropsychological tests for SLE This study was designed to provide validity and reliability for this proposed short battery. Pilot data by this investigator has suggested that diagnostic categories are complicated by a large proportion of systemic lupus erythematosus (SLE) patients being categorized as CNS-SLE (Central Nervous SystemSLE) due to symptoms of depression. We see these preliminary data as providing controversy and ultimately opportunity to better classify and understand the cognitive effects of depression in SLE. Information regarding the sensitivity and specificity of this type of battery will justify the use of a short battery for clinical evaluation in SLE, in SLE patients with depression, and potentially in patients with depression only. Specific Aims are 1) To compare the frequency of neuropsychological impairment in CNS-SLE, CNSSLE-depressed, non-CNSSLE and normal controls using the proposed American College of Rheumatology (ACR) Repeatable Neuropsychological Battery for SLE (ACR-RNBSLE); 2) To examine the frequency of SLE patients impaired on the ACR-RNB-SLE compared to a larger comprehensive battery; 3) To determine reliability of the proposed ACR-RNB-SLE; 4) To examine associations between performance on the ACR-RNB-SLE and proposed measures of depression, fatigue and pain; and 5) To compare cognitive performance of the CNS-SLE-depressed group to a control group of depressed-only patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NIAMS CLINICAL RESEARCH CENTER FOR RHEUMATOID DISEASE Principal Investigator & Institution: Silver, Richard M. Professer; Medicine; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2008 Summary: (provided by applicant): The Medical University of South Carolina will establish a Multidisciplinary Clinical Research Center (MCRC) for the Study of Rheumatic Diseases in African-Americans. This MCRC will focus on scleroderma (SSc) and systemic lupus erythematosus (SLE), two rheumatic diseases that disproportionately affect the African-American community. Outstanding leadership in three key areas - Rheumatology, Biometry/Epidemiology, and Health Services Research - provides a framework for successful design and implementation of meaningful clinical research in this understudied population of patients. Three projects and three supporting cores are proposed. Project A is designed to study the interactions between TGF-beta and sphingolipid signaling pathways in SSc and normal fibroblasts. The proposed studies will elucidate this heretofore-unknown interaction to shed light on the
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mechanism whereby TGF-beta signaling is integrated with other cellular signaling pathways leading to fibrosis. Project B addresses an important understudied area, namely psychosocial aspects of female adolescents with SLE, the majority of whom are African-American. MCRC investigators will assess the associations between adaptational processes and adjustment and health-related quality of life, and will conduct an interventional trial designed to enhance adjustment and quality of life for these patients. Project C will address the important issue of divergent racial trends in morbidity from lupus nephritis. Mortality has increased for African-American lupus patients while remaining stable in Caucasian lupus patients, and this divergence cannot be accounted for by differences in socioeconomic status alone. Utilizing the unique resources of the Carolina Lupus Study and the sea island Gullah population, MCRC investigators will address genetic and environmental influences on the development and progression of lupus nephritis. Each of these projects, as well as future pilot projects to be developed by the MCRC, will be served by two Cores: (1) a Methodology Core will provide rigorous methodological and biostatistical support; and (2) a Patient Resource Core will assure MCRC investigators access to a population of African-American patients who are clinically well characterized and from whom biological samples are obtained and stored. This MCRC will facilitate the translation of basic research into the clinical arena, support much needed behavioral research, and conduct epidemiology and health services research on rheumatic diseases affecting minorities and women disproportionately, thus exemplifying the "cross-cutting" nature of research proposed in NIAMS's strategic plan. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NITRIC OXIDE AND SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Gilkeson, Gary S. Professor; Medicine; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2001; Project Start 23-APR-1999; Project End 28-FEB-2003 Summary: Nitric oxide (NO) is a biologically pluripotent compound that is induced during immune responses and overproduced in murine models of lupus. We have found in murine lupus: 1) pharmacologic blockade of NO production ameliorates lupuslike disease, 2) nitration of tyrosine residues of several proteins including catalase with alternations in catalase function in the kidney, and 3) increased levels of NO in the spleens modulate splenocyte apoptosis. In our retrospective studies of human lupus, serum measures of NO (serum nitrate) correlated with clinical disease activity, although there was overlap between normals and lupus patients which may reflect effects of dietary nitrate intake. To definitively determine NO production in human lupus compared to controls and the effects of NO in disease pathogenesis, we propose a prospective study of lupus patients and murine studies to provide insight into immune factors promoting NO production in disease. These studies are incorporated in the following specific aims: 1. Prospectively follow 70 lupus patients with primarily early disease monitoring disease activity and systemic NO production via serum nitrate/nitrite and 3nitrated proteins quarterly for 3 years. 2. A. Measure apoptosis of PBMCs in lupus patients and controls both in vivo and in vitro, correlating apoptosis with disease activity and NO production. B. Determine NOS2 expression in human lupus kidneys and correlate NOS2 expression with disease class, activity, and chronicity. C. Identify serum factors in lupus sera that stimulate NO production by PBMCs. D. Identify human lupus serum and kidney proteins that are nitrated using mass spectrometry analysis. 3. A. Using genetically deficient mice, we will determine the role of immunoglobulin, C3, complement factor B, and CD40 in macrophage
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activation and NO production in MRL-lpr mice. B. We will assess if L-NIL, a specific inhibitor of NOS2, is effective in preventing lupus-like disease in MRL-lpr mice. C. We will determine if estrogen modulates NO production in MRL-lpr mice using ovariectomy studies and estrogen receptor knockout mice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: NOVEL PROTEINS ASSOCIATED WITH SS-A/RO IN TARGET ORGANS Principal Investigator & Institution: Chan, Edward K. Professor; Scripps Research Institute 10550 N Torrey Pines Rd La Jolla, CA 920371000 Timing: Fiscal Year 2001; Project Start 01-MAR-2001; Project End 28-FEB-2006 Summary: (Verbatim) Autoantibody reactivity with the SS-AIRo antigen is an important clinical serological marker for SLE, Sjogren's syndrome, subacute cutaneous lupus erythematosus and neonatal lupus erythematosus (NLE). Two cellular proteins, 60 and 52kDa, have been identified as the predominant targets of the autoimmune response. The long-term objectives of the current proposal are to understand both the origin of this specific autoreactivity and the cellular function of the cognate antigens. Such knowledge should provide critical insights into the pathogenesis of the associated diseases that may differ for each clinical phenotype. Recently a novel 75kDa phosphoprotein (pp75) has been identified as an interaction partner for the 6OkDa SSAIRo protein. In addition it has been identified as an autoantigen recognized by antibodies in sera from patients with Sjogren's syndrome and mothers of children with NLE. Accordingly, three Specific Aims are designed to examine the overall significance of SS-AIRo autoantibodies in the four disease entities and to evaluate if any candidate protein partner(s) of SS-AIRo may provide new clues to the autoimmune pathogenesis. Aim 1 will focus on the identification of pp75 and further define its relationship with 6OkDa SS-A/Ro. Additional experiments will examine whether pp75 is associated with additional proteins. Aim 2 will explore the association of SS-AJRo antigens with other tissue-specific and ubiquitously expressed proteins in the skin, heart, and salivary glands using yeast two-hybrid screen with respective cDNA libraries. The rationale is that each of the target organs may have unique proteins which are available to interact with SS-A/Ro proteins. Differences and similarities among interactions defined in the three affected organs should be highly informative. Aim 3 will address the prevalence of anti-pp75 and antibodies to other putative tissue-specific candidate interaction partners in sera from the four disease groups. Clinical correlations will strengthen the relationship of the antibodies to the pathogenesis of tissue injury. The proposed studies will significantly advance our current understanding of the SS-AIRo antigen/antibody system and its functional role in disease states which target specific organs. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: OKLAHOMA SPECIALIZED CENTER OF RESEARCH IN SLE Principal Investigator & Institution: Reichlin, Morris; Vice President of Research; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2002; Project Start 05-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): The Oklahoma Medical Research Foundation proposes a Specialized Center of Research (SCOR) in systemic lupus erythematosus which will focus on understanding the roles of autoantibodies in SLE. The overarching goal of this center will be to integrate clinical rheumatic disease research with basic science investigators from throughout campus thereby strengthening basic science and
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patient oriented research in this state. Dr. Morris Reichlin is the Director, and he brings a distinguished career's worth of rheumatic disease research activities to this new integrated program. Briefly, this SCOR has five key investigators, five collaborating investigators (in-state), five collaborating investigators (out-of-state), nine additional arthritis investigators, fourteen basic science investigators, a Data and Analysis Core Director, Advisory Committee and a host of adjunct faculty, post-doctoral fellows, clinical fellows, MD-PhD students and graduate students. The key projects for this proposal include various approaches to understand how autoimmune responses start in SLE and how autoantibodies could lead to pathogenesis. The major projects in this SCOR will focus on understanding the development of autoantibodies with regards to symptom and SLE disease onset, exploring the role of autoantibodies in the development of dyslipidemias and accelerated cardiac disease, evaluating the basic role of B cell tolerance in the accrual of lupus autoantibodies, identifying potential etiologic triggers of these aberrant autoimmune responses and assessing the genetic predisposition to a severe serological and clinical SLE phenotype. A Data and Analysis Core and Administrative Core will support each of these individual projects. The primary Oklahoma SLE SCOR objectives are fourfold. First, this SCOR will strengthen research through integration of basic and clinical science in finding key etiological factors for SLE by supporting the above mentioned research projects. Second, biostatistical/research design issues will be incorporated into all ongoing rheumatic disease research in order to raise the current level of research activity. Third, key unique patient resources will be shared by several of the projects. Finally, this SCOR will provide a multidisciplinary structure and resources to strengthen current clinical rheumatic disease research through the influence of strong basic scientists involved in cardiovascular biology, lupus genetics, inflammation, coagulation, molecular biology, immunobiology, immunogenetics, clinical pharmacology and protein studies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: OMEGA 3 LIPIDS AND CALORIES EFFECT ON SLE AND AGING Principal Investigator & Institution: Fernandes, Gabriel I. Professor; Medicine; University of Texas Hlth Sci Ctr San Ant 7703 Floyd Curl Dr San Antonio, TX 78229 Timing: Fiscal Year 2001; Project Start 15-APR-1998; Project End 31-MAR-2003 Summary: The autoimmune prone NZBxNZW F1 (B/W) mouse is a useful model of systemic lupus erythematosus (SLE). Moderate calorie restriction (CR) delays the onset of disease and doubles the life span of these animals. Both CR and diets enriched in fish oil ((w-3) fatty acids decrease inflammatory cytokines, maintain (CD4+ CD44Low) T cell subsets and prevent the rise of memory (CD4+/CD44High) T cells with age. The involvement of Th-1 and Th-2 T cells subsets in the pathogenesis of murine (and probably human) lupus raises the question of whether CR and w-3 supplementation produce their effects by modulating anti- inflammatory cytokines. We propose that CR delays differentiation of CD44High T cells into Th-0, and/or Th-1 and Th-2 subsets by maintaining higher levels of adrenocorticosteroid (CORT) levels and apoptosis. In addition w-3 lipids and CR may also influence co-stimulatory molecules by increasing expression of antioxidant enzymes and reducing chronic oxidative stress. We propose that supplementation with w-3 fatty acids combined with CR maintains a youthful immune system by decreasing formation of free radicals and inflammatory cytokines and decreasing B cells autoantibody production. We hypothesize that CR delays formation of Th-1/Th-2 subsets by co-stimulatory interaction including lowering CD40 ligand expression and by normalizing apoptosis in both lymphoid cells and target tissues. Studies in specific aim 1 will determine if corn oil vs. fish oil lipids with and
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without CR affect the differentiation pathways and functional activity of Th-1 and Th-2 cells by modulating the interaction between CD28/CTLA-4 and B7-1/B7-2 costimulatory molecules in CD4+ T cells and B cells. Experiments in specific aim 2 will determine if prolongation of life span by w-3 fatty acids and/or CR is accompanied by increased anti-CD3 or dexamethasone induced apoptosis in thymic and splenic cells. Studies in specific aim 3 will determine whether prolongation of lifespan by w-3 fatty acids and/or CR is accompanied by decreased autoantibody production in B/W mice. In summary, our studies will examine whether w-3 fatty acids combined with moderate CR can further ameliorate autoimmune disease in B/W mice. These studies are especially important in view of the significant rise in the consumption of vegetable oils in recent years which may promote obesity and autoimmune disorders in the USA. In contrast, the anti-inflammatory w-3 fatty acids are consumed in negligible amounts. It is possible that moderate dietary changes will delay the onset of autoimmune diseases as well as significantly decrease the required doses of cytotoxic and immunosuppressive drugs normally used to treat SLE and other autoimmune diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: MINORITIES
OUTCOME
OF
SYSTEMIC
LUPUS
ERYTHEMATOSUS
IN
Principal Investigator & Institution: Lisse, Jeffery R.; University of Texas Medical Br Galveston 301 University Blvd Galveston, TX 77555 Timing: Fiscal Year 2001 Summary: Systemic Lupus Erythematosus(SLE) is relatively common disease that presents with several different features. It is more prevalent in women and may be more prevalent and severe in African American and Hispanics. This study will determine whether socioeconomic and ethnic factors influence genetic factors that contribute to the development of SLE. Caucasians, Hispanics and African Americans with SLE for less than 5 years at entry will be recruited into the study. Demographic, socioeconomic and cultural features will be recorded. Laboratory findings will be followed. Disease outcome as manifested by number and severity of flares, responses to standardized questionnaires, over all functional status, kidney function and length of survival will be followed for 5 years. The study will contribute to out understanding of the impact of genetic associations on the outcome of SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PATHOGENESIS OF MURINE LUPUS NEPHRITIS Principal Investigator & Institution: Richards, Hanno B. Assistant Professor; Medicine; University of Florida Gainesville, FL 32611 Timing: Fiscal Year 2001; Project Start 01-SEP-2001; Project End 31-AUG-2006 Summary: (provided by candidate): Systemic Lupus Erythematosus (SLE) is an autoimmune disease of uncertain etiology that is influenced not only by genetic but also by environmental factors. Particularly the latter remain poorly understood. Development of nephritis is a prominent feature in SLE, contributing substantially to morbidity and mortality. Present treatment options for nephritis are limited and associated with significant toxicity. Better understanding of the pathogenesis of renal disease in SLE may lead to the development of new, less toxic therapies. Pristane injected intraperitoneally (i.p.) into mice genetically not prone to autoimmune disease induces the production of lupus specific autoantibodies, including antiDNA/chromatin, and immune complex nephritis. In preliminary studies we have found
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that IFNg and IL-6 are essential for both the production of these antibodies and the nephritis, but that nephritis may develop prior to the antibody response. We hypothesize that anti-DNA/chromatin autoantibodies are a marker for overproduction of inflammatory cytokines which promote nephritis, rather than the cause of renal disease in pristane-induced lupus. We propose to examine the temporal relationship between the production of autoantibodies and the development of nephritis in pristanetreated mice. Outcome of this study will answer whether anti-DNA/chromatin antibodies are obligatory for the development of nephritis or whether other factors are involved in development of renal disease. In an effort to determine if dysregulated cytokine homeostasis is sufficient to induce the production of autoantibodies and nephritis we plan to induce their constitutive expression by bone marrow derived cells. The proposed experiments will involve the transduction of hematopoietic stem cells with retroviral vectors encoding IFNg, IL-6 or both for later injection into mice. We predict that with this approach we will be able to induce autoantibody production and nephritis. Finally we aim to define the roles of ITAM vs ITIM containing Fc receptors for the development of nepliritis.We will initially study of the effect of FcgRI/III and FcgRII deficiency on pristane-induced lupus. Subsequently anti-DNA/chromatin antibody production and nephritis after pristane-treatment in FcgRI/III deficient mice over expressing IFNg or IL-6 and FcgRII deficient mice also deficient in IFNg or IL-6 will be studied. The out come of these experiments will determine if Fc receptor mediation is obligatory for the development of nephritis or whether a direct effect of cytokines alone is sufficient for the induction of renal disease. Based on our preliminary data we expect that Fc receptor signaling is the critical varialble for the induction of renal disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PATHOGENESIS ERYTHEMATOSUS
OF
MURINE
SYSTEMIC
LUPUS
Principal Investigator & Institution: Kono, Dwight H. Associate Professor; Scripps Research Institute 10550 N Torrey Pines Rd La Jolla, CA 920371000 Timing: Fiscal Year 2001; Project Start 30-SEP-1995; Project End 31-MAR-2004 Summary: (Adapted from the Investigator's abstract): Genetic susceptibility is the major predisposing factor thus far identified for spontaneous systemic lupus erythematosus (SLE) in both humans and animal models of the disease. Previously, the investigator identified in an (NZB x NZW)F2 intercross 8 loci designated Lbw1-Lbw8 on chromosomes 17, 4, 5, 6, 7, 18, 1, and 11, respectively, that exhibited evidence of linkage to one or more of four major SLE disease traits. Five of these loci have been confirmed in other crosses. The investigator has generated congenic lines for Lbw2, a locus on chromosomes 4 required for hemolytic anemia, and Lbw5, a locus on chromosome 7. Lbw5 is an autoimmune accelerator that increases the production of IgG autoantibodies and promotes hemolytic anemia, glomerulonephritis and early mortality in NZB mice. In this application the investigator proposes to: 1) define the genetics of immunoglobulin and autoantibody responses and the relationship of autoantibody specificity to other traits. This will be done by linkage analysis using an expanded set of BWF2 mice; 2) generate congenic lines for Lbw1, 2, 4, 5 and 7 to examine the effects of susceptibility and resistance alleles at these loci on clinical and immunologic manifestations; and 3) undertake positional cloning of Lbw2 and Lbw5. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PATHOGENIC ROLE OF THE COMPLEMENT SYSTEM IN MURINE LUPUS Principal Investigator & Institution: Quigg, Richard J. Professor; Medicine; University of Chicago 5801 S Ellis Ave Chicago, IL 60637 Timing: Fiscal Year 2001; Project Start 15-AUG-1999; Project End 31-JUL-2003 Summary: (Verbatim from Investigator's Abstract): Activation of the complement system by immune complexes (IC) leads to an inflammatory response. This occurs through the direct actions of complement proteins as well as indirectly via the stimulation of other mediator\systems. Furthermore, complement is necessary for an optimal humoral immune response to naive antigens and effective disposal of circulating ICs. The studies in this application will examine the role of the complement system in the prototypical IC disease, systemic lupus erythematosus (SLE). Here, the NZBNV F, murine model of SLE will be studied. These animals have pathological features similar to those of human SLE, including the development of a wide spectrum of auto-antibodies and diffuse proliferative glomerulonephritis that ultimately is fatal. The roles of complement will be dissected through its inhibition. Because C3 and C5 play pivotal roles in complement actions, the effects of inhibiting each will be compared in these studies. Inhibition of C3 will be achieved with the murine protein Crry (Complement receptor related protein y). Two different strategies of Crry administration will be used: 1) recombinant Crry containing a non-complement activating IgG1 "tail" (Crry-Ig) will be given chronically to animals; and, 2) transgenic mice constitutively producing endogenous soluble Crry will be studied (Crry-tg). C5 will be inhibited with an anti-mouse C5 monoclonal antibody that blocks the cleavage and activation of C5. As a parallel approach, the role of C3 in experimental SLE will also be determined by using mice made deficient in C3 through gene targeting (C3 -/- mice). Such studies will determine the effects of absolute C3 deficiency in experimental SLE. These studies will carefully evaluate how the complement system is involved in the pathogenesis of experimental SLE. The following variables will be measured: 1 ) clinical outcomes, including mortality and renal functional changes; 2) pathological alterations in kidney, including the progressive fibrogenesis occurring in glomeruli and the tubulointerstitium; 3) proinflammatory cytokines that are up regulated in these models; 4) amount and antigen specificity's of circulating and glomerular bound auto-antibodies; and, 5) the relative state of autoimmune activation of B lymphocytes. Dissecting the proand anti-inflammatory actions of the complement system in experimental SLE will provide important insights into the mechanisms of disease in human SLE, as well as in other IC diseases and may lead to viable therapeutic approaches that can be applied in practice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PHASE I/II TRIAL ON THE USE OF CD20 ANTIBODY FOR THE TREATEMENT OF SLE Principal Investigator & Institution: Albert, Daniel A.; University of Pennsylvania 3451 Walnut Street Philadelphia, PA 19104 Timing: Fiscal Year 2001 Summary: Systemic lupus erythematosis is a multi-system autoimmune disease characterized by numerous autoantibodies. These autoantibodies may be responsible for most, and possibly all, of the disease manifestations. While numerous defects and abnormalities of the immune system both, innate and acquired, and endogenous and exogenous, have been described the final common denominator is aberrant antibody
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production by B lymphocytes. There are compelling arguments to support interventions aimed at reducing B lymphocyte function and/or number. Observations that support this concept are that cyclophosphamide is a is a B cell predominant chemotherapeutic agent that is highly effective in SLE, that intravenous immunoglobulin is effective therapeutically for SLE and down regulates B lymphocyte activity, that both HIV disease and anti- CD4 reduce lupus activity, and that when patients with SLE develop common variable immunodeficiency their SLE is improved. A therapeutic approach that selectively diminished B lymphocyte function or number might be a useful intervention to treat patients with SLE. Because immunoglobulin deficiency is easily reversed by pooled immunoglobulin which is not toxic (and might be somewhat therapeutic) targeted B cell therapy might be moth effective and well tolerated. Current efforts in this same direction using antibodies to the CD40 ligand are underway in a multi-center trial sponsored by Biogen and a second trial at University of San Francisco sponsored by IDEC. The CD40/GP 39 is a co-stimulatory interaction necessary for an immune response. Bon contrast, the anti-CD20 antibody is directed at a pan B cell marker and a directly inhibitory to B cells in part by antibody dependent cellular cytotoxicity. In both in vitro and in vivo studies the anti-CD20 is more effective than CD40 antibody in B cell lymphoma and should be a more effective inhibitor of B cell function. We plan an open label phase 2 trial of rituximab a chimeric anti-CD20 antibody (IDEC-C2B8) in the treatment of severe but not life threatening systemic lupus erythematosis. Patents will have failed conservative management and be candidates for cytotoxic and chemotherapeutic intervention. We will monitor disease activity by convention indices (SLEDAI and SLAM), immunologic markers of disease activity such as complement components and acute phase reactants, hematological parameters and renal function. These outcome measures will permit us to make an initial assessment of the safety and efficacy of this agent for the treatment of systemic lupus erythematosis. With these finding we will be able to plan a randomized double blind placebo controlled trial of anti- CD20 therapy for SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PILOT STUDY--NZW DERIVED RESISTANCE TO MARINE LUPUS Principal Investigator & Institution: Ferguson, Polly J.; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2001; Project Start 28-SEP-2001; Project End 31-AUG-2006 Summary: Systematic Lupus Erythematosus (SLE) is a phenotypically diverse, multisystem autoimmune disorder that is caused by ill-defined interaction(s) between environmental and genetic factors. Genome wide linkage analysis of SLE multiplex families suggests there is significant racial and genetic heterogeneity, further complicating the genetic dissection of this disorder. Fortunately, there are phenotypically similar mouse models of lupus; including genetically engineered and spontaneous models. Most of the investigative work in this field has focused on identifying disease susceptibility loci; however, there are experimental data that data that suggest disease resistance genes are also important. The non-autoimmune New Zealand White (NZW) mouse does not develop lupus despite harboring the best characterized lupus susceptibility intervals and a disease permissive major histocompatibility locus [H-2z/z] suggesting that the NZW genome contains allelic polymorphisms that negatively regulate the phenotypic expression of lupus susceptibility gene(s). We hypothesize that experimental crosses between NZW and C57BL/6.FcgammaRIIB-deficient mice will allow the identification of NZW-derived intervals that attenuate the lupus phenotype(s) in this genetically engineered murine
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model of lupus. We will 1) map the quantitative trait loci (QTL) that modulate the FcgammaRIIB-/- lupus phenotype and 2) construct chromosome substitution strains (CSS) in order to dissect the genetic contribution of these individual QTL. Identification of QTL that modulate that lupus phenotype and the subsequent development of QTL containing CSS will lay the foundation for the future functional assessment of the lupusattenuating QTL and for candidate gene analysis. Understanding the genetic basis of disease resistance could facilitate the development of novel therapeutic approaches for treatment this devastating disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PILOT--INTERLEUKIN 15 IN HUMORAL AUTOIMMUNITY Principal Investigator & Institution: Peng, Stanford L.; Washington University Lindell and Skinker Blvd St. Louis, MO 63130 Timing: Fiscal Year 2003; Project Start 01-SEP-2003; Project End 31-AUG-2006 Summary: The pleiotropic cytokine interleukin (IL)-15 has been heavily implicated in the pathogenesis of autoimmune diseases like lupus and rheumatoid arthritis because it is found at significantly elevated levels in afflicted patients, and because it is known to play critical roles in the proliferation and homing of lymphocytes. Thus, IL-15 is a potential pathogenic and therapeutic target in the rheumatic diseases; however, definitive experimental evidence remains lacking. We propose in this pilot and feasibility study to explore the mechanisms by which interleukin-15 might participate in the pathogenesis of systemic humoral autoimmunity using mutant animal models. Specifically, we will: 1. Determine the role of interleukin-15 in murine lupus, and 2. Investigate the role of interleukin-15 in the regulation of autoreactive B cell proliferation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PKR/ DIFFERENTIAL TRANSLATIONAL CONTROL IN LUPUS T CELLS Principal Investigator & Institution: Beretta, Laura; Associate Professor; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 28-SEP-2001; Project End 31-JUL-2006 Summary: Despite numerous advances in systemic lupus erythematosis (SLE), the molecular pathogenesis of this disease remains poorly understood. This proposal takes advantage of recent findings we have uncovered that suggest defective regulation of translation initiation in SLE. It was previously known that T cells from patients with active SLE are refractory to mitogenic stimuli. An important T cell response to mitogenic stimuli is an increased rate of protein synthesis, regulated at translation initiation. It was previously known that T dells from patients with active SLE are refractory to mitogenic stimuli. An important T cell response to mitogenic stimuli is an increased rate of protein synthesis, regulated at translation control. We investigated the effect of activating signals on translation initiation factor activates, in T cells from SLE patients. Activation by mitogens resulting in a strong increase in protein synthesis in control T cells but not in T cells from SLE patients. This lack of response in SLE T cells was further associated with over-expression of the pr4oytein kinase PKR and with increased phosphorylation of its substrate, the initiation factor eIF2alpha. Interestingly, up-regulation of PKR impairs cell functions that are relevant to lupus, including: cell proliferation, Fasdependent apoptosis, cytokine gene expression and signaling and release of Ca2+ from intracellular stores. Therefore we propose to test in this application the hypothesis that high expression of PKR and subsequent eIF2alpha phosphorylation contribute to the
Studies 91
impaired responses to mitogens in T cells from SLE patients. The specific aims are: (i) to determine the role of PKR in lupus pathogenesis. We will produce PKR-/- lupus-prone mice by backcrossing PKR knockout mice with MRL-lpr breeders. We will analyze in these mice different parameters of relevance to this lupus-like disease including serum immunoglobulin production, anti-nuclear auto-antibody response, end-organ disease and mortality. (ii) to identify specific mRNAs whose translation is highly dependent on PKR and eIOOF2alpha phosphorylation in T cells, and that may be affected in SLE. We will analyze gene products in TG cells from control mice and from transgenic mice with either a dominant negative mutant of eIF2alpha or with a targeted disruption of the PKR gene. Gene products will be identified using two-dimensional gel electrophoresis and mass spectrometry. (iii) PKR regulates cytokine signaling mediated by the transcription factor IRF-1 and we reported that IRF-1 trans-activates PKR expression. We will investigate the function of IRF-1 in lupus T cells. It is anticipated that these studiers will provide novel and important insights into mechanisms contributing to SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: POST TRANSLATIONAL MODIFICATIONS AND AUTOIMMUNITY Principal Investigator & Institution: Mamula, Mark J. Senior Scientist; Internal Medicine; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2001; Project Start 01-APR-2001; Project End 31-MAR-2006 Summary: (provided by applicant): It is the principle purpose of the immune system to protect the host from infectious pathogenic microorganisms. With that purpose in mind, lymphocytes have been programmed to differentiate self tissue antigens from foreign antigen. However, the appearance of autoimmune diseases and the presence of autoreactive lymphocytes in the normal population illustrates that the regulation of immunity is far from perfect. Systemic autoimmune diseases are the product of a complex interaction of lymphocytes, soluble macromolecules, and self tissues leading to the pathology of disease. Autoimmune responses often target multiple determinants within an autoantigen. For example, in systemic lupus erythematosus (SLE), autoantibodies are directed at a number of determinants on small nuclear ribonucleoproteins (snRNPs) and on nucleosomes. The self or foreign proteins involved in the spontaneous initiation of these autoimmune responses is not known. We have recently identified the ability of a novel post-translational protein modification, termed isoaspartyl, to confer autoimmunity to otherwise immunologically inert self peptides. For example, the immunization with isoaspartyl forms of snRNPs can provoke autoantibody responses typical of human SLE. Isoaspartyl peptide modifications arise frequently in aged and stressed cells. This application will examine the presence of isoaspartyl forms of lupus autoantigens in resting, aged, and activated lymphocyte populations and determine their ability to circumvent immune tolerance. Second, we will examine the immunologic abnormalities, lymphokine responses and intracellular signalling, in murine models unable to repair isoaspartyl modifications (PIMT knockout mice). Finally, we will study the progression of spontaneous lupus autoimmunity and pathology in MRL Lpr/lpr-PIMT-/- mice, a murine model of human SLE. This application will define the biological and immunological implications of posttranslational protein modifications. Overall, our studies will address mechanisms important in the induction and perpetuation of autoimmune disease. A more thorough understanding of the earliest events in the genesis of autoimmunity may help identify important elements to exploit for the immunologic intervention of these diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: POSTTRANSLATIONAL MODIFICATIONS AND AUTOIMMUNITY Principal Investigator & Institution: Doyle, Hester A. Internal Medicine; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2001; Project Start 01-SEP-2001 Summary: The immune system has developed a number of mechanisms for recognizing foreign antigen while ignoring self-antigens. However, the presence of autoimmune diseases demonstrates these mechanisms can be bypassed. Our laboratory has demonstrated that the posttranslational modification of aspartic acid to isoaspartic acid in self-peptides induces immunogenicity to otherwise immunologically ignored peptides. Isoaspartyl residues occur spontaneously at physiological conditions and increase in aged or stressed cells. The proposed studies will examine how isoaspartyl containing self-peptides/proteins break tolerance and contribute to autoimmunity. This will be achieved by 1) examining T and B cell responses to isoaspartyl and normal forms of self-antigens, 2) determining the effect of isoaspartyl accumulation on immune function in mice deficient in the isoaspartyl repair enzyme PCMT, and 3) examining the spontaneous development of autoimmunity and pathology in PCMT -I- mice alone and backcrossed to a murine model of systemic lupus erythematosus (SLE). Results of these studies will be helpful in developing immunotherapies for the modulation of autoimmunity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PREDICTION OF LUPUS OUTCOME BY GENE EXPRESSION PATTERNS Principal Investigator & Institution: Winchester, Robert J. Professor; Pediatrics; Columbia University Health Sciences New York, NY 10032 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 31-DEC-2006 Summary: (provided by applicant): Support is sought for performing mechanistic studies to define the gene expression profiles of glomeruli isolated from biopsy sections of lupus glomerulonephritis with the overall goals of determining whether the glomerular gene expression phenotype will predict outcome and efficacy in an ongoing parent trial. The parent trial compares administration of CellCept versus IV Cytoxan for initiating control of biopsy-proven lupus nephritis. We have demonstrated the feasibility of studying gene expression profiles by microarray analysis in glomeruli isolated from frozen biopsy sections by laser microdissection t6 characterize the molecular pathologic mechanisms leading to lupus nephritis. This work revealed considerable heterogeneity in gene expression patterns in samples classified as proliferative glomerulonephritis, suggesting the feasibility of lupus renal biopsy subclassification by gene expression criteria. The expressed genes formed 8 main clusters and the presence or absence of genes comprising these clusters in a given sample divided the biopsies into 3 distinct types. The hypothesis underlying the proposed studies is that differences in molecular pathologic mechanisms revealed by the various transcriptional phenotypes will predict the heterogeneous natural history and therapeutic outcome of lupus. The first aim of the proposed mechanistic studies is to cluster glomerular gene expression patterns in diagnostic renal biopsies performed in the current trial and use them to extend understanding of pathways involved in the molecular pathogenesis of lupus glomerulitis. Parallel quantitative PCR for selected genes found through the microarray assessment will be performed to validate the patterns found and determine if their differential expression can be used as a surrogate. The second aim will correlate the clusters and pathways with conventional pathologic
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features to identify the molecular basis of the pathologic findings. The third aim will determine whether particular outcomes of the trial could be predicted by the gene expression phenotype of the initial renal biopsy. In particular we will address, first, whether one gene expression type, characterized by apoptosis, TNF signaling and fibrosis, is highly correlated with poor outcome and thus predict the subset of nonresponders to Cytoxan or CellCept. Second, whether CellCept will prove to be efficacious in a different gene expression subset, suggesting it could be used in these cases as a less toxic alternative to Cytoxan. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: PRESERVING OVARIAN FUNCTION IN LUPUS NEPHRITIS Principal Investigator & Institution: Dooley, Mary A. Professor; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, NC 27599 Timing: Fiscal Year 2001; Project Start 01-DEC-2000; Project End 30-NOV-2001 Summary: This abstract is not available. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PROGRAM PROJECT IN THE GENETICS OF SLE Principal Investigator & Institution: Kimberly, Robert P. Professor; Medicine; University of Alabama at Birmingham Uab Station Birmingham, AL 35294 Timing: Fiscal Year 2002; Project Start 24-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): Epidemiologic studies strongly support a genetic basis for susceptibility to human SLE, and genetically-defined murine models of systemic lupus indicate that lupus is a complex, polygenic disease with a threshold liability for inheritance. These studies in "lupus-like" mice suggest that different genes may control different aspects of the autoimmune phenotype. Most importantly, these studies, coupled with the rapid advances in knowledge available through the human genome project, underscore the feasibility of using the tools of modem genetics in defining human disease susceptibility and severity. The approach to the genetics of human SLE requires a multidisciplinary, team effort. Within this Program Project, we have assembled a unique and exceptionally strong multidisciplinary team, which leverages both fundamental and clinical investigations in SLE at the host institution and at the partner institutions. Our team expertise includes mastery of the theory and techniques of modem genetic mapping (linkage and association), full appreciation of the SLE clinical phenotypes and the proven ability to recruit and maintain cohorts of SLE patients (multiplex families, simplex families, case-control and longitudinal cohorts). Uniting this expertise is a broad appreciation for the pathophysiological processes in SLE in order to facilitate the selection of candidate genes and to translate findings into meaningful, mechanism-based clinical intervention. Through our team effort, we have an unprecedented power to accelerate the pace of discovery, to replicate and narrow regions of linkage, to pursue the structure and biology of candidate genes, and to test the relevance of these discoveries to clinical phenotype in a very large, meticulously studied cohort of SLE patients. Our efforts will advance our understanding of SLE and leverage both the application of current therapy and the development of new, mechanism-based therapies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PROTEIN KINASE A-1 DEFICIENCY IN LUPUS PATHOGENESIS Principal Investigator & Institution: Brown, Doris R. Internal Medicine; Wake Forest University Health Sciences Winston-Salem, NC 27157 Timing: Fiscal Year 2001; Project Start 01-AUG-2001 Summary: Systemic Lupus Erythematosus (SLA) Is an autoimmune disorder that predominantly afflicts females in the child-bearing years. The cause(s) of the disorder remain unknown; however, recent progress in understanding the role of T cell dysfunctions in SLE patients has lead to progress in unravelling the molecular pathogenesis of the disease. A generalized failure of the T cell to perform its functions has been characterized in SLE. This impairment in the T cell function contributes to the destructive autoimmune pathogenesis observed in SLE patients. This proposal will investigate one pathway that may contribute to impaired T cell function. We have defined the first disorder of a protein kinase in SLE T cells, characterized by a deficient type I isozyme of protein kinase A (PKA-1). Our working hypothesis is that the RI Betasubunit protein of PKA-I is deficient due to abnormal synthesis and/or stability of the protein. Our goal is to identify the step at which RI-Beta protein production is altered in SLE T cells. The identification of the cause of RI Beta- subunit deficiency may provide the rationale in the future to apply new therapies in SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: PROXIMAL AUTOIMMUNITY
DETERMINANTS
OF
NEPHRITOGENIC
Principal Investigator & Institution: Foster, Mary H. Associate Professor; Medicine; Duke University Durham, NC 27706 Timing: Fiscal Year 2001; Project Start 01-JAN-1995; Project End 31-DEC-2002 Summary: (Adapted from Investigator's Abstract): Self antigen is the target of most nephritogenic immune responses, yet little is known abut the proximal events that generate, regulate, and induce expression of autoreactive lymphocytes. Effective therapy for severe autoimmune disease thus remains limited to nonspecific and toxic immunosuppression. The goal of the proposed studies is to gain a better understanding of the immunologic mechanisms that normally regulate nephritogenic humoral autoimmunity, as a basis for developing more specific interventions to eliminate or inactivate autoreactive cells and/or reestablish tolerance. These studies emphasize autoantibodies that target renal basement membrane (BM) antigens because BM proteins are the only confirmed renal targets in human autoimmune nephritis, BM is targeted in both systemic and organ-restricted autoimmunity, and regular of immune responses to these complex matrix antigens has been virtually ignored to date. The proposed studies will test the hypothesis that unmutated nephritogenic anti-laminin and anti-DNA B cells similar to those activated in systemic lupus are present but tolerant in the normal host; that their expression can be induced by inherited autoimmune susceptibility and/or environmental challenge; and that nephritogenic anti-alpha3(IV)NC1 collagen B cells persist as nontolerant ("ignorant") lymphocytes in the periphery of nonautoimmune hosts until activated by specific immunization in a permissive genetic background. The Ig transgenic approach is used for this purpose because it permits enrichment for the autoreactive B cell population of interest while preserving complex immunologic microenvironments in vivo. Six established Ig-tg lines that encode lupus anti-laminin (LamH) or nephritogenic and anti-DNA (238H) Ig will be used to determine the presence, phenotype, functional state and pathogenecity of autoreactive B cells in the nonautoimmune B6 and autoimmune MRL genetic
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backgrounds. Laminin epitopes recognized by autoantibodies will be determined, using spontaneous Ig recovered from nontolerant mice and Ig rescued from tolerance mice by in vitro or in vivo bcl-2 expression. Finally, B6 mice will be rendered transgenic for an anti-alpha3(IV)NC1 Mab that recognizes Goodpasture's epitope, to determine the tolerance phenotype of B cells specific for a cryptic and organ-restricted BM antigen targeted in severe human nephritis. The autoantibody transgenic model thus provides a well defined experimental system in which to study immunologic mechanisms and etiologic influences and to test novel therapeutic interventions for autoimmune renal disease. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: RECEPTOR REVISION CREATES AUTOANTIBODIES IN LUPUS Principal Investigator & Institution: Monestier, Marc; Professor; Microbiology and Immunology; Temple University 406 Usb, 083-45 Philadelphia, PA 19122 Timing: Fiscal Year 2003; Project Start 25-SEP-2003; Project End 30-JUN-2005 Summary: (provided by applicant): Antibodies to DNA or nucleosomes possess numerous positively charged amino acids, such as arginines, in their heavy chain CDR3. These cationic residues are critical contact points with negatively charged sites on the DNA helix in chromatin. Atypical VH-D-J H rearrangements such as D-D fusions contribute to create these arginine codons. Based on our preliminary data, we propose that secondary immunoglobulin heavy gene rearrangements in lupus-prone mice are responsible for potentially selfreactive VH-D-J H junctions. An increased predisposition to generate such atypical VH-D-J H rearrangements can be an important contributor to the development of lupus. We propose to test our hypothesis by accomplishing the following objectives. In Specific Aim 1, we will investigate whether lupus-prone MRL mice differ from control strains in their levels of RAG1 and RAG2 mRNA during B cell ontogeny. We will evaluate whether RAG expression is differentially affected in MRL B cells after engagement of the B cell receptor. We will also characterize, at the DNA level, VH-D-J H and D-J H rearrangements as well as other evidence of receptor revision in MRL and control B cells at various stages of development. In Specific Aim 2, we will directly test this model in vivo by examining the presence of secondary heavy chain rearrangements and autoimmune manifestations in hemizygous MRL Jh +/- mice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: REGULATORY AND EFFECTOR T CELLS IN SLE Principal Investigator & Institution: Tung, Kenneth S. Professor; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2002; Project Start 27-SEP-2002; Project End 30-JUN-2003 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with protean clinical presentation and variable outcome; and glomerulonephritis (GN) is a serious manifestation of SLE. SLE patients and lupus prone mice exhibit abnormalities in B cell and T cell tolerance and their responsive state. They produce autoantibody (autoAb) response to multiple self antigens (Ag) and have immune complex (IQ deposits in tissues. The CD4+CD25+ regulatory T cells normally prevent organ specific autoimmune disease occurrence. Herein we investigate the role of the regulatory T cells in SLE by studying the effect of thymectomized on day 3 (d3tx) in lupus prone mice. The d3tx lupus prone NZM2328 mice exhibited earlier autoAb response, accumulated more glomerular IC, and developed accelerated acute GN. Remarkably, severe acute GN occurred in - 90% of male NZM2328 mice that are
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normally more resistant to lupus GN. These results have led to the following hypotheses. First, regulatory T cells can negatively influence development of SLE. Second, acute lupus GN is a T cellmediated autoimmune disease that targets the renal glomerulus. The CD4+CD25effector T cells participate in renal glomerular injury by targeting either renal Ag or Ag provided by the glomerular 1C. Thus, in SLE, autoreactive T cells: 1) drive an autoAb response, and 2) directly elicit acute GN. Third, acute GN is a checkpoint in lupus GN, from which the male mice regress and the female mice progress to chronic GN. Thus male NZM2328 mice may have less effective or more regulatory T cell function. The model will also permit the study on factors responsible for the progression and regression of lupus GN. We will investigate these hypotheses in Aim I of our proposal. A very different story emerged from the study on the lupus prone female SNF1 mice. Following d3tx, an accelerated autoAb response was also noted but this was associated with significant reduction in fatal GN. Analysis of their serum and glomerular autoAb isotypes revealed an autoimmune response with a strong Th2-bias. In Aim 2, we will test the hypothesis that d3tx of the SNF1 mice retains the nonpathogenic neonatal Th2 responsiveness. At the same time, they had a reduced Ag specific Th1 response that is required for pathogenic autoAb production and for lupus GN. Finally, we will seek an explanation for the different responses to d3tx between the two lupus prone mice. Accordingly, we will determine the outcome of CD4+CD25+ T cell depletion by methods other than d3tx in SNF1 mice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: REGULATORY T CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Bagavant, Harini; Pathology; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2003; Project Start 01-MAR-2003; Project End 31-JAN-2008 Summary: (provided by applicant): My graduate thesis was in the field of Reproductive Immunology specifically, the study of immune responses and ovarian function in primates immunized with egg protein antigens. My post-doctoral work was on the development of Contraceptive Vaccine in primates in Dr. Tung's laboratory, a part of the Center for Recombinant Gamete Contraceptive Vaccinogens at the University of Virginia (UVA). I had to take a break in my research for over one year due to family health problems. I returned to Dr. Tung's lab in July 2000 to complete and publish my post-doctoral work. At this time, Dr. Tung's lab was studying the loss of CD4+CD25+ regulatory T cell function induced by neonatal thymectomy between days 1-4 after birth (d3tx) as a cause of organ specific autoimmune disease. In addition, Drs. Fu, Tung and McDuffie had established a Specialized Center of Research for Systemic Lupus Erythematosus (SLE) at UVA. I got interested in the mechanisms of immunoregulation and initiated a project to study the role of regulatory T cells in SLE, which is a systemic autoimmune disease affecting multiple organs and characterized by the presence of circulating autoantibodies (Aab) to nuclear and cytoplasmic antigens. The project has given us exciting leads for future study and form the basis of the present proposal. We will use the d3tx model of CD4+CD25+ regulatory T cell depletion in murine SLE. Two mouse strains studied, SNF1 and NZM2328, spontaneously develop Aab and fatal glomerulonephritis (GN). D3tx in SNF1 mice accelerated Aab but protected from fatal GN. In contrast, d3tx exacerbated Aab and GN in NZM2328 mice compared to sham thymectomized (stx) mice. In addition, reconstitution of d3tx NZM2328 mice with CD25+T cells prevented exacerbation of SLE. These data suggest the hypothesis that CD4+CD25+ regulatory T cells influence induction of SLE in NZM2328 mice. Spontaneous SLE in NZM2328 has a gender bias, predominantly affecting females.
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However, depletion of regulatory T cells by d3tx results in comparable GN in both sexes. This leads to the second hypothesis that regulatory T cell function dictates the gender bias of GN in NZM2328 mice. I had a productive post-doctoral fellowship in the area of Reproductive Immunology, however, additional training in cellular immunology and renal pathology would be essential for my development as an independent scientist in lupus research. This award will help to overcome the lag period in my career induced by my leave of absence and return to a new field of study, and will facilitate my aim of becoming an independent scientist. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: REPEATED DOSES OF LPJ-394 IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Ballou, Stanley; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001 Summary: This is a prospective multi-center double-blind, placebo-controlled Phase IIIII trial in which an investigational new drug, LPJ 394, will be tested for its ability to reduce the production of antibodies to double stranded DNA (anti-dsDNA) and limit flare-ups of active renal disease in patients with lupus nephritis. The subjects will receive medication for 52 weeks, after which there will be a six month period of followup to assess clinical lupus activity, anti-DNA levels and health status. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ROLE OF ARGININE DEFICIENCY IN PATHOGENESIS OF LUPUS Principal Investigator & Institution: Satoh, Minoru; Medicine; University of Florida Gainesville, FL 32611 Timing: Fiscal Year 2003; Project Start 25-SEP-2003; Project End 30-JUN-2005 Summary: (provided by applicant): Immunological characteristics of systemic lupus erythematosus (SLE) include polyclonal B-cell activation and production of specific autoantibodies. However, the autoimmune response is highly restricted to only a few antigens, indicating that there are mechanisms to specifically select target antigens. Autoantibodies to small nuclear ribonucleoproteins (snRNPs: Sm and nRNP) are frequently produced in SLE, MRL mice, and pristane-treated normal mice. In pristanetreated mice, the U1-70K, which has an unusually high arginine content (21%), appears central to the autoimmune process. Arginine is consumed by activated macrophages and arginases in inflammatory sites. In addition, L-canavanine, a non-protein amino acid homologue of L-arginine present in higher plants, may be efficiently incorporated into proteins, producing aberrant proteins that could create cryptic epitopes capable of triggering autoimmunity. In this study, we will investigate why U1 snRNPs are selectively targeted in pristane-induced lupus. We hypothesize that the arginine-rich U1-70K is aberrant in arginine-deficient pristane granulomas, generating cryptic epitopes that initiate autoimmunity. In aim 1, whether arginine is deficient in pristanetreated mice will be examined by amino acids analysis. Arginine metabolism (uptake, consumption, and reconversion) also will be evaluated. In Aim 2, U1-70K synthesized in arginine-deficient conditions and in the presence of L-canavanine in vitro will be evaluated for modifications by immunoprecipitation, amino acid analysis, and mass spectrometry. U1-70K from cells in pristane-treated mice also will be examined. In Aim 3, whether aberrant UlsnRNPs (arginine-deficient, L-canavanine containing) can trigger
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a specific autoimmune response will be investigated by immunizing mice with purified UlsnRNPs or apoptotic cells derived from these conditions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ROLE OF NFAT PROTEINS IN CD154 GENE REGULATION AND LUPUS Principal Investigator & Institution: Cron, Randall Q.; Children's Hospital of Philadelphia 34Th St and Civic Ctr Blvd Philadelphia, PA 19104 Timing: Fiscal Year 2002; Project Start 23-SEP-2002; Project End 30-JUN-2004 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by pathogenic autoantibodies that are dependent on T lymphocyte interactions with autoantibody producing B- lymphocytes. This interaction is reliant on CD40-1igand (CD154), which is expressed on the surface of activated CD4 T cells and which engages its cognate cell surface receptor, CD40, on B cells. Because of its critical and pleiotropic role in the immune system, CD154 expression is normally tightly regulated. Several recent reports have described dysregulated (increased and prolonged levels) expression of CD154 in patients with SLE relative to normal controls. Similar findings have been made in lupus-prone mice, and treatment of these mice with a neutralizing anti-CD154 monoclonal antibody delays and reduces the incidence of glomerulonephritis, a hallmark of SLE. However, similar approaches in humans have not proven efficacious due to unanticipated side effects involving coagulation. The expression of CD154, like that of other T cell cytokine genes, is controlled at the level of gene transcription. We have previously characterized the CD154 transcriptional promoter and demonstrated its dependence on the nuclear factor of activated T cells (NFAT) for activation-induced expression in T cells. Others have shown that CD154 expression is markedly decreased in T cells from mice deficient in NFAT1, the most prominent NFAT family member in T cells of the peripheral immune system. Therefore, we predict that NFAT1 will be critically important for endogenous CD154 expression in peripheral CD4 T cells, and that increased expression of NFATI, or other NFAT family members, contributes to the hyper-expression of CD 154 in patients with SLE. We will test the role of NFAT1 on endogenous CD 154 expression by T cells in vivo under circumstances where NFAT1 is limiting using mice that conditionally express NFATI in T cells only. To examine the role that NFAT1 and other NFAT proteins play in the abnormal expression of CD154 in SLE, primary human CD4 T cells from SLE patients and controls will be evaluated for NFAT RNA and protein levels, as well as for functional NFAT activity using reporter gene assays. Lastly, inhibition of NFAT activity in primary lupus CD4 T cells will be attempted using a newly described NFAT-specific inhibitory peptide. Ultimately, these studies may lead to alternative approaches to inhibiting NFAT activity, and the subsequent over-expression of CD154, in SLE T cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ROLE OF NITRIC OXIDE AND EICOSANOIDS IN LUPUS NEPTHRITIS Principal Investigator & Institution: Oates, James C. Medicine; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2001; Project Start 25-APR-2001; Project End 28-FEB-2006 Summary: Lupus nephritis (LN) leads to renal failure in up to 50% of cases over 5 years despite aggressive immune suppressing therapies that are nonspecific and often
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contribute to significant morbidity and mortality. Further investigations into the pathogenesis of LN are necessary to develop targeted therapies. Elevated nitric oxide (NO), isoprostane, and thromboxane (TXA2) and reduced prostacyclin (PGI2) synthesis occur in LN. TXA2 synthase inhibitors, TXA2 receptor antagonists, and inducible NO synthase (iNOS) inhibitors abrogate murine LN, while the same TXA2 specific therapies improve renal function in human LN. No combines with superoxide to form peroxynitrite (ONOO), which can alter TXA2 and PGI2 metabolism. Thus, one mechanism by which NO may be pathogenic in LN is through the effect of ONOOinduced alternations in intra renal accessioned synthesis. To test this hypothesis, the following specific aims are proposed: 1) Correlate systemic ONOO production in humans with LN with urinary excretion of 8-is PGF2a ( an isoprostane) and the excreted renal metabolites of TXA2, PGI2 and PGE2. Alter ONOO production in murine LN using pharmacologic an genetic means and measure the renal synthesis and urinary excretion of the same eicosanoids, GFR, and nitration of renal TXA2 and PGI2 synthase. 3) Isolate glomeruli from murine models of LN and measure directly the effects of exogenous NO and ONOO donors and iNOS inhibitors on accessioned synthesis and determine if nitration of renal TXA2 and PGI2 synthase occurs. We will measure longitudinal SLE and LN disease activity (in 38 SLG and 38 LN subjects compared to 38 controls) and correlated these with serum 3NT and renal accessioned synthesis. To determine if NO and ONOO exert downstream effects on accessioned metabolism, we will measure the same parameters in 2 models of murine LN in response to iNOS inhibition (with iNOS inhibitors, vitamin E, and MRL/lpr iNOS -/- mice). Glomeruli from murine models of LN will be isolated to determine in vitro the effect of iNOS inhibitors and NO/ONOO- donors on accessioned synthesis. Determining the pathogenic mechanisms of ONOO production as it relates to accessioned synthesis in LN may lead to the use of specific iNOS inhibitors in humans with LN. If we disprove our hypothesis that ONOO induces alternations in eicosanoid synthesis in LN iNOS activity and eicosanoids synthesis may be triggered by common or parallel upstream mechanisms. Alternatively, eicosanoids could regulate NO synthesis in LN as they can in some experimental systems. In either event, future studies would address the use of selective COX-2 or TXA2 synthase inhibitors/receptor blockers, antioxidants, and/or PGI2/PGE2 agonists in conduction with iNOS inhibitors as therapies for LN. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: SIXTH ERYTHEMATOSUS
INT
CONFERENCE
ON
SYSTEMIC
LUPUS
Principal Investigator & Institution: Lockshin, Michael D. Professor; Hospital for Special Surgery 535 E 70Th St New York, NY 10021 Timing: Fiscal Year 2001; Project Start 01-APR-2001; Project End 31-MAR-2002 Summary: Support is requested for travel of participating American investigators to the Sixth International Conference on Systemic Lupus Erythematosus (SLE), to be held March 26-28, 2001, in Barcelona, Spain. This conference will bring together investigators and practitioners from around the world to summarize and reflect on a vast accumulation of new data in the three years since the Fifth Conference. Topics to be highlighted in plenary sessions are: pathogenesis, accelerated atheroma, autoimmune response, antiphospholipid syndrome, autoantibodies as cause, prognosis and future research. Topics featured as 'hot topics' are: prolactin, stem cell transplantation, and nucleosomes. Regular session topics include: epidemiology, lupus in childhood and the elderly, lymphocytes, nephritis, neonatal lupus, animal models, cerebral lupus, druginduced lupus, infection, immunogenetics, malignancy, autoantibodies (clinical),
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autoantibodies (immunology), autoantibodies (pathogenic mechanisms), skin, sex hormones, pregnancy, disease activity/damage, cardiovascular disease, geographical lupus, apoptosis, treatment, cytokines, overlap syndromes, future therapies, and vasculitis. Six sessions will be devoted to free papers and three to poster presentations. Each session will be chaired by a world leader in the topic and will focus on new information in each subfield (including pathogenesis and disease measurement), how this new information influences practice, animal models (including transgenics), and current and future therapies. Women and minorities are represented among session chairs, invited speakers, and organizers. This meeting brings together international investigators who seldom otherwise meet as a group to discuss this important disease. These investigators include basic scientists, clinical scientists, and practitioners from several specialties, including neurology, nephrology, hematology, obstetrics, internal medicine and family practice. The conference provides opportunities for collaborations and reagent exchange. Funding will allow participation of young investigators in this field. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: SLE DISEASE ACTIVITY--PREDICTORS OF MORBIDITY AND HEALTH Principal Investigator & Institution: Tucker, Brian; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: The Lupus Center at JHH is uniquely situated to address clinical research issues through prospective follow-up of a large number of SLE patients. During Dr. Petri's FIRST award, issues addressed included the risk of future thrombosis and coronary artery disease associated with antiphospholipid antibodies, routine cardiovascular risk factors, and prednisone use. The Lupus Cohort has now been refunded by the NIH. The specific aims of the RO-1, although a continuation of the FIRST award work, now include an emphasis on disease activity and disability. Ongoing disease activity exposes patients to the side effects of corticosteroids. Over onehalf of patients sustain permanent organ damage. Determination of predictors of disease activity should help to reduce unneeded corticosteroid treatment and allow institution of earlier treatment when it is needed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: SPECIALIZED CENTER OF RESEARCH IN SYSTEMIC LUPUS ERYTHE* Principal Investigator & Institution: Fu, Shu Man M. Professor; Internal Medicine; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2002; Project Start 01-JUL-1998; Project End 30-JUN-2007 Summary: (provided by applicant): Systemic lupus Erythematosus is a prototypic autoimmune disease affecting multiple systems. This disease causes significant morbidity and mortality. The University of Virginia Specialized Center of Research in Systemic Lupus Erythematosus (UVa SCOR in SLE) was established in July 1998 with funding from the NIAMS. The Center has successfully fostered a multi-discipline approach to study the pathogenesis of this disease. The consortiums between the University of Virginia and the Mayo Clinic and the Johns Hopkins Hospital have strengthened the SCOR. Studies on a new strain of lupus- prone mice NZM2328 have led to the identification of new SLE susceptibility loci, the distinction between acute and
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chronic glomerulonephritis, and most importantly the dissociation of ANA, anti-dsDNA and anti-nucleosome antibody production from chronic glomerulonephritis. Significant information has been obtained regarding the mechanism of epitope spreading in the autoantibody diversification to SLE-related autoantigens. The identification of crossreactive T cells is a major advance in the understanding of this process. Two 3-day thymectomy models to study the role of CD25+ regulatory T cells have been developed. Models for Sjogren's syndrome/autoimmune sialoadenitis have been created. Studies of the DR and DQ transgenic mice provide evidence for the importance of the DR antigens in determining the specificities of the autoantibodies produced. Studies involving serial patient samples provide evidence for fluctuation of autoantibody titers without clear correlation with patients' clinical courses. A collaboration between the University of Virginia and the National Institute of Immunology in India has established a program to study the role of DR in the generation of anti-Sm antibody responses by exploring the ethnic and environmental differences between the India and the U.S. populations. Studies on the role of estrogen receptors in the pathogenesis of SLE show interesting preliminary data. These advances form the basis for the competitive renewal. The competitive renewal application will continue to develop the interdisciplinary approach to study the immunological, genetic and environment factors important in the pathogenesis of SLE. The SCOR renewal application has four projects and three cores. The projects are 1) HLA-D Molecules, T Cell Epitopes and Autoantibody Specificities in SLE; 2) Regulatory and Effector T Cells in SLE; 3) Genetic Control in Lupus-prone NZM2328; and 4) Estrogen Receptors in SLE and the UVa Lupus Cohort. The proposed studies are to provide experimental evidence to support the stated hypothesis that molecular mimics (environmental factors) may initiate an autoimmune response, and the diversification of the autoimmune response with inflammation leads to end-organ damage in appropriate hosts. The three cores are 1) Administrative Core, 2) Cell Sciences and Immunochemistry Core, and 3) Mouse Genetics Core. These cores will serve all the projects and will continue to facilitate interaction among investigators within the UVa SCOR in SLE. The SCOR continues to represent both inter- and intrainstitutional collaboration. It is expected that this interactive approach will continue to provide new insights into the immunological and genetic factors important in SLE. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: SYNDROME
SPORADIC
MECHANISM
OF
THE
ANTIPHOSPHOLIPID
Principal Investigator & Institution: Merrill, Joan T. Member and Head; St. Luke's Roosevelt Hosp Ctr (New York) 1111 Amsterdam Ave New York, NY 10025 Timing: Fiscal Year 2001; Project Start 15-MAR-1999; Project End 31-JUL-2001 Summary: (Adapted from the applicant's abstract)-The APLS is an autoimmune disease of sporadic and unpredictable thrombosis which leads to miscarriages, venous clots, strokes, and sudden death of young people. The only clinical tests available are nonspecific screening assays which detect a lupus anticoagulant or antiphospholipid autoantibodies, but do not predict which patients will develop life- threatening disease. Because of this, patients do not receive anticoagulant therapy until after a serious thrombotic event occurs. Then they are left on potentially dangerous anticoagulant regimens for life, with no assurance that they need it. There is an obvious mandate for better predictive testing and for more specific therapies aimed at the underlying coagulation disorder in this syndrome. A critical aspect of the problem that must be addressed by a disease model is the sporadic nature of the thrombotic events that occur. This application will evaluate a likely model for sporadic thrombosis, transient
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functional deficiency of the anticoagulant protein S. Functional protein S deficiency results from excessive binding by a protein S inhibitor, the C4BP, a dual regulator of the complement and coagulation systems. Functional inhibition of protein S by C4BP has been recently found in patients with APLS by many authors and is temporally related to clotting events. The applicants determined that beta2-GPI, the major target antigen for antiphospholipid autoantibodies, binds protein S and reverses inhibition of protein S by C4BP. A monoclonal anti-beta2-GPI antibody inhibits its interaction with protein S and causes functional protein S deficiency in vitro. The applicants hypothesize that a subset of pathogenic antiphospholipid autoantibodies inhibit interaction between beta2-GPI and protein S, causing intermittent thrombosis during states of high complement activity and excess C4BP, such as are found during lupus flare and pregnancy. The applicants propose to define interactions between protein S, beta2- GPI, and C4BP on a molecular level. Beta2-GPI and C4BP share homologous regions associated with the complement control protein superfamily. These are likely protein S-binding sites and will be targeted in mutagenesis experiments aimed at finding epitopes to which pathogenic antibodies bind. Peptides derived from the protein S-binding region of beta2-GPI might serve as antigens for a more specific clinical assay for pathogenic antiphospholipid autoantibodies or as a basis for a novel therapeutic agent. They will also evaluate the significance of the sex hormone-binding globulin (SHBG) region on protein S. Pregnancy is a risk factor for thrombosis in APLS, and decrease in protein S function is associated with oral contraceptive use. Therefore, possible modulation by sex-hormones of interactions between these proteins will be addressed in this application. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: SRC TYROSINE KINASES AND AUTOIMMUNITY. Principal Investigator & Institution: Defranco, Anthony L. Professor and Chair; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 30-NOV-2006 Description (provided by applicant): The immune system must decide whether to mount an immune response to a particular entity or whether to ignore it. The latter outcome is referred to as immunological tolerance. A great deal has been learned about tolerance over the past decade, but our understanding is still fragmentary, despite its importance for understanding autoimmune disease, managing acceptance of organ transplants, and promoting cancer immunotherapy. We have unexpectedly found that mice deficient in the Lyn tyrosine kinase have B lymphocytes that exhibit elevated responsiveness to antigenic stimulation. In vivo, these mice make high levels of autoantibodies directed at nuclear components such as double-stranded DNA and some of them develop kidney disease. Double mutant mice defective in Lyn and another Srcfamily kinase, Fyn were found to develop a much more severe autoimmune lupus-like kidney disease, with 50% of the animals dying by 7 months of age. We hypothesize that the defect in Lyn makes B cells hyperresponsive and also defective in tolerance induction, resulting in production of antibodies directed at nuclear components released by apoptotic cells. We further hypothesize that the defect in fyn contributes to more rapid disease incidence, possibly by making the kidneys more susceptible to damage resulting from immune complex deposition. In addition, Fyn-deficiency may lead to defects in T cell tolerance. These hypotheses will be tested by three Specific Aims: 1) We shall determine the effects of Lyn and Fyn deficiencies on tolerance to double-stranded (ds) DNA using Ig- transgenic mice developed by Martin Weigert and coworkers. 2) We shall determine the role of helper T cells in IgG anti-dsDNA production by Lyn-/- mice
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and examine the effects of lyn and fyn defects on T cell tolerance, and 3) We shall define the cellular basis of defects leading to autoimmune disease in lyn-/-fyn-/- mice. This will be done by bone marrow transplantation and by adoptive transfer of mature lymphocytes. The proposed studies may lead to significant insights into the nature of the severe autoimmune disease in lyn-/-fyn-/- mice, which in turn may aid in understanding the causes of human autoimmune diseases such as systemic lupus erythematosus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: STUDIES OF SYSTEMIC LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Diamond, Betty A. Professor; Microbiology and Immunology; Yeshiva University 500 W 185Th St New York, NY 10033 Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-MAR-2007 Summary: Four physician-scientists will share model systems, reagents and methodologies to expand an interactive program for preclinical and clinical studies on the pathogenesis and treatment of systemic lupus erythematosis. Drs. Davidson, Diamond, Porcelli and Putterman are practicing rheumatologists with established research programs focused on aspects of the initiation of autoreactivity and the mechanisms of target organ damage in SLE. Three core facilities will provide essential support in administration, flow cytometry, and protein expression and tetramer production. Dr. Diamond has recently developed a novel approach for the identification and analysis of autoreactive B cells at the single cell level using fluorescent tagged tetramers of a peptide mimetope for DNA. She will use the methodology to determine which B cell subsets give rise to anti-DNA antibodies in patients with SLE. DR. Davidson proposes to examine the role of newly identified costimulatory molecules in the NZB/W F1 model of lupus and in the peptide antigen-induced model of lupus pioneered by Drs. Diamond and Putterman. Using novel reagents for costimulatory blockade, she will ask whether the memory of B cell compartment is critical in the progression of disease and in relapse after therapy in murine models of lupus. Dr. Porcelli is interested in CD1 restricted T cells. Based on novel data on the regulation of potentially autoreactive marginal zone B cells, he proposes to study the involvement of the CD1 system in the NZB/W F1 mouse and in the antigen-induced model of SLE mentioned above. Dr. Putterman is studying tissue damage in lupus, focusing on antiDNA antibody-mediated kidney disease. He has recently identified a glomerular antigen recognized by a significant fraction of anti-DNA antibodies; he will explore this antigenic specificity to develop a model for glomerular damage. Together these studies new information and new methodologies to address critical aspects of disease initiation and progression, and tissue damage in lupus. The strategy is to apply information obtained from murine studies to studies of the human disease; thus, the program includes several studies in mice and one study addressing the phenotypic analysis of human lupus B cells drawing on methodologies and hypotheses derived from murine studies. The goal is to develop "designer" therapies tailored to particular subsets of patients with particular patterns of disease induction and tissue injury. This effort is based on already existing interactions among the investigators and will be substantially strengthened by the resources provided by the Program Project. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: STUDY OF DEXAMETHASONE IN NEONATAL LUPUS CHB Principal Investigator & Institution: Buyon, Jill P. Professor; Hospital for Joint Diseases Ortho Inst Orthopaedic Institute New York, NY 10003
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Timing: Fiscal Year 2001; Project Start 30-SEP-2000; Project End 31-AUG-2005 Summary: (Taken from the application): Neonatal lupus is currently considered a model of passively acquired autoimmunity whereby immune abnormalities in the mother (who may have systemic lupus erythematosus, Sjogrens syndrome, or be entirely asymptomatic) lead to the production of antibodies to the SSA/Ro-SSB/La ribonucleoproteins which cross the placenta and presumably injure the developing fetus. The most serious manifestation is cardiac injury which includes varying degrees of atrioventricular (AV) block, most often third degree, and myocarditis. The mortality approaches 20% and the majority of children require lifelong pacing. This application focuses both on the clinical approach to diagnosed congenital heart block (CHB) [interventional study] and the search for an early echocardiographic marker of injury [observational study]. In Specific Aim 1, a randomized double-blind placebo-controlled trial will examine the effect of daily oral dexamethasone (4 mg) on the outcome of CHB. The rationale rests on the hypothesis that CHB is the consequence of an inflammatory process mediated by maternal anti-Ro/La antibodies. Mothers, irrespective of disease activity but requiring less than 10 mg prednisone/day, identified before 30 weeks of gestation to be carrying a fetus with CHB, will be randomized to receive dexamethasone or placebo (50 patients per arm) for a minimum of 6 weeks. Primary outcome measures include neonatal ventricular rate and ejection fraction at birth, and presence or absence of abnormal fluid collection as assessed on the final fetal echocardiogram before delivery. Secondary outcome measures include the degree of block at birth, gestational age, birth weight, and cardiothoracic ratio. In Specific Aim 2, we will attempt to identify the earliest noninvasive echocardiographic marker of AV nodal dysfunction and/or myocardial injury. At present it is not known whether injury to the AV node progresses through stages with the final outcome being fibrosis of the node and irreversible third degree block. It has been proposed that global inflammation of the working myocardium and surrounding pericardium may precede or accompany AV nodal injury. One hundred pregnant women considered at high risk for having a child with CHB, as defined by presence of Ro/La antibodies documented prior to pregnancy (regardless of whether or not they have had a previous child with neonatal lupus), will be followed by weekly echocardiograms from 16 weeks of gestation, with special attention to prolongation of the mechanical PR interval and/or development of myocardial dysfunction. Mothers whose fetuses develop 1st, 2nd or 3rd degree block will then be randomized to receive either dexamethasone or placebo as part of Specific Aim 1. The importance of this second aim is twofold: first, it will identify whether the subclinical incidence of tissue injury exceeds overt injury manifest as advanced AV dissociation; and second, it will provide the best chance for reversibility of block given identification of early lesions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: T PNEUMONITIS
CELL
ADHESION
MOLECULES
IN
MURINE
LUPUS
Principal Investigator & Institution: Curtis, Jeffrey L. Professor of Internal Medicine; Internal Medicine; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 01-DEC-1998; Project End 30-NOV-2002 Summary: Pulmonary involvement in systemic lupus erythematosus (SLE) is common, often incapacitating, and occasionally lethal. Current therapies are less effective for pulmonary involvement than for other organ systems. To define the molecular pathogenesis of SLE, we have developed a murine model system that depends on
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adoptive transfer of syngeneic activated CD4+ T cells treated with DNA methyltransferase (DNA MTase) inhibitors such as procainamide (Pca). Normal AKR mice receiving cells of the cloned T cell line D10 that have been treated with Pca (D10Pca) develop high-titer anti-DNA autoantibodies, nephritis, liver disease resembling biliary cirrhosis, and lymphoid interstitial pneumonitis (LIP). Splenectomy abrogates disease activity in all organs except the lungs, indicating that pathology in this organ does not depend of autoantibody production. Treatment with DNA MTase inhibitors increases expression of the Beta2 integrin LFA-1 (CD11a/CD18). T cells transfected with CD18 are also autoreactive and induce lupus on transfer to syngeneic mice. Lymphocyte DNA hypo-methylation and LFA-1 over-expression is also seen in patients with active lupus. These findings imply that T cell overexpression of LFA-1 is sufficient to initiate SLE, and that the T cell-dependent lung lesion may be the earliest stage in the process. This proposal will examine the molecular mechanisms involved in lung pathology in this model system, utilizing a variety of techniques and lessons learned from the study of other models of lung lymphocyte trafficking. Central Hypothesis: Increased LFA-1 expression by autoreactive T cells mediates adhesion both to lung endothelial cells and to lung antigen-presenting cells (APCs), especially macrophages (Mphis) (resulting in apoptosis and release of autoantigens). These interactions initiate recruitment of other activated T cells to the lung via VLA-4/VCAM and selectin-dependent interactions, inducing LIP. Specific Aim 1: To verify the lung localization of D10Pca is required to induce drug-induced murine LIP. Specific Aim 2: To determine the adhesive interactions mediating lung localization of D10Pca and other lung lymphocytes during development of LIP. Specific Aim 3: To determine whether inhibiting pulmonary retention of D10Pca via monoclonal antibody (mAb) treatment prevents development of LIP. Our long-term goal is to develop effective therapies to treat established SLE based on anti-adhesive strategies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: T CELL COST IMMULATION ON MURINE LUPUS Principal Investigator & Institution: Daikh, David I. Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 01-SEP-1997; Project End 31-AUG-2003 Summary: Systemic Lupus Erythematosis is a systemic autoimmune disease of unknown cause characterized by the production of antibodies directed against a variety of self antigens. A spontaneous form of lupus which closely parallels the human disease also occurs in the lupus-prone B/W mouse. Production of autoantibodies in humans and the B/W mouse is dependent on activated CD4+ helper T cells. Activation of these T-cells requires interactions between the T-cell antigen receptor and antigen presented by an antigen-presenting cell, as well as a second signal which can be provided by interactions between B7 molecules on antigen- presenting cells and the CD28 molecule on the T-cell. Blockade of the first signal by antibodies against the CD4 molecule is effective in preventing or reversing lupus in the B/W mouse, an observation which in part led to therapeutic trials with anti-CD4 antibodies in humans with autoimmune disease. Blockade of the second activation signal by a soluble molecule called aLA4Ig has recently been shown to dramatically slow the progression of lupus in B/W mice as well as treat established disease. The mechanism of this effect, however, is unknown. T cell costimulation can also be provided by interactions between CD4O and its cognate ligand gp39, but the role of these molecules in autoimmunity is also unknown. The objective of the current proposal is to further define the mechanism by which interruption of T-cell costimulation can block the development of autoimmunity. This
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objective encompasses five specific aims: Specific Aim 1. To determine the relative contributions of B7-1 and B7-2 to the development of autoimmunity in lupus-prone NZB/NZW (B/W) mice. Specific Aim 2. To determine if blockade of CD28 and/or CTLA4 early in life can induce tolerance to autoantigens. Specific Aim 3. To determine the effects of costimulation blockade on T cell cytokine production in lupus-prone mice Specific Aim 4. To determine the role of Th1 and Th2 cells in the development and maintenance of murine lupus. Specific Aim 5. To determine whether interruption of multiple costimulation signals can have a synergistic effect on murine lupus. These studies will not only define the critical costimulation signal(s) necessary for the development of murine lupus, but should point to new, more specific therapeutic approaches in humans. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: T CELLS AND THE INDUCTION OF LUPUS Principal Investigator & Institution: Richardson, Bruce C. Professor; Internal Medicine; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, MI 481091274 Timing: Fiscal Year 2001; Project Start 30-SEP-1993; Project End 30-JUN-2002 Summary: (Adapted from the applicant's abstract)-Decreased T cell DNA methyltransferase (MTase) activity has been causally linked to human lupus. The principal investigator's group has reported that inhibiting DNA MTase in dividing T cells results in DNA hypomethylation, LFA-1 overexpression, and autoreactivity, and that adoptive transfer of the autoreactive cells is sufficient to cause a lupus-like disease. They have also shown that some agent which induce lupus, including procainamide, hydralazine, and UV light, inhibit T cell DNA methylation, increase LFA-1 expression, and induce autoreactivity, and they have used the adoptive transfer model to demonstrate a mechanism by which these agents can trigger a lupus-like disease. They and others have shown that T cells from patients with active lupus have diminished levels of DNA MTase, hypomethylated DNA, and overexpress LFA-1 on an autoreactive T cell subset, indicating that a similar mechanism could contribute to idiopathic SLE Together, these results suggest that abnormally decreased T cell DNA MTase enzyme activity may directly contribute to the development of drug- induced and idiopathic lupus by modifying T cell gene expression. The mechanisms regulating human DNA MTase are unknown. This group has established that levels of DNA MTase normally increase following T cell stimulation. They have also obtained evidence that human DNA MTase levels may be regulated through the ras-MAPK signaling pathway. In other studies the principal investigator found that the mitogen-stimulated increase in DNA MTase is impaired in T cells from patients with active lupus, and the Ha-ras mRNA levels and ras-MAPK signaling are diminished in these T cells, suggesting a mechanism for the decreased DNA MTas response. Finally, the principal investigator has evidence for multiple isoforms of human DNA MTase, the function and expression of which are unknown. The principal investigator hypothesizes that decreases in the levels of DNA MTase, due to decreased Ha-ras expression, may contribute to the development of lupus. The principal investigator also hypothesizes that the different isoform of DNA MTase serve distinct roles within the cell. The specific aims are to: 1 Determine the role of the ras-MAPK signaling pathway in the regulation and function of human T cell DNA MTase; 2) determine the pathologic significance of decreased Ha-ras pathway signaling using a novel model of drug-induced lupus; 3) determine the significance of the decreased Ha-ras levels observed in T cells from patients with active lupus, and 4) characterize expression of DNA MTase isoforms. The principal
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investigator anticipates that these studies will clarify mechanisms contributing to the development of human lupus. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS Principal Investigator & Institution: Kammer, Gary M. Professor; Internal Medicine; Wake Forest University Health Sciences Winston-Salem, NC 27157 Timing: Fiscal Year 2001; Project Start 01-SEP-1994; Project End 31-AUG-2002 Summary: (Adapted from investigator's Abstract): An important defect in SLE is T cell dysfunction, manifested as reduced proliferation to mitogens or antigens, reduced release of IL-2 and impaired generation of suppressor T cells. In this proposal, the Principal Investigator builds on his carefully established observation that the activity of the adenyl cyclase/cyclic AMP/protein kinase A isozyme phosphotransferase signal transduction pathway (specifically of the R1 subunit of PKA-1), is defective in 88% of people with SLE. PKA is the only cytosolic pathway for cAMP-mediated phosphorylation of multiple membrane and cytosolic proteins. The Principal Investigator has isolated the abnormality to the R1 subunit of the holoenzyme, and has shown that quantities of R1 are normal in SLE patients, but activity is reduced. His hypotheses are 1) that the defect is due to point mutations in R1a and/or R1b isoforms comprising the regulatory subunits of PKA-1, and 2) that these defects are inherited. A large body of previous work and preliminary data are presented. Specific Aims are: 1. To study a cohort of unrelated people with SLE to estimate the prevalence of deficient PKA-1 activity in DLE, SCLE and SLE, determine whether activity of enzyme relates to activity of disease, whether it differs in different races and sexes, and if it is a heritable trait in the families of these patients; 2. To identify mutations of the R1a or R1b subunit by SSCP and sequencing of cDNA from cloned PCR products and genomic DNA. Preliminary data show 2 examples of such mutations - both are T to A with F replacing I - one in R1a and the other in R1b, both located in regions likely to impair folding of the A and B subunits which permits full cAMP binding. Screening for mutations will use gel shift in SSCP or competitive PCR; bands of interest will be sequenced. 3. To prepare mutant and wild-type recombinant R1a and/or R1b subunit proteins from lupus subjects to quantify isozyme kinetics and phosphotransferase activities to determine if and how the mutations affect function. 4. To examine transcriptional and posttranscriptional regulation of R1a and R1b subunit mRNA in T cells from active and inactive SLE vs controls by quantifying the mRNA content, cytoplasmic mRNA turnover and amounts of each isoform protein. Transcription will be inhibited at various stages by actinomycin D and dichloro-b-D-ribouranosylbenzimidazole). Amounts will be measured by immunoblotting of 35S-labeled material. 5. To screen lupus families to determine whether the mutation is passed through lupus families and is associated with a PKA-1 isozyme deficiency. The Principal Investigator will study 14 families with 3 generations available - beginning with a lupus proband with a mutation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: TARGETED COMPLEMENT INHIBITORS Principal Investigator & Institution: Tomlinson, Stephen; Associate Professor; Microbiology and Immunology; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2001; Project Start 01-MAY-1994; Project End 31-AUG-2004
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Summary: (Adapted from the Investigator's abstract): The broad long term objective of the proposed research is to develop novel complement inhibitory proteins for the effective and safe treatment of autoimmune and inflammatory disease. The specific aims of this application are to: 1. Prepare and characterize complement inhibitory proteins that can be targeted to specific tissues or sites of disease. Different classes of complement inhibitor will be fused to a targeting component consisting of either an antibody variable region or a receptor ligand. 2. Evaluate the therapeutic potential of targeted complement inhibitors in a murine model of spontaneous systemic lupus erythematosus (SLE). 3. Determine the effect of systemic complement inhibition on host ability to control an infection during chronic disease. Soluble CD59 or DAF functional units will be fused to either antibody fragments containing a variable region specific for a cell surface molecule, or to a soluble ligand recognizing selectins. Selectins are cell adhesion molecules that are expressed on inflamed endothelium and are involved in leukocyte recruitment. Various construct designs will be evaluated in vitro for selective targeting to a cell surface, and for their functional effectiveness at inhibiting complement and complement-dependent inflammatory processes. Complement inhibitory proteins based on antibody fusions that are effective in vitro will be tested in vivo in a murine model. A characterized murine anti-DNA antibody that has been shown to target to the kidney will be used for the preparation of IgG-complement inhibitor fusion proteins. Anti-DNA antibodies of the kind that will be used are relatively specific for lupus and have a pathogenic role in disease. Studies will determine the relative effects of targeted vs. untargeted (systemic) complement inhibitors, as well as the effect of selectively blocking the generation of different complement activation products. In addition to therapeutic evaluation, the proposed studies will allow testing of certain hypotheses relating to complement-associated disease mechanisms and the clinical use of complement inhibitors. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: THE GENETIC EPIDEMIOLOGY OF RA AND SLE Principal Investigator & Institution: Criswell, Lindsey A. Associate Professor; Medicine; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 27-SEP-2001; Project End 31-MAY-2006 Summary: (Taken from the applicant's abstract): Dr. Lindsey Ann Criswell has demonstrated a strong commitment to patient-oriented research since the beginning of her rheumatology fellowship training at the University of California, San Francisco (UCSF). Dr. Criswell's track record in peer-reviewed publications and her ability to successfully compete for NIH and other sources of research funding provide compelling evidence of her ability to conduct high quality patient-oriented research. Furthermore, Dr. Criswell has successfully mentored a large number of junior clinical investigators who are now establishing their own independent patient-oriented research programs. During the past six years, Dr. Criswell's research program has focused on the genetic epidemiology of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This work has led to several important findings, including the association of tumor necrosis factor polymorphism with RA severity through an interaction with the HLADRB 1 shared epitope. Due to the rapid pace of advances in analytic methods for genetically complex diseases such as RA and SLE, Dr. Criswell must continue to devote a substantial amount of time and effort to mastering these rapidly evolving methods. The K24 award offers a unique opportunity to provide support for this essential ongoing training. Dr. Criswell's immediate research goals are to successfully complete the research that is now funded and underway within her research unit. This application
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describes 3 projects that utilize state of the art methods to identify genetic and nongenetic risk factors for these complex diseases. The expertise of the study investigators in the areas of rheumatology, genetics and statistics, in conjunction with the progress of these studies to date, ensures a high likelihood of success for this work. The research environment at UCSF provides an outstanding environment in which Dr. Criswell can continue to develop as a leader in patient-oriented clinical research and as a mentor to junior clinical investigators. As a result of Dr. Criswell's role as the Associate Director of the UCSF General Clinical Research Center (GCRC), she is ideally positioned to make a major impact at UCSF as a mentor for young clinical investigators. Dr. Criswell has precisely the skills and career goals for which this program was developed, and the resources of the K24 award would greatly enhance Dr. Criswell's ability to accomplish her goals as a patient-oriented clinical researcher and mentor through the provision of essential resources and relief of patient care and other activities. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: THE MANNOSE-BINDING LECTIN AND AUTOIMMUNITY Principal Investigator & Institution: Ezekowitz, Alan B. Professor & Chief of Pediatrics Services; Cbr Institute for Biomedical Research 800 Huntington Ave Boston, MA 02115 Timing: Fiscal Year 2002; Project Start 01-JUL-2002; Project End 30-JUN-2007 Summary: The predominant interest of our laboratory is to study the innate immune system in vertebrates and insects. Our focus is to define the structure and function of pattern recognition molecules that appear to selectively recognize the patterns of oligosaccharides that decorate microorganisms from self-glycoproteins. The mannosebinding lectin (MBL) is one such molecule which functions like an ante-antibody and is regarded as a prototypic mammalian pattern recognition molecule. MBL appears to play a role in first line host defense in the lag period that is required to generate a long lasting adaptive immune response. A hallmark of innate immunity is that it is now recognized as a necessary antecedent for the development of an adaptive immune response. Little or no attention has been paid as to whether MBL is able to interact and prime the development of adaptive immunity, in particular systemic lupus erythematosis (SLE), we believe that MBL plays a role B cell homeostasis. The goal of this proposal is to explore this question by making use of unique MBL null mou7se models that we have generated in our laboratory in conjunction with other known lupus prone animal models. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: THE ROLE OF COMPLEMENT IN PROTECTION AGAINST SLE Principal Investigator & Institution: Carroll, Michael C. Professor; Cbr Institute for Biomedical Research 800 Huntington Ave Boston, MA 02115 Timing: Fiscal Year 2002; Project Start 01-JUL-2002; Project End 30-JUN-2007 Summary: Systemic lupus erythematosis (SLE) is a relatively common and incurable autoimmune disorder that affects women 10 fold more than men. The etiology is unknown, but the disease is characterized by autoantibodies specific for nuclear antigens, such as dsDNA histones and ribonuclear proteins. Genetic studies support a critical protective role for the innate immune system, as humans deficient in complement components C1q or C4 almost always develop this disease. To dissect the importance of self-antigen and the complement system in negative regulation of selfreactive B cells, mice bearing a targeted deletion in complement proteins C1q, C4 or the receptor C42 will be examined alone, or in combination with immunoglobulin (Ig)
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transgenes (tg), in three interrelated aims described below. The first aim will test the hypothesis that self-antigen alters the development of B-lymphocytes. Two sub-aims will explore the questions: (i) What is the relative importance of antigen concentration and affinity on B cell receptor (BCR) editing and apoptosis? (ii) Does receptor editing represent re- initiation of RAG gene expression (induction), or is it a dye product of selection or continued expression of the rearrangement complex? The second aim will test the hypothesis that complement C1q, C4 and CD21/CD35 participate in negative selection of developing B cells. The two sub-aims will address: (i) Does deficiency in C1q, C4 or CD21/CD35 alter negative selection? (i) Is CD21/CD35 expression on stroma or B cells important in negative selection? The third aim will test the hypothesis that complement protects from autoimmunity by enhancing clearance of apoptotic material. The two sub-aims will address the questions: (ii) Does expression of C4 in adult C4-/mice restore clearance and protect against auto-antibodies? The overall significance of this project is that it directly addresses a fundamental question regarding the role of complement in protection against autoimmune disease. The results should distinguish whether complement protects by enhancing negative selection of self-reactive Blymphocytes; or if its importance is more in sequestration and clearance of lupus antigens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: THE ROLE OF FC GAMMA RECEPTORIIB IN B CELL TOLERANCE Principal Investigator & Institution: Mcgaha, Tracy L. Lab/Molecular Genetics; Rockefeller University New York, NY 100216399 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2006 Summary: (provided by the applicant): Fc gamma receptor IIB is an important regulator of immuno-reactivity. Genetic disruption of the Fc gamma receptor results in a fulminate, lupus-like disease in the C57BL/6 murine background. The goal of this project is to more closely examine the lupus which arises in these animals to determine the effect that Fc gamma receptor IIB disruption has on the B cell repertoire and on silencing of auto-reactive B cells in the periphery. In addition, these studies investigate the nature of chromatin tolerance in these animals (i.e. is the loss of tolerance reversible or is it a permanent physiological change) by reestablishment of the Fc gamma receptor IIB regulatory pathway in animals with established autoimmunity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: THROMBOCYTOPENIA AND THE GENETICS OF SEVERE LUPUS Principal Investigator & Institution: Scofield, R Hal. Associate Professor; Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Timing: Fiscal Year 2002; Project Start 05-SEP-2002; Project End 31-AUG-2007 Summary: (provided by applicant): Systemic lupus erythematosus (SLE) is a complicated autoimmune disease with a definite genetic predisposition. However, the exploration of SLE genetics is in its infancy. SLE is an extremely complicated clinical illness with a wide range of manifestations. Thus, far the genome scans have been performed using a general SLE phenotype. Use of stratification of a disease population has proven very useful in other diseases. For example, the BCR1 gene was found only when early onset breast cancer was considered among families that also had ovarian cancer. We hypothesize (and the preliminary data demonstrate) that the, as yet unexplored clinical phenotypes of SLE, will prove extremely valuable in uncovering the genetics of SLE. Thrombocytopenia predicts severe disease and death in SLE, making
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the identification of the related genetic risk factors especially important. We selected the 38 pedigrees that had an SLE patient with thrombocytopenia (platelets