Atlas of Immunology, Third Edition

THIRD EDITION

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THIRD EDITION Julius M. Cruse, B.A., B.S., D.Med.Sc., M.D., Ph.D., Dr. h.c., F.A.A.M., F.R.S.H., F.R.S.M Professor of Pathology, Vice Chair, Department of Pathology Director of Anatomic Pathology Director of Immunopathology and Transplantation Immunology Director of Graduate Studies in Pathology Professor of Medicine and Professor of Microbiology Distinguished Professor of the History of Medicine University of Mississippi Medical Center Jackson, Mississippi

Robert E. Lewis, B.S., M.S., Ph.D., F.R.S.H., F.R.S.M. Professor of Pathology Director of Immunopathology and Transplantation Immunology Department of Pathology University of Mississippi Medical Center Jackson, Mississippi

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-1-4398-0269-4 (Ebook-PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Dedicated in Celebration of the Election of the 16th Chancellor of the University of Mississippi

Whose life and work is guided by selfless dedication to our university, state, and nation. A gifted educator and administrator with a clear vision for the university’s future, Dr. Jones is the quintessential representative of what is and has always been the very best of America. Steadfast always for social and moral justice, he is truly “a man for all seasons.”

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Editorial Staff Jeanann Lovell Suggs, Ph.D. (M.D. candidate) Coordinating Editor

Bradley J. Suggs, M.D. Contributing Editor

Venkat K.R. Mannam, M.B.B.S (Ph.D. candidate) Managing Editor, Co-author of Chapter 18, and Lexicographic Researcher

Will Singleterry (Ph.D. candidate) Contributing Editor

Joshua Ryan Goodin, B.S., B.S.N., R.N., H.T. (ASCP) (M.S.N. candidate) Contributing Editor

G. Reid Bishop, B.S., Ph.D. Formerly Assistant Professor of Chemistry Mississippi College Silicon Graphics Molecular Models Illustrator

Julia C. Peteet Principal Transcriptionist Melissa Howard (M.D. candidate) Principal Transcriptionist

CRC Press Editorial Staff Barbara Ellen Norwitz Executive Editor

Jonathan Pennell Art Director

Pat Roberson Production Coordinator

Pamela Morrell Prepress Manager

Amy Rodriguez Project Editor

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Authors Julius M. Cruse, B.A., B.S., D.Med.Sc., M.D., Ph.D., Dr.h.c., is a Guyton Distin­ guished Professor, Professor of Pathology, Director of Immunopathology and Trans­ plantation Immunology, Director of Anatomic Pathology, ViceChair Department of Pathology, Director of Graduate Studies in Pathology, Professor of Medicine, Professor of Microbiology, and Distinguished Professor of the History of Medicine at the University of Mississippi Medical Center in Jackson. Formerly, Dr. Cruse was Professor of Immunology and of Biology in the University of Mississippi Graduate School. Dr. Cruse graduated in 1958, earning B.A. and B.S. degrees in chemistry with honors from the University of Mississippi. He was a Fulbright Fellow in the University of Graz (Austria) medical faculty, where he wrote a thesis on Russian tickborne encephalitis virus and received a D.Med.Sc. degree summa cum laude in 1960. On his return to the United States, he entered the M.D./Ph.D. program at the University of Tennessee College of Medicine, Memphis, completing his M.D. degree in 1964 and Ph.D. in pathology (immunopathology) in 1966. Dr. Cruse also trained in pathology at the University of Tennessee Center for the Health Sciences, Memphis, where he was a USPHS Postdoctoral Fellow. Dr. Cruse is a member of numerous professional societies, including the American Association of Immunologists (historian), the American Society for Investigative Pathology, the American Society for Histocompatibility and Immunogenetics (historian; member of council, 1997–1999; formerly chairman, Publications Committee [1987–1995]), the Societé Francaise d’Immunologie, the Transplantation Society, and the Society for Experimental Biology and Medicine, among many others. He is a Fellow of the American Academy of Microbiology, a Fellow of the Royal Society of Health (U.K.) and a Fellow of the Royal Society of Medicine (London). He received the Doctor of Divinity, honoris causa, in 1999 from The General Theological Seminary of the Episcopal Church, New York City. Dr. Cruse’s research has centered on transplantation and tumor immunology, autoimmunity, MHC genetics in the pathogenesis of AIDS, and neuroendocrine immune interactions. He has received many research grants during his career, including 12 years of support from the Wilson Research Foundation for neuroendocrine–immune system interactions in patients with spinal cord injuries. He is the author of more than 300 publications in scholarly journals and 45 books,

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and has directed dissertation and thesis research for more than 50 graduate students during his career. He is author or editor of the international journals Immunologic Research, Experimental and Molecular Pathology, and Transgenics. He was chief editor of the journal Pathobiology from 1982 to 1998 and was founder of Immunologic Research, Transgenics, and Pathobiology. Robert E. Lewis, B.A., M.S., Ph.D., is Professor of Pathology and Director of Immunopathology and Transplantation Immunology in the Department of Pathology at the University of Mississippi Center in Jackson. Dr. Lewis received his B.A. and M.S. degrees in microbiology from the University of Mississippi and earned his Ph.D. in pathology (immunopathology) from the University of Mississippi Medical Center. Following specialty postdoctoral training at several medical institutions, Dr. Lewis has risen through the academic ranks from instructor to professor at the University of Mississippi Medical Center. Dr. Lewis is a member of numerous professional societies, including the American Association of Immunologists, the American Society for Investigative Pathology, the Society for Experimental Biology and Medicine, the American Society for Microbiology, the Canadian Society for Immunology, and the American Society for Histocompatibility and Immunogenetics (chairman, Publications Committee; member of Board of Directors), among numerous others. He is a Fellow of the Royal Society of Health of Great Britain and a Fellow of the Royal Society of Medicine (U.K.). Dr. Lewis has been the recipient of a number of research grants in his career, including 12 years of support funded by the Wilson Research Foundation for his research on neuroendocrine–immune system interaction in patients with spinal cord injuries. Dr. Lewis has authored or coauthored more than 150 papers and 150 abstracts and has made numerous scientific presentations at both the national and international levels. In addition to neuroendocrine–immune interactions, his current research includes MHC genetics and disease associations and immunogenetic aspects of AIDS progression. Dr. Lewis is a founder, senior editor, and deputy editor-in-chief of Immunologic Research and Transgenics, and is senior editor and deputy editor-in-chief of Experimental and Molecular Pathology. He was senior editor and deputy editor-in-chief of Pathobiology from 1982 to 1998.

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Preface The splendid reception of the first and second editions of this book convinced both the authors and the publisher to prepare a third edition. The 11 years since this atlas first appeared have witnessed an exponential increase in immunological information emanating from more than 130 journals devoted to the subject. The Journal of Immunology is published twice monthly in an effort to accommodate an ever-increasing demand for immunological information among researchers spanning all fields of biomedicine. Besides the unprecedented advances in knowledge of cell receptors and signal transduction pathways, an avalanche of new information has been gleaned from contemporary research concerning cytokines and chemokines, with special reference to their structure and function. This edition has not only been thoroughly updated but also contains two new chapters, Immunophenotyping of Hematopoietic Malignancies, and Immunomodulators. The Atlas of Immunology is designed to provide a pictorial reference and serve as a primary resource as the most up-to-date and thorough illustrated treatise available in the complex science of immunology. The book contains more than 1300 illustrations and depicts essentially every concept of importance in understanding immunology. It is addressed to immunologists and nonimmunologists alike, including students, researchers, practitioners, and basic biomedical scientists. Use of the book does not require prior expertise. Some of the diagrams illustrate basic concepts, while others are designed for the specialist interested in a more detailed treatment of the subject matter of immunology. The group of illustrations is relatively complete and eliminates the need

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to refer to another source. The subject matter ranges from photographs of historical figures to molecular structures of recently characterized cell receptors, chemokines and cytokines, the major histocompatibility complex molecules, immunoglobulins, hematopoietic cells in leukemia, and molecules of related interest to immunologists. The subject matter is divided into chapters that follow an outline which correlates with standard immunology textbooks. This provides for a logical and sequential presentation and gives the reader ready access to each part of the subject matter as it relates to the other parts of the publication. These descriptive illustrations provide the reader with a concise and thorough understanding of basic immunological concepts that often intersect the purview of other basic and clinical scientific disciplines. A host of new illustrations, such as cellular adhesions molecules, is presented in a manner that facilitates better understanding of their role in intercellular and immune reactions and immunophenotyping of hematopoietic malignancies. Figures that are pertinent to all of the immunological subspecialties, such as transplantation, autoimmunity, immunophysiology, immunopathology, antigen presentation, the T cell receptor, and flow cytometric diagnosis of leukemia and lymphoma, to name a few, may be found in this publication. Those individuals with a need for ready access to a visual image of immunological information will want this book to be readily available on their bookshelf. No other publication provides the breadth or detail of illustrated immunological concepts that may be found in the Atlas of Immunology, Third Edition.

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Acknowledgments Although many individuals have offered help or suggestions in the preparation of this book, several deserve special mention. We are very grateful to Dr. Steven Bigler, Chairman of the Department of Pathology, University of Mississippi Medical Center, Jackson, for his support of our academic endeavors at this institution. Dr. Fredrick H. Shipkey, professor emeritus of pathology at the University of Mississippi Medical Center, provided valuable assistance in selecting and photographing appropriate surgical pathology specimens to illustrate immunological lesions. We express genuine appreciation to Dr. Edwin Eigenbrodt and Dr. Marsha Eigenbrodt for many photomicrographs. We thank Professor Albert Wahba for offering constructive criticism related to a number of the chemical structures, and express genuine appreciation to Dr. Virginia Lockard for providing the electron micrographs that appear in the book. We also thank Dr. Robert Peace for a case of Job’s syndrome, Dr. Ray Shenefelt for the photomicrograph of cytomegalovirus, Dr. Jonathan Fratkin for photomicrographs of eye and muscle pathology, Dr. C.J. Chen for VKH photographs, Dr. Howard Shulman for GVH photographs, Dr.  David DeBauche for providing an illustration of the Philadelphia chromosome, and Dorothy Whitcomb for the history photographs. We thank Dr. G. Reid Bishop, formerly of Mississippi College, for his generous contribution of molecular models of cytokines and other configurations critical to immunology. We wish to thank specially Dr. Richard D. Brunning, Dr. Koichi Ohshima, Dr. Kathryn Foucar, Prof./ Dr. Stefano A. Pileri, Prof./Dr. PhM. Kluin, Dr. Daniel A. Arber, Dr. Peter Isaacson, Dr. Bharat N. Nathwnai, Dr. Estella Matutes, Dr. Roger A. Warnke, Dr. Wing C. (John) Chan, and Dr. Rein Willemze for providing us with histopathology trinkets from their personal collections. They are individually acknowledged in the credits for their contributions.

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We express genuine appreciation to Dr. Venkat K.R. Mannam, Dr. Jeanann Suggs, and Ms. Julia Peteet for their dedicated efforts in helping us to complete this publication in a timely manner and making valuable editorial contributions. We also appreciate the constructive criticisms of Patsy Foley, B.S., M.T., C.H.T., C.H.S.; Susan Touchstone, B.S., M.T., S.B.B., C.H.T., C.H.S.; Jay Holliday, B.S., M.T., C.H.T.;  Josh Hammons, B.S., M.T., C.H.T.; Heather Jones, B.S., M.T.; Wendy Thomson, C.H.S.; Angie Bond, B.S., M.T., C.H.T.; and Maxine Crawford, B.S., C.H.T. We are most grateful to Joanna LaBresh of R&D Corp. for providing the artwork and for sharing with us a number of schematic diagrams of immunological molecules and concepts owned by R&D. It is a pleasure also to express our genuine gratitude to INOVA Diagnostics Inc., especially to Carol Peebles, for permitting us to use their photomicrographs of immunological concepts for the autoimmunity chapter and to Cell Marque Corp., especially to Michael Lacey, M.D., and Ms. Roshel Aghassi, for providing figures for the chapter on diagnostic immunology. We would also like to commend the individuals at Taylor & Francis Group: Barbara Ellen Norwitz, Executive Editor; Pat Roberson, Production Coordinator; Amy Rodriguez, Project Editor; and all members of their staff—for their professionalism and unstinting efforts to bring this book to publication. To these individuals, we offer our grateful appreciation. Special thanks are expressed to Dr. Daniel W. Jones, Chancellor, The University of Mississippi, and formerly Dean of Medicine and Vice Chancellor for Health Affairs, University of Mississippi Medical Center, and to Dr. James E. Keeton, Vice Chancellor for Health Affairs and Dean, School of Medicine, for their unstinting support of our research and academic endeavors.

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Illustration Credits Structural image for the front cover, crystal structure of TNF alpha: x-ray diffraction image of TNF alpha provided through the courtesy of Research Collaboratory for Structural Bioinformatics, 2009. Yang, Z., West Jr., A.P., Bjorkman, P.J., Nat. Struct. Mol. Biol.: The Protein Data Bank. H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H.  Weissing, I.N. Shindyalov, P.E. Bourne: The Protein Data Bank, Nucleic Acids Research 28 pp. 235–242 (2000). Photograph of Dr. Daniel W. Jones on dedication page furnished through the courtesy of Jeanee Shell, Executive Assistant to the Vice Chancellor, University of Mississippi Medical Center, Jackson. Figure 1.6 courtesy of World Book Encyclopedia. Figure 1.12 courtesy of the Mary Evans Picture Library. Figure 1.11 courtesy of the Harvey Cushing/John Hay Whitney Library, Yale University, New Haven, CT. Figure 1.15 courtesy of the Wellcome Library, London. Figure 1.19 courtesy of Dr. Jason Weisfeld. Figure 1.33 Marquardt M. Paul Ehrlich als Mensch und Arbeiter, 1924. Figure 1.34 courtesy of George Mackenzie Collection, American Philosophical Society, Philadelphia, PA. Figure  1.35 Cruse Collection, courtesy of Dr. M.W. Chase. Figure 1.36 Felix Haurowitz, an early proponent of the instructive theory of antibody formation. Figure 1.37 courtesy of The Linus Pauling Institute. Figure 1.40 courtesy of Dr. D.W. Talmage. Figure 1.41 courtesy of Sir Gustav Nossal. Figure 1.42 courtesy of Dr. Susumu Tonegawa. Figure 1.43 courtesy of Dr. Leroy Hood. Figure 1.52 Ann Rev Immunol, Annual Reviews, Inc., Palo Alto, CA. Figure 1.55 courtesy of Dr. Gerald Edelman. Figure  1.71 courtesy of Mount Sinai Medical Center, New York, NY. Figure 1.77 courtesy of Dr. Robert A. Good. Figure  1.75 courtesy of Prof. Nicole Suciu-Foca, Columbia University, New York, NY. Figure 1.93 courtesy of Simon and Schuster Publishers. Figure 1.95 Burgos Cathedral Museum, Burgos, Spain. Figure 1.96 courtesy of The Royal College of Surgeons of England, London. Figure  1.97 Gaspare Tagliacozzi, Du Curtorum Chirurgia Per Institonem, 1597. Figure 1.98 Putti’s Donation of the Instituto Ortopedico Rizzoli, Bologna, Italy.

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Figure 1.106 courtesy of Dana Farber Cancer Institute, Boston, MA. Figure 1.107 courtesy of Dr. Paul Terasaki. Figure 1.109 courtesy of Jerry Berndt. Figure 1.122 courtesy of Mrs. Marka Webb Stewart. Figure 1.123 courtesy of the Bentley Historical Library, University of Michigan. Figure  1.81 compliments of Professor Dr. Rolf Zinke­ rnagel, Institute of Pathology, University of Zurich. Figures  1.22, 1.27, and 1.44 Parish H.J., History of Immunization; (plate 16) E&S Livingstone Ltd., Edinburgh and London, 1965. With permission of Elsevier Publishers. Figures  1.16, 1.17 and 1.18 courtesy of World Health Organization. Figures  1.3, 1.8, 1.51, 1.56, 1.63, 1.72, 1.73, 1.88, and 1.100 courtesy of National Library of Medicine. Figures  1.105 and 1.108 courtesy of UCLA Tissue Typing Laboratory. Figures  1.1, 1.121, and 1.124 courtesy of New York Academy of Medicine. Figure  2.4 adapted from Lachman, P.J., Keith, P.S., Rosen, F.S., and Walport, M.J., Clinical Aspects of Immunology, 5th ed., Vol. 1993, p. 203, Fig. 112. With permission. Figures 2.6, 7.86, and 11.18 redrawn from Lachmann, P.J., Clinical Aspects of Immunology, Blackwell Scientific Publications, Cambridge, MA, 1993. Reprinted by permission of Blackwell Science, Inc. Figures 2.7, 2.8, 2.11, 2.21, 2.54, 4.4, 4.15, 4.18, 7.80, 7.83, 9.24, 9.25, 9.26, 9.29, 9.31, 9.39, 10.6, 10.7, 10.8, 10.12, 10.21, 10.26, 10.28, 10.32, 10.37, 10.39, 10.41, 10.42, 20.8, 20.9, 20.10, 21.11, 21.13, 21.15, 22.4, 22.5, 22.6, 22.7, 23.25, 24.25, 24.29, 24.40, and 24.41 reprinted from Protein Data Bank. Abola, E.E., Bernstein, F.C., Bryant, S.H., Koetzle, T.F., and Weng, J., In: Crystallographic Database: Information Content, Software Systems, Scientific Applications, Allen, F.H., Bergerhoff, G., and Wievers, R., Eds. Data Commission of the International Union of Crystallography, Bonn/ Cambridge/Chester, 1987, pp.  107−132. Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F. Jr., Bride, M.D., Rogers, J.R., Kennard, O., Simanouchi, T., and Tasumi, M., The protein data bank: A computer-based archival file for macromolecular structures, Journal of Molecular Biology, 112:535−542, 1977. These images are part of the Swiss-3D Image Collection. Manuel C. Peitsch, Geneva Biomedical

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Research Institute, Glaxo Wellcome R&D, Geneva, Switzerland. Figures 2.17, 2.41, 2.80, 2.87, 4.19, 4.20, 9.22, 9.23, 9.28, 9.40, and 9.41 redrawn and adapted from Barclay, A.N., Birkeland, M.L., Brown, M.H., Beyers, A.D., Davis, S.J., Somoza, C., and Williams, A.F., The Leucocyte Antigen Facts Book, Academic Press, Orlando, FL, 1993. Figures 2.32, 2.33, 2.43, 2.52, 2.70, 2.71, 2.73, 2.77, 2.78, 2.86, 2.87, 2.92, 2.96, 2.97, 2.100, 2.102, 2.105, 9.7, and 22.33 through 22.47 compliments of Marsha L. Eigenbrodt, MD, MPH, formerly assistant professor, Department of Medicine, and Edwin H. Eigenbrodt, MD, formerly professor of pathology, University of Mississippi Medical Center. Figure 2.35 Marder, O., et al., Effect of interleukin-1α, interleukin-1β, and tumor necrosis factor-α on the intercellular fluorescein fluorescence polarization of human lung fibroblasts, Pathobiology 64(3): 123–130, p 103. Figure 2.46 redrawn from Ravetch, J.V. and Kinet, J.P., Fc receptors, Annual Review of Immunology 9:462, 1991. Figures 2.81, 7.23, 7.25, 7.28, 12.2, 12.18, 12.22, 12.28, and 15.9 redrawn from Murray, P.R., Medical Microbiology, Mosby-Yearbook, St. Louis, MO, 1994. Figure 2.88 redrawn from Tedder, T.F., Structure of the gene encoding the human B lymphocyte differentiation antigen CD20(B1), Journal of Immunology 142(7):2567, 1989. Figures 3.2, 6.19, 21.20, and 22.5 redrawn and adapted from Bellanti, J.A., Immunology II. W.B. Saunders, Philadelphia, PA, 1978. Figure 4.13 reprinted from Janeway, C.A. Jr. and Travers, P., Immunobiology: The Immune System in Health and Disease, 3rd ed., pp. 4−5, 1997. Reprinted by permission of Routledge/Taylor & Francis. Figure 4.22 reprinted with permission from Bjorkmam, P.J., Saper, M.A., Samraoui, B., Bennet, W.A.S., Strominger, J.L., and Wiley, D.C., Structure of the human class I histocompatibility antigen, HLA-A2, Nature, 329:506−512. ©1987 Macmillan Magazines. Figures 6.6, 6.9, and 27.50 redrawn and adapted from Paul, W.E., Fundamental Immunology, 3rd ed., Raven Press, New York, 1993. Figure  7.1 redrawn and adapted from Hunkapiller, T. and Hood, L., Diversity of the immunoglobulin gene superfamily, Advances in Immunology 44:1−62, 1989. Figure 7.11 courtesy of Mike Clark, PhD, Division of Immunology, Cambridge University. Figure  7.12 reprinted with permission from Harris, L.F., Larson, S.E., Hasel, K.W., Day, J., Greenwood, A., and McPherson, A., The three-dimensional structure of an intact monoclonal antibody for

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canine lymphoma, Nature 360(6402):369−372. 1992 Macmillan Magazines. Figures 7.20, 7.22 through 7.25, 7.30 through 7.32, and 6.52 for immunoglobulins redrawn and adapted from Oppenheim, J., Rosenstreich, D.L., and Peter, M., Cellular Function Immunity and Inflammation, Elsevier Science, New York, 1984. Figure 7.35 redrawn from Capra, J.D. and Edmundson, A.B., The antibody combining site, Scientific American 236:50−54, 1977. ©George V. Kelvin/ Scientific American. Figure  7.43 courtesy of Dr. Leon Carayannopoulos, Department of Microbiology, University of Texas, Southwestern Medical School, Dallas. Figure  7.48 adapted from Kang, C. and Kohler, H., Immunoregulation and Autoimmunity, Vol. 3, Cruse, J.M. and Lewis, R.E., Eds., 226 S. Karger, Basel, Switzerland, 1986. Figure 7.66 reprinted with permission from Raghavan, M. et al., Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants, Biochemistry 34:14,469–14,657. ©1995 American Chemical Society; and from Junghans, R.P., Finally, the Brambell receptor (FcRB), Immunologic Research 16:29−57. Figure  7.67 reprinted with permission from Brambel, F.W.R., Hemmings, W.A., and Morris, I.G., A theoretical model of gamma globulin catabolism, Nature 203:1352-1355. ©1987 Macmillan Magazines. Figure 7.68 reprinted from Brambell, F.W.R., The transmission of immunity from mother to young and the catabolism of immunoglobulins, The Lancet, ii:1087−1093, 1966. Figure  7.74 adapted from Haber, E., Quertermous, T., Matsueda, G.R., and Runge, M.S., Innovative approaches to plasminogen activator therapy, Science 243:52–56. ©1989 American Association for the Advancement of Science. Figure 7.84 redrawn from Conrad, D.H., Keegan, A.D., Kalli, K.R., Van Dusen, R., Rao, M., and Levine, A.D., Superinduction of low affinity IgE receptors on murine B lymphocytes by LPS and interleukin-4, Journal of Immunology 141:1091–1097, 1988. Figures  8.7, 8.13, 8.16, 8.18, and 8.21 redrawn and adapted from Eisen, H., Immunology, pp. 371, 373, 385–386, 1974, Lippincott-Raven, New York. Figure  8.23 redrawn and adapted from Kabat, E.A., Structural Concepts in Immunology and Immunochemistry, Holt, Rinehart & Winston, New York, 1968 with permission of Dr. E.A. Kabat. Figure 8.24 illustration drawn by Dr. Reid Bishop. Figure 9.2 reprinted from Atlas of Tumor Pathology, 2nd Series, Fascicle 13, Armed Forces Institute of Pathology. Figures  9.3 and 9.8 reprinted from Atlas of Tumor Pathology, 3rd Series, Fascicle 21, Armed Forces Institute of Pathology. ©

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Figures  9.4, 9.5, and 9.6 reprinted from MullerHermelink, H.K., Marina, M., and Palestra, G., Pathology of thymic epithelial tumors. In: The Human Thymus. Current Topics in Pathology. Muller-Hermelink, H.K., Ed., 75:207−268. 1986. Figure 9.10 reprinted from van Wijingaert, F.P., Kendall, M.D., Schuurmann, H.J., Rademakers, L.H., Kater, L., Heterogeneity of epithelial cells in the human thymus. An ultrastructural study, Cell and Tissue Research 227−237, 1984. Figure  9.13 reprinted from Lo, D., Reilly, C.R., DeKoning, J., Laufer, T.M., and Glimcher, L.H., Thymic stromal cell specialization of the T cell receptor repertoire, Immunologic Research 16(1):3−14, 1997. Figure  9.27 adapted from Werner, K. and Ferrara, J., Immunologic Research 15(1):52, 1996. Figures 9.35 and 17.95 redrawn from Davis, M.M., T cell receptor gene diversity and selection, Annual Review of Biochemistry 59:477, 1990. Figure  10.30 redrawn from Ealick, S.E., Cook, W.J., and Vijay-Kumar, S., Three-dimensional structure of recombinant human interferon-γ, Science 252:698–702. ©1991 American Association for the Advancement of Science. Figure  10.39 adapted from Rifkin, D.B. et al., Thrombosis and Haemostasis 1993:70, 177–179. Figure 11.4 redrawn from Arlaud, G.J., Colomb, M.G., and Gagnon, J., A functional model of the human C1 complex, Immunology Today 8:107–109, 1987. Figures  11.9 and 11.11 redrawn and adapted from Podack, E.R., Molecular mechanisms of cytolysis by complement and cytolytic lymphocytes, Journal of Cellular Biochemistry 30:133–70, 1986. Figure 11.12 redrawn from Rooney, I.A., Oglesby, T.J., and Atkinson, J.P., Complement in human reproduction: Activation and control, Immunologic Research 12(3): 276−294, 1993. Figure 11.19 redrawn from Kinoshita, T., Complement Today. Cruse, J.M. and Lewis, R.E., Eds., 48 S. Karger, Basel Switzerland, 1993. Figure  12.26 courtesy of Dr. Julius M. Cruse Immunology Collection, University of Wisconsin, Madison. Figures  14.2, 14.3, 14.6, and 14.8 through 14.54 furnished courtesy of INOVA Corp. and Ms. Carol Peebles, San Diego, CA. Figures  16.5 and 16.22 adapted from Vengelen-Tyler, V., Ed., Technical Manual, 12th ed., American Association of Blood Banks, Bethesda, MD, pp. 231, 282, 1996. Figures 16.11 and 16.33 adapted from Walker, R.H., Ed., Technical Manual, 11th ed., American Association of Blood Banks, Bethesda, MD, pp. 242, 281, 1993. Figures 16.12, 16.24, and 16.26 reprinted from Daniels, G., Human Blood Groups, Blackwell Science, Oxford, UK, pp. 13, 271, 432, 1995.

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Figure  16.33 reprinted from Clemetson, K.J., Glycoproteins of the platelet plasma membrane, in Platelet Membrane Glycoproteins, George, J.N., Norden, A.T., and Philips, D.R., Eds., Plenum Press, New York, pp. 61−86, 1985. With permission. Figures  17.9 and 17.74 redrawn and adapted from Cotran, R.S., Kumar, V., and Robbins, S.L., Robbins, Pathologic Basis of Disease, B. Saunders, Philadelphia, PA, 1989. Figures  17.28 and 17.92 redrawn and adapted from Valenzuela, R., Bergfeld, W.F., and Deodhar, S.D., Immuno-fluorescent Patterns in Skin Diseases, American Society of Clinical Pathologists Press, Chicago, IL, 1984. With permission of the ASCP Press. Figures  17.32 and 17.33 redrawn and adapted from Edmundson, A.B., Ely, K.R., Abola, E.E., Schiffer, M., and Panagotopoulos, N., Rotational allomerism and divergent evolution of domains in immunoglobulin light chains, Biochemistry 14:3953–3961. ©1975 American Chemical Society. Figure  17.51 redrawn and adapted from Stites, D.P., Basic and Clinical Immunology, Appleton & Lange, East Norwalk, CT, 1991. Figure  17.117 redrawn and adapted from Dieppe, P.A., Bacon, P.A., Bamji, A.N., and Watt, I., Atlas of Clinical Rheumatology, Lea & Febiger, Philadelphia, PA, 1986. Figure 18.2, 18.4 through 18.7, 18.9, 18.11, 18.12, 18.15 through 18.20, 18.22, 18.25, 18.26, 18.28, and 18.29 courtesy of Richard D. Brunning, MD, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis. Figures  18.32 through 18.39 courtesy of Kathryn Foucar, MD, Department of Pathology UNMHSC, Albuquerque, NM. Figures 18.42 through 18.49, 18.51, and 18.52 courtesy of Dr. Peter Isaacson, Department of Histopathology, Royal Free and University College Medical School, University College London, Hospital Trusts, London, UK. Figures  18.54 through 18.56 courtesy of Dr. Bharat N. Nathwnai, Department of Hematopathology, University of Southern California, Keck School of Medicine, Los Angeles, CA. Figures 18.03 and 18.14 courtesy of Daniel A. Arber, MD, Professor of Pathology, Director of Anatomic Pathology and Clinical Laboratory Services, Associate Chair for Clinical Services, Department of Pathology, Stanford University Medical Center, Stanford, CA. Figure  18.23 courtesy of Dr. Estella Matutes, Reader/ Consultant Haematologist, Section of HaematoOncology, Institute of Cancer Research, London, UK. Figure  18.59 courtesy of Dr. Roger A. Warnke, Department of Pathology, Stanford University School of Medicine, Stanford, CA.

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Figures  18.61, 18.76, and 18.77 courtesy of Prof. Dr. Stefano A. Pileri, Full Professor of Pathology, Director of the Haematopathology Unit, Bologna University School of Medicine, St. Orsola Hospital, Massarenti, Bologna, Italy. Figures  18.62 and 18.63 courtesy of Prof. Dr. PhM. Kluin, head of the Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands. Figure  18.66 courtesy of Wing C. (John) Chan, MD, Amelia and Austin Vickery, Professor of Pathology, Co-Director, Center for Lymphoma and Leukemia Research, University of Nebraska Medical Center, Omaha, NE. Figures  18.68 through 18.73 courtesy of Dr. Koichi Ohshima, Department of Pathology, School of Medicine, Kurume University, Asahimati, Kurume, Japan. Figure  18.75 courtesy of Dr. Rein Willemze, Depart­ ment of Dermatology, Leiden University Medical Center, Leiden, The Netherlands. Figure 22.17 adapted from Splits, Associated Antigens and Inclusions, PEL-FREEZE Clinical Systems, Brown Deer, WI, 1992. Figures 22.48 through 22.62 compliments of Howard M. Shulman, MD, Professor of Pathology, University of Washington, Member Fred Hutchinson Cancer Research Center, Seattle. Figures 23.1 through 23.3 reprinted and adapted from Monoclonal Antiadhesion Molecules, Nov. 8, 1994. Seikagaku Corp. Figure  23.23 adapted from Immunoscintigraphy (nude mouse) with a 131I-labelled monoclonal antibody, photographs prepared by Hachmann, H. and Steinstraesser, A., Radiochemical Laboratory, Hoechst, Frankfort, Germany. With permission.

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Figure  24.17 reproduced from Tilney, L.G. and Portnoy, D.A., Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes, Journal of Cell Biology, 109:1597−1608, 1989. With permission of the Rockefeller University Press. Figures 24.31 reprinted from Seifer, M. and Standring, D.N., Assembly and antigenicity of hepatitis B virus core particles, Intervirology 38:47−62, 1995. Figure 24.32 Hatten, T. et al: RNA- and DNA-binding activities in hepatitis B virus capsid protein: A model for their roles in viral replication. J Virol 1992; 66:5232–5241, published by the American Society for Microbiology. Figure  24.36 courtesy of Farr-Jones, S., University of California at San Francisco. Figure  25.06 Adapted from Immunofacts: Vaccines and Immunologic Drugs. Facts and Comparisons, St. Louis, MO, a Wolters Kluwer Company, 1996. Figures  28.3 and 28.4 redrawn and adapted from Hudson, L. and Hay, F.C., Practical Immunology, Blackwell Scientific Publications, Cambridge, MA, 1989. Figure  28.10 redrawn and adapted from Miller, L.E., Manual of Laboratory Immunology, Lea & Febiger, Malvern, PA, 1991. Figure  28.19 redrawn and adapted from Elek, S.D., Staphyloccocus pyogenes and Its Relation to Disease, E&S Livingstone, Edinburgh and London, 1959. Figures 29.3 through 29.69 furnished courtesy of Cell Marque Corp. and Dr. Michael Lacey, Hot Springs, AR. Cell Marque Corp. and Roshel Aghassi, Rocklin, CA.

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Contents Chapter 1. History of Immunology............................................................................................................................................ 1 Chapter 2. Molecules, Cells, and Tissues of the Immune Response....................................................................................... 77 Chapter 3. Antigens and Immunogens....................................................................................................................................161 Chapter 4. Major Histocompatibility Complex...................................................................................................................... 183 Chapter 5. Antigen Processing and Presentation................................................................................................................... 201 Chapter 6. B Lymphocyte Development and Immunoglobulin Genes.................................................................................. 215 Chapter 7. Immunoglobulin Synthesis, Properties, Structure, and Function........................................................................ 233 Chapter 8. Antigen–Antibody Interactions............................................................................................................................ 285 Chapter 9. The Thymus and T Lymphocytes........................................................................................................................ 307 Chapter 10. Cytokines and Chemokines................................................................................................................................. 343 Chapter 11. The Complement System..................................................................................................................................... 383 Chapter 12. Types I, II, III, and IV Hypersensitivity............................................................................................................... 407 Chapter 13. Immunoregulation and Immunologic Tolerance.................................................................................................. 437 Chapter 14. Autoimmunity...................................................................................................................................................... 447 Chapter 15. Mucosal Immunity............................................................................................................................................... 495 Chapter 16. Immunohematology............................................................................................................................................. 505 Chapter 17. Immunological Diseases and Immunopathology................................................................................................. 525 Chapter 18. Immunophenotyping of Hematopoietic Malignancies......................................................................................... 591

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Chapter 19. Immunodeficiencies: Congenital and Acquired....................................................................................................617 Chapter 20. Acquired Immune Deficiency Syndrome (AIDS)................................................................................................ 637 Chapter 21. Immunomodulators.............................................................................................................................................. 651 Chapter 22. Transplantation Immunology............................................................................................................................... 665 Chapter 23. Tumor Immunology.............................................................................................................................................. 699 Chapter 24. Immunity against Microorganisms.......................................................................................................................717 Chapter 25. Vaccines and Immunization................................................................................................................................. 769 Chapter 26. Therapeutic Immunology..................................................................................................................................... 785 Chapter 27. Comparative Immunology.................................................................................................................................... 799 Chapter 28. Immunological Methods and Molecular Techniques........................................................................................... 815 Chapter 29. Diagnostic Immunohistochemistry...................................................................................................................... 867 Index.......................................................................................................................................................................................... 897

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History of Immunology

The metamorphosis of immunology from a curiosity of medicine associated with vaccination to a modern science focused at the center of basic research in molecular medicine is chronicled in the following pages. The people and events that led to this development are no less fascinating than the subject itself. A very great number of researchers in many diverse areas of medicine and science contributed to building the body of knowledge we now possess. It will be possible to name only a few, but we owe a debt to them all. We are standing on the shoulders of giants, and in remembering their achievements we come to understand better the richness of our inheritance. Resistance against infectious disease agents was the principal concern of bacteriologists and pathologists who established the basis of classical immunology in the latter half of the 19th and early 20th centuries. Following the brilliant investigations by Pasteur on immunization against anthrax and rabies and Koch’s studies on hypersensitivity in tuberculosis, their disciples continued research into the nature of immunity against infectious disease agents. Emil von Behring and Paul Ehrlich developed antitoxins, while Metchnikoff studied phagocytosis and cellular reactions in immunity. Buchner described complement and Bordet discovered complement fixation. With the studies of Landsteiner on immunochemical specificity, the discovery of immunological tolerance by Medawar, the announcement of Burnet’s clonal selection theory of acquired immunity and the elucidation of immunoglobulin structure by Porter and Edelman, modern immunology emerged at the frontier of medical research. This eclectic science, with foundations in clinical medicine, molecular biology and biophysical chemistry, has relevance today for essentially every biomedical discipline, ranging from molecular recognition to organ transplantation. For many years it was considered a part of what has come to be known as microbiology, and in some universities it is still taught in the same department. The discipline has acquired a number of subspecialties as knowledge about the action of the living body has been expanded, so that now research and teaching are progressing in molecular immunology (immunochemistry), immunobiology, immunogenetics, immunopathology, tumor immunology, transplantation, comparative immunology, and the related fields of serology, virology and parasitology. The New Oxford Dictionary gives as the origin of the word immune, the Latin word immunis, from im plus munis, ready to be of service: hence the meaning of exemption from a service or obligation to certain duties or

to taxation. We still use this meaning when we speak of diplomatic immunity. The contemporary medical usage of the word is cited as being found in literature in England starting in 1879 with the meaning of freedom from infection, resistance to poison or to contagion. Immunology, therefore, becomes the study of the biological mechanisms producing nonsusceptibility to the invasive or pathogenic effects of foreign microorganisms or to the toxic effects of antigenic substances, or, more vividly phrased, the capacity to distinguish foreign material from self. The ability of the human body to resist infection and disease must have been recognized in very ancient civilizations. There are references to this recognition in early literature. Thucydides of Athens is quoted as observing that, during a plague epidemic in about 430 BC, those who recovered were used to tend the sick, “as the same man was never attacked twice, never at least fatally.” Procopius is also quoted as recording during the plague of Justinian in AD 541 that those who recovered were not liable to a second attack. King Mithridates of Pontus was a particularly bloodthirsty Middle Eastern ruler who systematically exterminated all whom he thought might usurp his throne and feared that he might be poisoned, so he protected himself with repeated small doses of poison. His name survives in the modern dictionary as the noun Mithridatism for the tolerance produced in the body as a result, or in the universal antidote “theriac” of the medieval formularies.

Smallpox This disease was known in India and China very early. Writings in the sacred canon of Hinduism describe inoculation with pus from the lesions of smallpox using thread soaked in the matter, which was introduced into small incisions in the skin, traditionally done by members of the priestly Brahmin caste. A different custom was reported from possibly the year AD 1000 or thereabouts, in China, where crusts from smallpox vesicles were wrapped in cotton or powdered and introduced into the nostrils of those who were to be protected, thereby producing a mild form of the disease. Muhammed ibn Zakariya, Abu Bakr al-Razi, better known to the world as Rhazes (Figure  1.1), wrote the first modern description of smallpox, which he described as a mild childhood disease caused by excess moisture in the blood, which fermented and escaped from the body through the 1

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Figure 1.1  Rhazes (865–932), Persian philosopher and alchemist who described measles and smallpox as different diseases. He also was a proponent of the theory that immunity is acquired. Rhazes is often cited as the premier physician of Islam.

pustules (Figure  1.2). Smallpox probably spread in Europe between the sixth and ninth centuries, and certainly had become widespread by the Crusades. It was introduced into the Americas by the Spaniards. Epidemics were of frequent occurrence thereafter. Probably fewer than 20% of the population escaped infection. At best, it was disfiguring and could cause blindness. In the 17th century in London, about 10% of all deaths were caused by this one disease. Folk medicine recorded in Europe in the 16th and 17th centuries shows that some sort of inoculation was frequently practiced. Thomas Bartholin, who was physician to Christian V of Denmark and Norway, wrote of the practice in 1675. It was reported from rural France and Wales. The universal folk expression seems to have been “buying the smallpox,” and a widely held superstition was that the inoculation would not be successful without a symbolic transaction, of giving a small coin, fruit, nut or sweetmeat for each “purchase.” Historically, the intracutaneous inoculation of pus from lesions of smallpox victims into healthy, nonimmune subjects to render them immune to smallpox was known as variolation. In China, lesional crusts were ground into a powder and inserted into the recipient’s nostrils. These procedures protected some individuals, but often led to life-threatening smallpox infection in others. The first scholarly observation of this custom may be one made by a Greek physician, Emmanuele Timoni, living in Constantinople, who had taken a medical degree at Oxford. He circulated several copies of a manuscript describing inoculation of smallpox matter and sent a copy to the Royal Society of London in 1713, which was published in the Philosophical Transactions in 1714. Two years later, another report, the first separately published treatise on inoculation,

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Figure 1.2  Rhazes’ famous treatise of the smallpox and measles. Title page from work translated by William Alexander Greenhill.

was sent by Jacob Pylarini, who was the Venetian Consul in Smyrna. It also appeared in the Philosophical Transactions. Perhaps because her actions were publicized by Voltaire, or because of her own literary talents, Lady Mary Wortley Montagu (Figure 1.3) is often cited as the first to report the custom in England. She had suffered a disfiguring case of smallpox as a young woman in England, before accompanying her husband to Constantinople, where he was posted as Ambassador to the Turkish court from 1716 to 1718. She had her young son inoculated and reported enthusiastically on the success of the treatment in a letter to a friend in England in April 1717. On her return to England, she also had her 4-year-old daughter inoculated by Mr. Charles Maitland in April 1721. She was a friend of the Prince and Princess of Wales, according to some reports, and they lent their prestige to popularizing the treatment in England. It is very possible that Sir Hans Sloane, who was editor of the Philosophical Transactions, president of the Royal Society, and physician to George II, had more influence in interesting the Princess of Wales in investigating the worth of inoculation than did Lady Mary. Sir Hans supervised the original experiments which were conducted in England with the inoculation, using

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and famous for their “method” of inoculation from about 1757. Their technique called for a period of preparation by rest and special light diet, and segregation during the course of the disease in one of their nursing facilities, an expensive process. They set up practices in different parts of southern England. Inoculation went through the same slow process of growth and promotion in France.

Figure 1.3  Lady Mary Wortley Montagu (1689–1762), often credited as the first to introduce inoculation as a means of preventing smallpox in England in 1722.

six criminals from Newgate Prison, who were inoculated by Mr. Maitland in 1721. Educated people began to use the term inoculation, from the Latin inoculare, to graft, or variolation (Latin varus, a pimple), the scholarly name for smallpox. In the American colonies, Cotton Mather, who regularly received communications from the Royal Society, read the reports of Timoni and Pylarini in the Philosophical Transactions. He also heard of the practice of inoculation from a slave who told of similar folk customs in southern Tripoli, in Africa. In 1721, when smallpox broke out in Boston, he enlisted the aid of Zabdiel Boylston, a physician, who first inoculated his own 6-year-old son and two slaves. Being successful, he went on to inoculate about 240 Bostonians. In spite of the opposition of other doctors, inoculation continued to be used, particularly when epidemics occurred. There was a storm of controversy about inoculation in England. Disease in general was often viewed as punishment meted out by God, who might see fit to chastise and mold his servants by various tribulations, and to take matters into human hands was seen as sinful. Since the disease produced was true smallpox, it could cause a more virulent attack of the disease than expected. The patient could certainly pass the smallpox on to others in the natural way if not isolated, giving rise to another epidemic. The practice took hold slowly, first among the nobility and wealthy class. The first inoculation hospital was opened in London in 1746, at the time of a serious epidemic. A pronouncement was made by the College of Physicians in 1755, commending the practice. James Kirkpatrick, a Scottish physician who had practiced in the Carolinas, was a successful and vocal promoter of inoculation who represented himself as reviving the practice in England in the 1740s. Robert Sutton and six of his seven sons took advantage of a good opportunity and became wealthy

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Several members of the nobility of Europe were attacked by smallpox. Maria Theresa of Austria survived a case in 1759. Queen Mary of England died in 1694 and the Duke of Gloucester died in 1700. Voltaire, who was in exile in England during the time when inoculation was first introduced, 1726– 1729, and who himself had had smallpox, was a correspondent of Catherine the Great of Russia. He encouraged her to try the new treatment to protect herself. British physicians had been popular in the Russian court since the 16th century. Dr. Thomas Dimsdale of Hertford wrote a popular and successful book, The Present Method of Inoculating for the Smallpox, which must have helped bring him to the attention of the Russian ambassador Mussin-Pushkin, who arranged for him to go to Russia to attend Catherine. Dr.  Dimsdale performed the inoculation successfully, and later also inoculated the Grand Duke, Catherine’s young son, and several others. He was given the title of Baron, a gift of £10 000, and an annuity of £1500 for life. The episode led to a period of westernization and modernization in Russian medicine in addition to popularizing inoculation there. The contemporary literature on inoculation was extensive. The arguments for and against it raged in pamphlets, sermons by the clergy, and communications to the learned societies of England and France. Sir Hans Sloane and Voltaire wrote accounts in the 1730s. Charles La Condamine published a Memoire sur l’inoculation de la petite verole, an excellent summary of the introduction of inoculation in France in 1754. William Woodville, of the Smallpox and Inoculation Hospital of London, wrote a detailed history of the introduction of inoculation into Britain, A History of the Inoculation of the Smallpox in Great Britain, London, 1796. After a thorough discussion of the antiquity of the folk customs, he gave a complete account of the various reports on early trials, the process of experimentation and investigation in Great Britain, including detailed lists of the patients and their various doctors, and reviewed a number of books and pamphlets on the subject. His Volume I ends with a promise to discuss the Suttons and their business venture in a second volume. He is said to have prepared a draft of the second part, but unfortunately abandoned it when Jenner published his Inquiry in 1798. Edward Jenner (1749–1823) (Figure 1.4) is often credited as the founder of immunology for his contribution of the first reliable method of conferring lasting immunity to a major contagious disease. As a young man, he studied under John Hunter and continued to collaborate with him in various natural history studies for many years. He spent most of his

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Figure 1.6  Dr. Edward Jenner inoculating pus from the hand of Sarah Nelmes into the arm of James Phipps. Figure 1.4  Edward Jenner, credited as the founder of immunology.

career as a country doctor in Berkeley, in southern England. It was fairly common knowledge in the country then that an eruptive skin disease of cattle, cowpox, and a similar disease in horses called grease, conferred immunity to smallpox on those who milked and cared for the animals and caught the infection from them. A few other people had also experimented with inducing an experimental infection of cowpox in humans to produce this immunity. However, it is to Jenner, with his careful observation and record of 23 cases and his initiative in describing his work in a small privately printed work, that we must give credit for initiating the technique of vaccination. He vaccinated an 8-year-old boy, James Phipps, with matter taken from the arm of the milkmaid Sarah Nelmes (Figure 1.5), who was suffering from cowpox. After the infection subsided, he inoculated the child with smallpox (Figure 1.6) and found, as he expected, that the inoculation had no effect. He observed the several related viruses causing similar eruptive diseases in animals and termed them collectively viruses, which in time led to the use of this word in its modern sense (Figure 1.7). He observed and described

Figure 1.5  The hand of Sarah Nelmes, a milkmaid infected with cowpox.

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an anaphylactic reaction in one of his patients, and made a correct observation in another case that a concurrent infection with herpes interfered with vaccination. His excellent colored illustrations of the typical lesion were accurate and showed that cowpox was a separate disease. The new procedure was known as vaccination (Latin vacca: cow). Jenner’s discovery was soon adopted by many physicians (Figure 1.8 to Figure 1.12). A Royal Commission was set up

Figure 1.7  Jenner’s famous report of his studies on immunization against smallpox.

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Figure 1.8  Dr. Dimsdale, a pioneer in smallpox vaccination.

to investigate his claim, and ruled in his favor. The British Parliament voted for him to receive two large grants in gratitude. He spent a great portion of his life thereafter in defending and publicizing his new method. He modified his views in the face of experience, for example, coming to the view that revaccination might become necessary and that

Figure 1.10  Dr. William Woodville’s famous history of inoculation against smallpox.

Figure 1.9  Kine pock inoculation: published observations.

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Figure 1.11  Smallpox cartoon, artist unknown, from the Clement C. Fry collection. Yale Medical Library, contributed by Jason S. Zielonka, published in J. Hist. Med. 27:477, 1972. Legend translated: Smallpox-disfigured father says “How shameful that your pretty little children should call my children stupid and should run away, refusing to play with them as friends…” Meanwhile, the children lament: “Father dread, it appears to be your fault that they’re avoiding us. To tell the truth, it looks as though you should have inoculated us against smallpox.”

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Figure 1.13  The collection of lymph from a cow infected with cowpox.

was officially banned in England. Edward Jenner’s introduction of vaccination with cowpox to protect against smallpox rendered variolation obsolete. Figure 1.12  L. Gillray cowpox cartoon, “The cowpox or the wonderful effects of the new inoculation.”

absolutely total protection could not be produced. It was soon learned that the vaccine could be dried and transported from place to place in quills, or using threads soaked in the matter (Figure 1.13). For distant transmittal by ocean voyage, a group of immigrant children were sent and the disease passed serially among them (Figures 1.15 and 1.16). By 1801, the vaccine had been sent overland as far as Basra and thence by ship to India, where many thousands were vaccinated (Figures 1.17 through 1.19). In Massachusetts, Benjamin Waterhouse introduced vaccination to the United States with vaccine sent by Jenner in 1800. President Jefferson vaccinated his family and neighbors with vaccine from Waterhouse, and wrote, encouraging the adoption of the practice elsewhere in the country. By 1840, the practice of inoculating with smallpox

Vaccination had its opponents into the 20th century. It is one of the outstanding achievements of modern medicine that, through the efforts of the World Health Organization, finally eradicated smallpox and continues to exist only in cultures in a few reference laboratories. Even though naturally occurring smallpox was eradicated in 1977, recent terrorist attacks around the world, including the United States, have raised the specter that deadly biological weapons might be crafted from stocks of the virus that remain in known or clandestine laboratories.

The Advent of Classical Immunology Although not very well understood, diseases were perceived as being contagious from very early times. The Bible contains many references to epidemics and the spread of disease, and the quarantine regulations which were enforced in

Vaccination

‘Take’ Antigens

Delayed skin reaction

Lymphatic system

Scar

Venule Lymph node Release of sensitized lymphocytes into circulation

Figure 1.14  Vaccination against smallpox.

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History of Immunology

Figure 1.15  Small eruptions on the face of a smallpox patient (a) and the same child following recovery (b).

an attempt to control them. In Europe, with the rise of trade, public health measures were instituted which were aimed at halting the spread of infection. Because the mechanisms of disease transmission were unknown, many of the regulations were useless and caused unnecessary hardship, which made them very unpopular.

Figure 1.17  Smallpox surveillance.

An early understanding of the concept of specific infections was shown by Girolamo Fracastoro, who in 1546 wrote De Contagione, Contagionis Morbis et Eorum Curatione. He speaks in this work of imperceptible particles which cause disease, some by contact, some through secondary objects

Figure 1.18  Rahima Banu of Bangladesh, the world’s last case of naturally occurring variola major. Photo courtesy of World Health Organization. Rahima’s rash began on October 16, 1975.

Figure 1.16  Smallpox eradication map.

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Figure 1.19  Ali Maow Maalin, the world’s last case of naturally occurring smallpox. Photo courtesy of Dr. Jason Weisfeld. Maalin’s rash began on October 26, 1977, in Merka Town, Somalia.

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such as clothing, and some at a distance. He understood that the “seeds” or “germs” of infection generate and propagate other germs precisely like themselves, and recognized that there was a similarity, not well understood, between putrefaction and contagion. Best known for his poem Syphilis, he also recognized typhus, rabies, diphtheria, and tuberculosis. Leeuwenhoek was the first to see and describe microscopic organisms, although he did not speculate on their origin or their relationship to disease. J.B. von Helmont in 1662 recognized that recovery from disease produced what he described as “balsamic” blood, which had some mysterious power of resisting infection. Spallanzani studied the properties of microscopic living things and the effect of heat in killing them. His demonstrations effectively demolished the theories of spontaneous generation, although there continued to be speculation about the subject up to Pasteur’s day. Jacob Henle (1809–1885) in 1840 wrote a small essay on miasmic, contagious, and miasmic–contagious diseases. He understood that something was introduced into the body which produced disease and which was eliminated from the body upon recovery. He thought it would be necessary to separate the contagious material and culture it, thus showing that it had an independent existence. The French physiologist Francois Magendie (1783–1855) showed that decomposing blood was lethal when injected intravenously and innocuous when taken by mouth. He proved by experiment that gas from decomposing material did not produce disease in laboratory animals. He observed the phenomenon of anaphylaxis in rabbits in 1839, although he had no means of interpreting what had happened. Agnostino Bassi (1773–1856) was the first to prove that microscopic living organisms, fungi, were the cause of a disease of silkworms. Villemin in 1865 demonstrated that tuberculosis was a specific disease entity, transmissible from man to rabbits by inoculation. Davaine observed the rod-shaped organism of anthrax, and transmitted the disease using blood containing the bacteria. In 1870, Klebs observed bacteria in wounds. He postulated that there was a single organism which caused all sorts of pathological conditions. Ferdinand Cohn (1828–1898) attempted a classification of bacteria into four main tribes, laying a foundation for bacteriology. It was one of Cohn’s students, Robert Koch (1843–1910) (Figure 1.20), who brilliantly proved that a specific organism caused a specific disease in an animal. He instituted many of the procedures which are common in bacteriology today. His postulates, arising out of the criteria implicit in Henle’s essay on contagion, became part of the central dogma of bacteriology. The most valuable outgrowth of his extensive research in tuberculosis for immunology was the concept of delayed hypersensitivity, an allergy of infection upon which the principles of skin testing were founded. As valuable as his many discoveries in relation to disease was the training of a large group of students and coworkers, who went on to make some of the major discoveries in early immunology. By common

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Figure 1.20  Robert Koch.

consent the greatest pure bacteriologist, he was awarded the Nobel Prize in 1905 for his research in tuberculosis. Robert Koch (1843–1910), German bacteriologist awarded the Nobel Prize in 1905 for his work on tuberculosis. Koch made many contributions to the field of bacteriology. Along with his postulates for proof of etiology, Koch instituted strict isolation in culture methods in bacteriology. He studied the life cycle of anthrax and discovered both the cholera vibrio and the tubercle bacillus. The Koch phenomenon and Koch– Weeks bacillus both bear his name. In the meantime, in France, Louis Pasteur (1822–1895) (Figure 1.21), who was trained as a chemist, had solved problems of economic importance to France in taking up the study

Figure 1.21  Louis Pasteur.

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of selected silkworm diseases and fermentation in relation to beer and wine. He turned his attention to the study of anthrax and confirmed the findings of Koch, then investigated chicken cholera, whose bacteriological cause was already known. When by chance he allowed some of his cultures to grow old, he found that the chickens treated with this culture did not die, and were subsequently able to withstand injections of virulent cultures. He had discovered that attenuated bacterial cultures could produce immunity. He also observed that the anthrax bacillus would grow but not produce spores at 42°C, and that this resulted in a nonvirulent form of anthrax which would produce immunity in animals. In May of 1881, he carried out a large-scale public demonstration of anthrax immunization of sheep, a goat, and several cattle, with an equal number of control animals, proving the correctness of his deductions. He paid tribute to Jenner by naming the new technique vaccination. He went on to produce a vaccine for rabies by drying the spinal cords of rabbits and using the material to prepare a series of 14 injections of increasing virulence. To attenuate the rabies virus, spinal cords of rabbits that had died from “fixed virus” injection were suspended in dry, sterile air. The spinal cords became almost nonvirulent in approximately 14 days. Dogs injected with emulsions of progressively less attenuated spinal cord were protected against inoculation with the most virulent form of the virus. The injection of dogs with infected spinal cord dried for 14 days, followed the next day by 13-dayold material, and so on until fresh cord was used, protected against contraction of rabies, even when the most virulent virus was injected into the brain. Thus, immunity against rabies was established in 15 days. Pasteur’s first human subject was a child, Joseph Meister, whose life was saved by the treatment. Meister later spent many years as the door porter of the Institut Pasteur in Paris, which was built with funds contributed from all over the world in order that the control of this terrifying disease might be facilitated.

Figure 1.22  Theobald Smith.

Department of Agriculture in 1886, when they found that pigeons inoculated with heat-killed cultures of hog cholera bacilli developed immunity. They reasoned from this discovery that immunity could be induced by exposure of the body to chemical substances or toxins produced by bacteria causing the disease. George Nuttal (1862–1937) (Figure  1.23), in studying the blood of various animals, found that it and other body fluids were able to cause bacteria to disintegrate. Hans Buchner (1850–1902) carried the study further and discovered that the bactericidal properties were to be found in cell-free serum, and that this property was destroyed by heat. He named the substance alexine (Greek: to ward off or protect). Experiments at the Institut Pasteur by Emile Roux (1853–1933) (Figure 1.24) and Alexandre Yersin (1863–1943) showed that the bacterium-free filtrate of the diphtheria

Louis Pasteur (1822–1895), French, “Father of Immunology.” One of the most productive scientists of modern times, Pasteur’s contributions included the crystallization of L- and O-tartaric acid, disproving the theory of spontaneous generation, studies of diseases in wine, beer and silkworms, and the use of attenuated bacteria and viruses for vaccination. He used attenuated vaccines to protect against anthrax, fowl cholera, and rabies. He successfully immunized sheep and cattle against anthrax, terming the technique vaccination in honor of Jenner. He produced a vaccine for rabies by drying the spinal cord of rabbits and using the material to prepare a series of 14 injections of increasing virulence. A child’s (Joseph Meister’s) life was saved by this treatment. Les Maladies des Uers a Soie, 1865; Etudes sur le Vin, 1866; Etudes sur la Biere, 1876; Oeuvres, 1922–1939. A step forward in the production of vaccines was made by Daniel E. Salmon (1850–1914) and Theobald Smith (1859– 1934) (Figure  1.22), in the Veterinary Division of the U.S.

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Figure 1.23  George H.F. Nuttal, author of Blood Immunity and Blood Relationships.

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Figure 1.24  Emile Roux, close associate of Louis Pasteur.

bacillus culture contained an exotoxin which produced the disease. While these insights into the humoral mechanisms of immunity were being developed, an original and dynamic personality appeared in the scientific community in Paris. Elie (Illya) Metchnikoff (also spelled Mechnikov) (1845– 1916) (Figure 1.25), was born at Avanovska, Ukraine, on May 15, 1845. He showed an interest in natural sciences from childhood, and completed his 4-year university studies in 2 years. The son of an officer in the Imperial Guard, he attended the University of Kharkov, graduating in 1864. He studied with several researchers in Germany and Italy and began publishing original observations at a young age. He returned to Russia in 1867 as professor of zoology and comparative anatomy at the University of Odessa. He was plagued with eye trouble and bouts of depression, and lost his young wife to tuberculosis despite his devoted efforts to save her life. His second marriage, to a much younger neighbor, was happy, and the world is indebted to her for a biography of her famous husband. He was not temperamentally suited to a teaching career and left to do private study in Messina, where he first studied phagocytes, examining the transparent larvae of starfish. He observed that cells surrounded and engulfed foreign particles that had been introduced into the larvae. He further discovered that special amoeba-like cells surrounded bacteria introduced into starfish larvae and fungal spores introduced into water fleas (Daphnia) and engulfed them. He observed the digestive powers of the mesodermal cells in starfish larvae and connected the process with the cause of immunity against infectious disease. He returned to Odessa, and while there, made further studies of the water flea, Daphnia, infected by a parasite, from which he built a theory of cellular immunity involving phagocytosis. He was appointed director of the Bacteriological Institute in Odessa, but being unhappy in this position, again traveled to the West, first visiting the Hygiene Institute of Robert Koch, who showed no interest. When he visited Pasteur in Paris,

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Figure 1.25  Elie Metchnikoff.

he was invited to remain, and spent the rest of his life at the Institut Pasteur. He found that many of the white blood cells or leukocytes are phagocytic, defending the body against acute infection by engulfing invading microorganisms. After discovering the role of phagocytes in host defense, he devoted much of his subsequent career to elaborating and championing his cellular theory of immunity. He attracted a number of students to his laboratory, including Jules Bordet (Figure 1.29 and 1.30), and published many papers, a series of Lectures on the Comparative Pathology of Inflammation, and a thorough and balanced review of the whole field of comparative and human immunity, L’immunité dans les maladies infectieuses. He was a colorful and influential personality in the early attempts to understand the mechanisms of immunity. He shared the Nobel Prize in Medicine or Physiology for 1908 with Paul Ehrlich for his work in immunology and made many more contributions to immunity and bacteriology. Metchnikoff became interested in longevity and aging in his later years, and advocated the consumption of yogurt. He believed that lactic acid-producing bacteria in the gut prolonged the life span. A new human bacterial pathogen was discovered every year from 1879 to 1888 and beyond. It was the golden age of bacteriology. In the laboratory of Robert Koch, a whole circle of younger men were trained and set to work on important research. Diphtheria toxin, demonstrated by Roux and Yersin,

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Behring published a further paper on immunization against diphtheria. The first child treated with diphtheria antitoxin was in von Bergmann’s clinic in Berlin; the injection was given on Christmas night, 1891, by Geissler. Von Behring was awarded the first Nobel Prize for Medicine or Physiology for his development of antiserum therapy in 1901. Emil Adolph von Behring (1854–1917), German bacteriologist who worked at the Institute for Infectious Diseases in Berlin with Kitasato and Wernicke in 1890 to 1892 and demonstrated that circulating antitoxins against diphtheria and tetanus toxins conferred immunity. He demonstrated that the passive administration of serum containing antitoxin could facilitate recovery. This represented the beginning of serum therapy, especially for diphtheria. He received the first Nobel Prize in Medicine in 1901 for this work. Die Blutserumtherapie, 1902; Gesammelte Abhandlungen, 1914; Behring, Gestalt und Werk, 1940; Emil von Behring zum Gedächtnis, 1942.

Figure 1.26  Emil Adolph von Behring.

was the first molecule identified as an antigen. Diphtheria was a serious disease at that time, and several people attempted to create artificial immunity to the disease. Carl Fraenkel (1861– 1915) induced immunity with heat-killed broth cultures, and published his finding in December, 1890. One day later, Emil von Behring (1854–1917) (Figure  1.26) and Shibasaburo Kitasato (1852–1931) (Figure 1.27) published their account of raising immune serum in rabbits and mice against tetanus toxin, which not only protected them from lethal doses but could be transferred by means of the immune serum to the bodies of other animals. In a footnote, the word antitoxic was used and was immediately adopted. A week later, von

Figure 1.27  Shibasaburo Kitasato.

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There followed a period of productive studies on the properties of blood serum. Richard Pfeiffer (1858–1945) (Figure  1.28) observed that cholera vibrios were lysed in the peritoneum of immunized guinea pigs and showed the same process in vitro. This became known as the Pfeiffer phenomenon. Jules Bordet (1870–1961) (Figures  1.29 and 1.30), studying in Metchnikoff’s laboratory, found that he could repeat the experiment and that there were two distinct substances in the serum, one heat-labile, which seemed to be similar to Buchner’s alexine, and a second component which was heat-stable. The lytic action was found by Bordet to be applicable to cells other than bacteria. Thus, red blood cells were also lysed, and studies of hemolysis continued to reveal fundamental facts about the immune response. Bordet was awarded the Nobel Prize for Medicine or Physiology in 1919

Figure 1.28  Richard Pfeiffer.

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Figure 1.31  Herbert Durham. Figure 1.29  Jules Bordet.

for his studies on complement. In 1889, A. Charrin (1856– 1907) and J. Roger had observed that immune sera agglutinated a suspension of bacteria. In 1896, Herbert Durham (1866–1945) (Figure  1.31), an English graduate student working in Max von Grüber’s laboratory in Vienna, made a thorough study of the agglutination of bacteria by the blood; von Grüber (1853–1927) (Figure  1.32) and Durham published several papers on the subject. Within a few months, Fernand Widal (1862–1929), at the Faculté de Médecine in Paris, turned the process around and used the agglutination of known cultures of bacteria to diagnose disease using patient blood serum, later to be

Figure 1.30  Jules Bordet in old age, portrait inscribed to Dr. Julius M. Cruse.

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known as the Grüber–Widal test for typhoid fever. In 1901, Bordet with a colleague, Octave Gengou, developed the complement-fixation reaction, the basis of many subsequent tests for infection, notably the Wassermann test for syphilis, which von Wassermann (1866–1915) and associates developed in 1906. Another of the scientists who received early training in Koch’s laboratory was Paul Ehrlich (1854–1915) (Figure  1.33), the pioneer in immunochemistry and one of the most original scientific minds of his time. His research had a profound influence on the direction taken by immunology. Although educated as a physician, Ehrlich had considerable expertise in chemistry. He was one of the first to recognize the limitations of the available knowledge of his day in solving the complex biochemical and genetic aspects of antibody formation and solving the riddle of cancer, which is only now

Figure 1.32  Max von Grüber.

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Figure 1.33  Paul Ehrlich.

beginning to be elucidated. One of his early projects was a study of the vegetable toxins abrin and ricin. He raised antibodies to the two poisons in laboratory animals, and from his studies concluded that the antibody–antigen union was essentially a chemical process with constant amounts combining. He was able to assist von Behring by working out a satisfactory method of standardizing diphtheria antitoxin, a problem von Behring had not been able to solve. Ehrlich began then to visualize the way antibody and antigen combine and developed his famous side-chain theory, which postulated that a toxin had two combining sites, the toxophore and the haptophore groups. Molecules of protoplasm were equipped with side-chains with a nutritive function. When they were blocked by the haptophoric group of a toxin, excess side-chains were secreted by the cell, constituting what was known as antitoxin in the peripheral blood. His theory stimulated much debate and a vast amount of research. We perceive his uncanny intuition upon noting diagrams such as those shown in these figures by observing the Y-shaped receptor on a cell, which is remarkably similar to the modern-day model for immunoglobulin such as IgG. Ehrlich’s life work fell into three phases: an early histology period when he worked mostly with biological stains to differentiate cells and tissues, a middle period comprising his studies on immunity, and a late period at the end of his career, which he devoted mostly to chemotherapy. He discovered a specific treatment for syphilis in the arsenical compound numbered 606, Salvarsan, with Sachahiro Hata (1873–1938), who had come to his laboratory in Frankfurt to teach the transmission of syphilis in laboratory animals and learn some principles of chemotherapy. His entire attention was drawn to supervising the production and use of this important drug, the “magic bullet” that could selectively destroy bacteria without harming the body. Ehrlich also trained a whole generation of younger men who continued to do research in immunology. He shared the 1908 Nobel Prize in Medicine with Metchnikoff for their studies on immunity. Fruits of these labors led to treatments for trypanosomiasis and syphilis.

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Paul Ehrlich presented his famous side-chain theory in the Croonian Lecture before the Royal Society of Great Britain in 1900. According to this theory, the cell was believed to possess highly complex chemical aggregates with attached groupings, or “side-chains,” the normal function of which was to anchor nutrient substances to the cell before internalization. These side-chains, or receptors, were considered to permit cellular interaction with substances in the extracellular environment. Antigens were postulated to stimulate the cell by attachment to these receptors. Because antigens played no part in the normal economy of the cell, the receptors were diverted from their normal function. Stimulated by this derangement of its normal mechanism, the cell produced excessive new receptors, of the same type as those thrown out of action. The superfluous receptors shed by the cell into the extracellular fluids constituted specific antibodies with the capacity to bind homologous antigens. Ehrlich endowed each of his receptors with a special chemical grouping, the haptophore group, which entered into chemical union with a corresponding group of the antigen, as in neutralization of a toxin by antitoxin. However, when the antigen was altered in some other recognizable way, as in agglutination or precipitation, Ehrlich postulated another grouping in the receptor, the ergophore group, determined the particular change in the antigen after the antibody was anchored by its haptophore group. In certain cases it was necessary to postulate receptors that united an antigen to complement. Ehrlich proposed receptors with two haptophore groups, one of which became attached to the antigen to be acted upon, and one to the complement that was the acting substance. Both of these groups were to be regarded as strictly specific in their chemical affinities. The one that combined with the cell or other antigen was called the cytophilic group, and the one that combined with complement the complementophilic group; Ehrlich named this type of receptor an amboceptor, because both groups were supposed to be of the haptophore type. Ehrlich regarded these receptors as definite chemical structures. When it was discovered that antigens such as toxins could be detoxified without losing their antitoxin-binding capacity, he assumed that a toxophore group had been altered, whereas the haptophore group remained intact. Similarly, he postulated the existence of a modified complement, in which an intact haptophore group was associated with an ergophore group that had lost its efficacy. Ehrlich varied the functional activity of the various groups to account for new phenomena. The quantitative aspects of toxin neutralization by antitoxin could not be reconciled with the side-chain theory in its simple form. To retain the concept of firm union between antigen and antibody in constant proportions, Ehrlich postulated a large number of different toxin components with varying degrees of affinity for antitoxin. Similarly, some of his studies on hemolysins made it necessary to assume the intervention of receptors of considerable structural complexity. The consequent coining of a host of new terms for components whose existence was extremely doubtful served to confuse the problem rather than to clarify it. In spite of

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these failings, Ehrlich’s theory had the merit of emphasizing chemical specificity as the essential feature of the antigen– antibody reaction. Obermayer and Pick, two Viennese chemists, in 1903 investigated the effects on specificity of coupling various chemical groupings to proteins. Nitric acid, nitrous acid, or iodine treatment of proteins altered their specificity to the point that they were rendered antigenic in the species of origin. Later experiments demonstrated that alteration of protein specificity by such treatment was based upon modification of aromatic amino acids, as opposed to alteration of protein specificity by the introduced groups alone. This work fired the imagination of Karl Landsteiner (Figure  1.34), who with some associates in 1918 coupled organic radicals to proteins. The ability of a particular chemical to act as a determinant of specificity was tested by coupling it to an aromatic amine such as aniline. The product of this reaction was diazotized and coupled to a protein to form a conjugated antigen or azoprotein. From these studies the chemical basis for serologic specificity was proved. Not only the nature of radicals coupled to proteins but also the position of the attachment site on the ring (ortho, meta, or para) was shown to be of paramount importance for specificity. Ehrlich had attempted to develop a selective theory of antibody formation with his side-chain theory, which required that every cell involved in antibody production be capable of reacting against every known antigen in nature. However, it was precisely this weakness in the hypothesis that allowed Landsteiner to discredit it by raising antibodies against haptens manufactured in the chemical laboratory, and which had never appeared before in nature. It was inconceivable that there would be preformed receptors for antigens that the animal body would never see. As Landsteiner had invalidated the first cell selection theory of antibody formation, it is ironic that his work subsequently contributed to the reemergence of a selective hypothesis. He proposed natural

Figure 1.34  Karl Landsteiner.

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Figure 1.35  Merril W. Chase, Landsteiner’s colleague at the Rockefeller Institute for Medical Research, New York.

diversity and accidental affinity. Together with Merrill Chase (Figure 1.35), he initiated a return to cellular immunology in 1942. Merrill Chase (1905–2004), American immunologist who worked with Karl Landsteiner at the Rockefeller Institute for Medical Research, New York. He investigated hypersensitivity, including delayed-type hypersensitivity and contact dermatitis. He was the first to demonstrate the passive transfer of tuberculin and contact hypersensitivity and also made contributions in the fields of adjuvants and quantitative methods. With the abandonment of Ehrlich’s selective theory, Breinl and Haurowitz (Figure  1.36) and Mudd proposed an instructive hypothesis, which the chemists favored. Pauling (Figure 1.37) proposed a template theory of antibody formation that required that the antigen must be present during the process of antibody synthesis. According to the refolding template theory, uncommitted and specific globulins could become refolded upon the antigen, serving as a template for it. The cell thereupon released the complementary antibodies, which thenceforth rigidly retained their shape through disulfide bonding. This theory had to be abandoned when it became clear that the specificity of antibodies in all cases is a result of the particular arrangement of their primary amino acid sequence. De novo synthesis template theories that recognized the necessity for antibodies to be synthesized by amino acid, in the proper and predetermined order, still had to contend with the serious objection that proteins cannot serve as informational models for the synthesis of other proteins. Felix Haurowitz (1896–1988), a noted protein chemist from Prague who later came to the United States. He investigated the chemistry of hemoglobins. In 1930 (with Breinl)

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Figure 1.38  Sir Macfarlane Burnet. Figure 1.36  Felix Haurowitz, an early proponent of the instructive theory of antibody formation.

he advanced the instruction theory of antibody formation. Chemistry and Biology of Proteins, 1950; Immunochemistry and Biosynthesis of Antibodies, 1968. Burnet (Figure 1.38) and Fenner at the Walter and Eliza Hall Institute were beginning to take a view of antibody production that was different from that proposed by chemists adhering to the template theory of antibody synthesis. The second edition of their classic monograph entitled The Production of Antibodies, published in 1949, contains an exposition of their developing concepts. Burnet advocated but later abandoned a self-marker hypothesis to explain antibody production.

Frank Macfarlane Burnet (1899–1975), Australian virologist and immunologist who shared the Nobel Prize with Peter B. Medawar in 1960 for the discovery of acquired immunological tolerance. Burnet was a theoretician who made major contributions to the developing theories of self-tolerance and clonal selection in antibody formation. Burnet and Fenner’s suggested explanation of immunologic tolerance was tested by Medawar et al., who confirmed the hypothesis in 1953 using inbred strains of mice. The Production of Antibodies (with Fenner), 1949; Natural History of Infectious Diseases, 1953; Clonal Selection Theory of Antibody Formation, 1959; Autoimmune Diseases (with Mackay), 1962; Cellular Immunology, 1969; Changing Patterns (autobiography), 1969. The template theory of antibody production that had been popular with the chemists and prevailed for so many years could no longer explain new biologic revelations that included immunologic tolerance, and it had never explained the anamnestic (memory) immune response. The coup de grâce to this hypothesis was the observation that mature antibody-synthesizing cells contained no antigen. Burnet proposed lymphoid cells genetically programmed to synthesize one type of antibody. As pointed out by Talmage, events in the 1950s that made the template theories of Breinl and Haurowitz and of Pauling untenable included four significant developments. The first was Jerne’s demonstration in 1951 that antibody avidity increased rapidly in an anamnestic response. The greater the avidity of antibody produced on an antigen template, the slower its rate of turnover on the template should be.

Figure 1.37  Linus Pauling, proponent of the template theory of antibody formation.

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David Wilson Talmage (1919– ), American physician and investigator who in 1956 developed the cell selection theory of antibody formation. His work was a foundation for Burnet’s subsequent clonal selection theory. After training in

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forming cells. In 1966 he was appointed Director of the Paul Ehrlich Institute at the University of Frankfurt, Germany, where he began formulating a theory on the role of self-antigens in the generation of antibody diversity. Jerne became director of the Basel Institute for Immunology in 1969, where he developed a network theory of the immune system. He was elected a fellow of the Royal Society in 1980, and after retiring from the Basel Institute, served for a year at the Institut Pasteur in Paris. He then took up residence in France.

Figure 1.39  Niels K. Jerne, author of the “natural selection theory of antibody formation” and of the “network theory of immunity.”

immunology with Taliaferro in Chicago where he became a professor in 1952, Talmage subsequently became Chairman of Microbiology, 1963; Dean of Medicine, 1968; and Director of the Webb–Waring Institute in Denver, 1973. In addition to his investigations of antibody formation, he also studied heart transplantation tolerance. The Chemistry of Immunity in Health and Disease (with Cann), 1961. Niels Kaj Jerne (1911–1994), (Figure 1.39) Danish immunologist who proposed a selection theory of antibody formation, the functional network of antibodies and lymphocytes, and distinction of self from nonself by T lymphocytes. He shared the 1984 Nobel Prize in Medicine or Physiology with Georges Köhler and Cesar Milstein. Niels Kaj Jerne was born in London to Danish parents. His ancestors were from western Jutland. He studied at Leiden, Holland, where he completed his baccalaureate when he was 16, but spent the next 12 years deciding on a profession. At the age of 28, he began the study of medicine in Copenhagen with the plan to become a village doctor. After taking a part-time job in a scientific laboratory, he became interested in science, especially immunology. Jerne followed in his father’s footsteps by moving frequently. His doctoral dissertation was on the theoretical foundation for the study of antibody avidity. He earned his MD in 1951. Jerne worked at the State Serum Institute in Copenhagen between 1943 and 1954. Thereafter, he left for America, working at the California Institute of Technology in Pasadena, where he wrote a paper that was the death-knell for the instructionist theories of antibody synthesis and a forerunner of the clonal selection theory. He next accepted a staff position with the World Health Organization in Geneva, Switzerland, where he coined new immunological terms such as epitope and idiotype. He also served as a professor of biophysics at the University of Geneva between 1960 and 1962. Thereafter, he went to the United States to serve for 4 years as professor and chairman of the Department of Microbiology at the University of Pittsburgh, where he developed a plaque assay for antibody

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Burnet and Fenner showed the logarithmic increase in antibody synthesis, and Barr and Glenny and William and Lucy Taliaferro demonstrated the logarithmic normal distribution of peak antibody titers induced in rabbits. These findings indicated that a replicating antigenic substance induced antibody formation. Owen demonstrated chimerism in dizygotic cattle twins in which blood cells of one twin were tolerated immunologically by the other. Billingham and associates demonstrated actively acquired immunologic tolerance in mice by exposing a fetus to antigen before or at birth. Taliaferro and Talmage and Roberts and Dixon demonstrated that the capacity to synthesize antibody could be transferred passively from an immune to a previously nonimmune animal with viable lymphoid cells. This weight of evidence rendered the antigen template theory obsolete, and necessitated the proposal of alternative explanations. These included Burnet and Fenner’s and Schwect and Owen’s indirect template theories. Jerne (Figure  1.39) proposed a natural selection theory of antibody formation in 1955 based on various antibody populations. Substituting replicating cells for the antibody populations, Talmage published a cell selection theory in 1956 (Figure  1.40). He communicated his ideas to Burnet in Australia, who had independently formulated a similar

Figure 1.40  David W. Talmage, who proposed the cell selection theory of antibody formation in 1956.

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concept. Clearly acknowledging Talmage’s contribution, Burnet named his own version of the cell selection hypothesis the “clonal selection theory of acquired immunity.” Burnet postulated the presence of numerous antibody-forming cells, each capable of synthesizing its own predetermined antibody. One of these cells, after having been selected by the best-fitting antigen, multiplies and forms a clone of cells that continue to synthesize the same antibody. Provided that one accepted the existence of very many different cells, each capable of synthesizing an antibody of a different specificity, all known facts of antibody formation were easily accounted for. An important element of the clonal selection theory as proposed by Burnet was the hypothesis that the many cells with different antibody specificities arise through random somatic mutations, during a period of hypermutability, early in the animal’s life. Also early in life, the “forbidden” clones of antibody-forming cells (i.e., the cells that make antibody to the animal’s own antigens) are destroyed after encountering these autoantigens. This process accounts for an animal’s tolerance of its own antigens. Antigen would have no effect on most lymphoid cells but would selectively stimulate those cells already synthesizing the corresponding antibody at a low rate. The cell surface antibody would serve as receptor for antigen and proliferate into a clone of cells producing antibody of that specificity. Burnet introduced the “forbidden clone” concept to explain autoimmunity. Cells capable of forming antibody against a normal self-antigen were forbidden and eliminated during embryonic life. Since that time various modifications of the clonal selection hypothesis have been offered. Burnet suggested a concept of “clonal deletion” as a means to eliminate precursor lymphocytes capable of reacting with self-antigens before birth. This concept provided for the permanent removal of self-reactive lymphocytes with the possibility that so-called forbidden clones might develop by spontaneous mutation in the later life of the individual. Therefore, tolerance to some self-antigens is maintained even when the antigen is removed, whereas tolerance to other self-antigens may be terminated. Thus, natural tolerance or unresponsiveness could result from the elimination of immunocompetent cell clones specific for self-antigens, or clones of immunocompetent cells rendered unresponsive by early exposure to self-antigenic determinants. Nossal and Pike demonstrated “clonal anergy” by functionally inactivating B-lymphocyte precursors from the bone marrow with excessive but critical concentrations of antigens bearing appropriate numbers of carrier determinants. This phenomenon may occur in vivo as well as in vitro. B cells suppressed functionally by these carefully adjusted concentrations of antigen persist but do not proliferate or form antibody. For clonal anergy to be able to explain self-tolerance could require the maintenance of significant concentrations of antigen during an individual animal’s lifetime, and should be true for T as well as B cells.

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Early criticism of the cell selection theory was based on its failure to explain antibody specificity for the limitless antigens in nature. Studying the specificity of antibodies against synthetic haptens, Landsteiner had shown that antisera demonstrated the features of specificity, universality, cross-reactivity, and diversity. Thus, it became apparent that an animal could synthesize antibodies in great numbers. Considering the astronomical number of antigens in nature and the fact that a single antiserum contained multiple antibodies for each antigen against which the animal was immunized, it appeared necessary for each antibody molecule to be tailormade using the antigen template as a pattern. To explain this problem, a different concept of specificity was developed in which antibodies were suggested to be derived from a large set of natural globulins with some reactivities in common. The chance reactivity of each globulin with a few molecules of all antigenic determinants would endow each individual animal with a group of globulin molecules that could react with essentially every antigenic determinant possible. Fifty thousand separate globulins were suggested to be able to explain the observation of specificity and cross-reactivity. The demonstration of immunoglobulin structure and its genetic basis also contributed to solving the riddle of Landsteiner’s observations. Separate antibodies are encoded by a unique combination of a few genes just as antisera consist of a unique mixture of antibodies. Several hundred genes could encode several million antibodies rather than 50,000 genes encoding 50,000 antibodies. Gustav Joseph Victor Nossal (1931– ) (Figure  1.41), Australian immunologist whose seminal works have concentrated on antibodies and their formation. He served as

Figure 1.41  Sir Gustav Nossal.

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director of the Walter and Eliza Hall Institute of Medical Research in Melbourne. Antibodies and Immunity, 1969; Antigens, Lymphoid Cells and the Immune Response (with Ada), 1971. Evidence in support of the cell selection theory accumulated rapidly. Nossal and Lederberg found that individual antibody-synthesizing cells could produce antibody of only one specificity. In addition, surface immunoglobulins were demonstrated on circulating lymphocytes in newborn and fetal pigs. Spleen cells responding to a single antigen could be eliminated without altering the response to a second antigen. Of special significance was the demonstration by Raff and colleagues that all surface immunoglobulin on antigen-binding lymphocytes formed caps when incubated with a single antigen. This showed that all of the immunoglobulin on the surface of a single lymphocyte was of one specificity. Joshua Lederberg (1925–2008), American biochemist who made a significant contribution to immunology with his work on the clonal selection theory of antibody formation. He received a Nobel Prize in 1958 (with Beadle and Tatum) for genetic recombination and organization of genetic material in bacteria. Even though criticism of the cell selection theory continued, the subsequent accumulation of scientific evidence in its favor facilitated its acceptance. A principal source of support was Köhler and Milstein’s demonstration in 1975 of hybridoma technology for the formation of monoclonal antibodies. Even though monoclonal antibodies had been well known for years in myeloma patients, the widespread use of hybridoma technology by multiple investigators synthesizing monoclonal antibodies to all sorts of antigens led to their popular use in research. Clearly, monoclonal antibodies provided convincing evidence of the validity of the cell selection hypothesis. In addition to the rapid advances in cellular immunology in the 1970s was the progress in molecular immunology. Immunoglobulin structure and the genes encoding these molecules were defined. Diversity was shown to be due to the random rearrangement of numerous separate variable genes in different cells. Thus, the clonal selection theory has proved compatible with the accumulated scientific evidence. The Ehrlich side-chain theory (historical) was the first selective theory of antibody synthesis developed by Paul Ehrlich in 1900. Although elaborate in detail, the essential feature of the theory was that cells of the immune system possess the genetic capability to react to all known antigens, and that each cell on the surface bears receptors with surface haptophore side-chains. On combination with antigen, the side-chains would be cast off into the circulation, and new receptors would replace the old ones. These cast-off receptors represented antibody molecules in the circulation. Although far more complex than this explanation, the importance of the theory was in the amount of research stimulated to try to disprove it. Nevertheless, it was the first effort to account

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for the importance of genetics in immune responsiveness at a time when Mendel’s basic studies had not even yet been “rediscovered” by de Vries. Ehrlich’s theory assumed that each antibody-forming cell had the ability to react to every antigen in nature. The demonstration by Landsteiner that antibodies could be formed against substances manufactured in the laboratory that had never existed before in nature led to abandonment of the side-chain theory. Yet its basic premise as a selective hypothesis rather than an instructive theory was ultimately proved correct. The indirect template theory (historical) was a variation of the template hypothesis, which postulated that instructions for antibody synthesis were copied from the antigen configuration into the DNA encoding the specific antibody. This was later shown to be untenable and is of historical interest only. The self-marker hypothesis (historical) was a concept suggested by Burnet and Fenner in 1949 in an attempt to account for the failure of the body to react against its own antigens. They proposed that cells of the body contained a marker that identified them to the host’s immunologically competent cells as self. This recognition system was supposed to prevent the immune cells of the host from rejecting its own tissue cells. This hypothesis was later abandoned by the authors and replaced by the clonal selection theory of acquired immunity, which Burnet proposed in 1957. The template theory (historical) was an instructive theory of antibody formation that required that the antigen must be present during the process of antibody synthesis. According to the refolding template theory, uncommitted and specific globulins could become refolded on the antigen, serving as a template for it. The cell thereupon would release the complementary antibodies, which thenceforth would rigidly retain their shape through disulfide bonding. This theory had to be abandoned when it became clear that the specificity of antibodies in all cases is due to the particular arrangement of their primary amino acid sequence. The template theory could not explain immunological tolerance or the anamnestic (memory) immune response. Susumu Tonegawa (Figure  1.42) received the 1987 Nobel Prize in Medicine or Physiology for his research on the generation of antibody diversity. Doubting that all genes needed for antibody diversity could be present in the germ line, most scientists postulated somatic mutation involving a very limited number of germ line genes. Dreyer and Bennett fostered a two gene/one polypeptide chain theory in 1965, which proposed that the combination of multiple variable-region genes with a single constant region gene for a particular isotype might require less DNA. Susumu Tonegawa (1939– ), Japanese-born immunologist working in the United States. He received the Nobel Prize in 1987 for his research on immunoglobulin genes and antibody diversity. Tonegawa and many colleagues were responsible

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Figure 1.42  Susumu Tonegawa.

for the discovery of immunoglobulin gene C, V, J, and D regions and their rearrangement. In 1976, Tonegawa and Hozumi demonstrated that embryonic DNA contained separate constant (C)-region and variable (V)-region genes. Tonegawa and his associates observed that an intron separates these two genes when joined in a differentiated cell. Following the finding by Tonegawa and by Leder that more amino acids were present in the variable region polypeptide chain than the V-region light-chain DNA could encode, a J (joining) segment was discovered to account for the missing DNA. The union of a single constant region segment with one of several J segments and one of numerous V-region segments could generate a broad variety of different light chains.

Figure 1.43  Leroy Hood, pioneer in immunogenetics and molecular immunology.

according to the ordinary law of mass action. Evidence to support this had come from Jan Danysz (1860–1928), who found that the toxicity of antigen was neutralized by a stated quantity of antibody when they were mixed but that the mixture remained toxic if half the antigen was added later, the “Danysz phenomenon.” Bordet had a still different view of the way the antigen–antibody union took place, considering that the two substances acted like colloids, having varying proportions akin to adsorptive phenomena. A solution to the problem had to await more sophisticated understanding of the structure and nature of macromolecules and the forces responsible for their behavior.

Tonegawa and Leroy Hood (Figure  1.43) proved that three separate DNA segments must be combined to complete the heavy-chain variable-region sequence. They found yet another diversity (D) group of DNA segments in addition to the V and J segments. Besides the numerous V, D, and J elements, splicing in the middle of a triplet codon, leading to a translational shift, contributed to even greater heavychain variability. Hood went on to demonstrate mutations in these gene segments. This elegant body of scientific evidence elucidated finally one of immunology’s most mystifying conundrums. Other workers were also interested in concepts of how antigen and antibody combine. Svante Arrhenius (1859–1927), of Stockholm, visited Ehrlich. He and one of Ehrlich’s colleagues, the Danish bacteriologist Thorvald Madsen (Figure  1.44), formed an opinion from their studies that the reaction was a reversible equilibrium between the substances as when a weak acid and weak base are combined

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Figure 1.44  Thorvald Madsen.

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Figure 1.45  Sir Almroth E. Wright.

The humoral–cellular immunity controversy had occupied much attention through the last decade of the 19th century. In 1903, the English pathologist Sir Almroth Wright (1861–1947) (Figure 1.45) and his co-worker Stewart Douglas (1871–1936) showed the existence of thermolabile substances in both normal and immune serum, which they termed opsonins (Greek opsono: I provide food for), which attach themselves to bacteria and facilitate their phagocytosis. This finding is often cited as reconciling the two schools of thought. However, the mainstream of research for many years continued to be the study of antibodies as the renaissance in cellular immunology emerged only in the 1960s.

Immunochemistry The fundamental position that immunology occupies among the natural sciences is no better demonstrated than by the fact that distinguished chemists outside the field of biomedical science have been fascinated by the basic biological significance of such topics as antibody formation and antigen–antibody interaction. Indeed, it was the Nobel Laureate in Chemistry Svante Arrhenius who coined the term immunochemistry in 1905 when he was invited to the University of California at Berkeley to present a series of lectures on the chemistry of immune reactions. Svante Arrhenius (1859–1927) (Figure  1.46) coined the term immunochemistry and hypothesized that antigen–antibody complexes are reversible. He was awarded the Nobel Prize for Chemistry, 1903. Immunochemistry. New York: MacMillan Publishers, 1907. Obermayer and Pick, two Viennese chemists, in 1903 investigated the effects on specificity of coupling various chemical groupings to proteins. Nitric acid, nitrous acid, or iodine treatment of proteins altered their specificity to the point that they were rendered antigenic (i.e., immunogenic) in species to which they were not originally alien. Later experiments

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Figure 1.46  Svante Arrhenius (left) and Paul Ehrlich (right).

demonstrated that alteration of protein specificity by such treatment was based upon modification of aromatic amino acids as opposed to alteration of protein specificity by the introduced groups alone. There can be little doubt that this work fired the imagination of Karl Landsteiner, who, with some associates in 1918, coupled organic radicals to proteins. Landsteiner devised a method whereby an immune response could be directed against small molecules of known structure. He referred to these substances as haptens, which by themselves were too small to initiate an immune response but were capable of reacting with the products of an immune response. He chemically coupled these haptens to large biological macromolecules, such as ovalbumin, which he termed carriers, producing conjugated antigens capable of stimulating an immune response. The monovalent hapten in pure form, together with serum antibodies, could then be used to study antibody–hapten interactions without the complications of multideterminant macromolecular antigens. The ability of a particular chemical to act as a determinant of specificity was tested by coupling it to an aromatic amine such as aniline. The product of this reaction was diazotized and coupled to a protein to form a conjugated antigen or azoprotein. From these studies, which dominated Landsteiner’s research activities until his death in 1943, the chemical basis for serological specificity was proved. Not only the nature of the radical coupled to a protein, but also the position of the attachment site on the ring (ortho, meta, or para), was shown to be of critical importance with respect to specificity. Landsteiner found cross-reactivity with immune antibodies to related haptens. This represented the “golden age of immunochemistry,” when the views of chemists prevailed over those of biologists.

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Figure 1.48  David Pressman.

Figure 1.47  Harry Gideon Wells.

Ehrlich had attempted to develop a selective theory of antibody formation with his side-chain theory, which required that every cell involved in antibody production be capable of reacting against every known antigen in nature. However, it was precisely this weakness in the hypothesis that allowed Landsteiner to deal it a death blow by raising antibodies against haptens (partial antigens) which he had manufactured in the chemical laboratory and which had never appeared before in nature. He pointed out that it was inconceivable that nature would provide receptors for antigens which the animal body would never see. It is important to remember the limitations not only of chemical knowledge of the period, but also that Hugo de Vries had rediscovered Mendel’s basic work in genetics only 23 years earlier. Arrhenius was not the only famous chemist to become interested in immunological phenomena. Wells (Figure  1.47) authored an important book on chemical aspects of immunity in the 1920s. The British chemical pathologist J. R. Marrack published a critical review of the chemistry of antigens and antibodies in 1934 (revised 1938) and proposed a lattice theory of antigen– antibody interaction. The description of forces involved in antigen–antibody interaction and the demonstration of the necessity of stereophysical complementarity of reaction sites were made in 1940 by Linus Pauling, another Nobel laureate in Chemistry. As had Felix Haurowitz and Breinl in 1930 and Mudd in 1932, Pauling proposed a template theory of antibody formation which required that the antigen be present during the process of antibody formation. This view prevailed among many immunologists until the demonstration that it was inadequate to explain such newly discovered phenomena as immunological tolerance, which represented the basis of successful tissue transplantation. Working with Pressman (Figure  1.48) and Campbell, he provided strong

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evidence for the bivalence of antibodies and the significance of molecular shapes in binding of antibody to antigen. The development of precise methods of quantitation of antibody and the discovery and characterization of nonprecipitating antibodies (incomplete or functionally univalent antibodies) was made between 1930 and 1935 by Heidelberger and Kendall. To O.T. Avery’s (Figure 1.49) nitrogen-free “specific soluble substance” derived from pneumococcal polysaccharide, Heidelberger added homologous antibody in a precipitin reaction to yield a precipitate. This permitted him to measure antibody, since the precipitate contained antibody nitrogen exclusively. Heidelberger, Kendall, and Kabat resolved the question of whether antibodies were globulins and whether precipitins and agglutinins were the same or separate entities. In fact,

Figure 1.49  Oswald T. Avery.

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specific precipitation, agglutination, and complement fixation were demonstrated to be different manifestations of a single antibody. Heidelberger and associates were able to isolate and analyze antibodies. In 1934, they developed a 70% pure antibody preparation which proved to be a globulin with the aid of a newly developed ultracentrifuge in Sweden. The Heidelberger team proved that complement was a real nitrogen-containing substance, or substances, with weight. This discovery, together with Pillemer and Ecker’s independent purification of the first component, enabled other investigators to purify and analyze the complement system. In his long and distinguished career, Professor Heidelberger served as the dean of American immunochemists who trained Elvin Kabat, Manfred Mayer, and a host of other investigators. The electrophoretic method of separating serum proteins and the assignment of antibody activity to the globulin fraction was proposed by Tiselius. In the same year Kabat and Tiselius demonstrated by electrophoresis and ultracentrifugation that 19S antibody is found early and 7S antibody late in the immune response. In 1959, Rodney Porter working in England demonstrated that antibody molecules could be split into fragments by the enzyme papain. He found that those fragments which remained in the supernatant of his reaction mixture retained the capacity to interact with antigen, whereas that part of the molecule which crystallized had no antigen-binding property. He subsequently designated these as Fab and Fc fragments (i.e., fragment antigen-binding and fragment crystallizable, respectively). In 1969, Gerald Edelman reported the results of his primary sequence analysis of a human myeloma protein (namely immunoglobulin G, IgG). The isolation of sufficient homogeneous antibody from the serum of a patient with multiple myeloma over a significant period of time had overcome the previous difficulty of attempting to analyze heterogeneous antibodies. For their

Figure 1.50  Henry G. Kunkel.

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work in demonstrating the fundamental structure of immunoglobulin molecules, Porter and Edelman received the Nobel Prize in Medicine in 1972. However, many other investigators including Henry Kunkel (Figure 1.50), Hugh Fudenberg, and Frank Putnam had laid the groundwork for this discovery. Valentine demonstrated immunoglobulin structure by electron microscopy. Alfred Nisonoff’s fundamental studies on immunoglobulin structure resulted in part from his preparation of F(ab′)2 fragments of IgG as a consequence of pepsin digestion. Kunkel was Edelman’s mentor at the Rockefeller Institute for Medical Research. Much activity was then initiated to sequence the heavy and light chains comprising immunoglobulin molecules. It was shown that man possesses three major and two minor classes of immunoglobulin, each determined by the specificity of the heavy chain. These were designated as IgG, IgM, IgA, IgD, and IgE. After this period of activity in the 1960s, interest in immunochemistry began to wane, and cellular immunology came to the forefront of research. Even the journal entitled Immunochemistry was renamed Molecular Immunology. Jan Gosta Waldenström (1906–1996), Swedish physician who described macroglobulinemia, which now bears his name. He received the Gairdner Award in 1966. Michael Heidelberger (1888–1991) (Figure 1.51), a founder of immunochemistry, was born on April 29, 1888, in New York City. Following completion of his PhD in organic chemistry at Columbia and postdoctoral training with Richard Willstätter in Zurich, he returned to a position at the Rockefeller Institute for Medical Research in New York City. He and Jacobs discovered tryparsamide, which proved very effective in the treatment of African sleeping sickness. In Van Slyke’s laboratory at the Rockefeller Institute, he perfected the technique for the preparation of crystalline oxyhemoglobin.

Figure 1.51  Michael Heidelberger.

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This brought him into contact with Karl Landsteiner, with whom he published two classic articles comparing immunologic and solubility techniques for distinguishing hemoglobins of different species. Professor O.T. Avery requested his assistance with the biochemical analysis of “specific soluble substance” of pneumococci. He purified Avery’s broth concentrate until the components were free of nitrogen, showing that it was indeed specific soluble substance. He precipitated the preparation with antiserum and recovered the same polysaccharide. This led to extensive future investigations by Heidelberger, Goebel, Avery, and others on the specificity of naturally occurring antigens. Precipitin analyses permitted estimation of the quantities of numerous antigens in native materials without the need for tedious isolation and purification. Heidelberger accepted an appointment as associate professor of medicine at Columbia Physicians and Surgeons, where he and Kendall developed a quantitative theory defining the precipitin reaction of polysaccharides. Together with Kabat and Kendall, he demonstrated that antibodies were globulins and that specific precipitation, agglutination, and complement fixation were different manifestations of a single antibody. They also performed classic experiments proving complement to be a real nitrogen-containing substance, or substances, with weight. Purified antibodies developed in rabbits against types I, II, and III pneumococci proved very effective in the treatment of pneumonia caused by pneumococci of the corresponding type, significantly reducing mortality. During the post–World War II decade, accolades at home and abroad were showered upon him. He twice served as president of the American Association of Immunologists in the 1940s. Among his many honors are the Lasker, Behring, and Hurwitz Awards, the Pasteur Medal of the Swedish Medical Society, the Order of Leopold II, membership in the Légion d’honneur and the Académie de Médecine of France, and the National Medal of Science, as well as a spate of honorary degrees from the world’s leading universities. After 27 years at Columbia Physicians and Surgeons, at 67 years of age, he accepted Dr. Selman Waksman’s invitation to join the Institute of Microbiology at Rutgers where, for nearly a decade, he inspired the Immunochemistry Group to pursue novel and innovative research. Upon leaving Rutgers, at the age of 76, Professor Heidelberger continued his research in the Department of Pathology at New York University. He began a series of challenging investigations on cross-reactivity of microorganisms and of plant polysaccharides, publishing many papers on immunologic relationships among their structures. Professor Heidelberger’s life work was aimed at the development of rigorous quantitative techniques for purification of antibody molecules and investigations on the specificity of naturally occurring antigens. Elvin Abraham Kabat (1914–2000) (Figure  1.52), American immunochemist. With Tiselius he was the first to separate immunoglobulins electrophoretically. He also demonstrated that globulins can be distinguished as 7S or 19S. Other contributions include research on antibodies to carbohydrates, the antibody combining site, and the discovery

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Figure 1.52  Elvin A. Kabat.

of immunoglobulin chain variable regions. He received the National Medal of Science. Experimental Immunochemistry (with Mayer), 1948; Blood Group Substances: Their Chemistry and Immunochemistry, Structural Concepts in Immunology and Immunochemistry, 1956, 1968. Arne W. K. Tiselius (1902–1971) (Figure 1.53), Swedish chemist who was educated at the University of Uppsala, where he also worked in research. In 1934 he was at the Institute for Advanced Study in Princeton. He worked for the Swedish National Research Council in 1946 and he became president of the Nobel Foundation in 1960. Awarded the Nobel Prize in Chemistry in 1948, he perfected the electrophoresis technique and classified antibodies as K globulins

Figure 1.53  Arne Tiselius.

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Figure 1.56  Rodney R. Porter.

Figure 1.54  John Richardson Marrack.

together with Elvin A. Kabat. He also developed synthetic blood plasmas. John Richardson Marrack (1899–1976) (Figure  1.54), British physician who served as professor of chemical pathology at Cambridge and at the London Hospital. He hypothesized that antibodies are bivalent, labeled antibodies with colored dyes and proposed a lattice theory of antigen–antibody complex formation in fundamental physicochemical studies.

Herman Nathaniel Eisen (1918– ), American physician whose research contributions range from equilibrium dialysis (with Karush) to the mechanism of contact dermatitis. Gerald Maurice Edelman (1929– ) (Figure 1.55), American investigator who was professor at the Rockefeller University and shared the Nobel Prize in 1972 with Porter for their work on antibody structure. Edelman was the first to demonstrate that immunoglobulins are composed of light and heavy polypeptide chains. He also did pioneering work with Bence Jones protein, cell adhesion molecules, immunoglobulin amino acid sequence, and neurobiology. Rodney Robert Porter (1917–1985) (Figure  1.56), British biochemist who received the Nobel Prize in 1972, with Gerald Edelman, for their studies of antibodies and their chemical structure. Porter cleaved antibody molecules with the enzyme papain to yield Fab and Fc fragments. He suggested that antibodies have a four-chain structure. Fab fragments were shown to have the antigen-binding sites, whereas the Fc fragment conferred the antibody’s biological properties. He also investigated the sequence of complement genes in the major histocompatibility complex (MHC). Defense and Recognition, 1973.

The Complement System

Figure 1.55  Gerald M. Edelman.

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Throughout the ages, man has been fascinated and, at times, obsessed by the marvelous, mysterious, and even baffling qualities of the blood. A ferment of research activity in the latter 1800s was designed to elucidate mechanisms of host immunity against infectious disease agents. Elie Metchnikoff’s demonstration that blood cells could phagocytize (ingest) invading microorganisms led to the cellular theory, the first of two opposing concepts of bacteriolysis. The other, the humoral theory, was based on Fodor, Nuttall, and Buchner’s detection of the heat-labile constituent of fresh,

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cell-free serum, which could include bacteriolysis. Hans Buchner named this principle alexin (Greek, meaning without a name). In 1889, Buchner described a heat-labile bactericidal principle in the blood, which he termed alexine. This was later identified as the complement system. In 1894, Pfeiffer discovered specific in vivo lysis of bacteria by observing that cholera vibrios injected into the peritoneum of immune guinea pigs were lysed. In 1895, Jules Bordet, working at the Institut Pasteur in Metchnikoff’s laboratory, extended this finding by demonstrating that the lytic or bactericidal action of freshly drawn blood, which had been destroyed by heating, was promptly restored by the addition of fresh, normal, unheated serum. Paul Ehrlich called Bordet’s alexine das Komplement. In 1901, Bordet and Gengou developed the complement fixation test to measure antigen–antibody reactions. Von Wassermann applied this principle to the serologic diagnosis of syphilis. Hans Buchner (1850–1902) (Figure  1.57), German bacteriologist who was a professor of hygiene in Munich in 1894. He discovered complement. Through his studies of normal serum and its bactericidal effects, he became an advocate of the humoral theory of immunity. Jules Jean Baptiste Vincent Bordet (1870–1961) (Figure  1.58), Belgian bacteriologist and immunologist, who discovered Bordetella pertussis, the causative agent of whooping cough, immune hemolysis, and complement fixation. He received the Nobel Prize in Medicine or Physiology in 1919 for his studies on complement. Bordet was born at Soignies, Belgium, and at age 16 entered the University of Brussels, graduating as doctor of medicine in 1892. By 1890, Jules was enhancing the virulence of Vibrio metschnikovii by passage in immunized animals. He received a travel grant in 1894 and served as preparateur in Metchnikoff’s laboratory at the Institut Pasteur in Paris from 1894 to 1901. In 1897 he traveled to the Transvaal to investigate rinderpest. He left the Institut Pasteur in 1901 to accept the post as

Figure 1.57  Hans Buchner.

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Figure 1.58  Jules Bordet.

director of the Rabies and Bacteriology Institute of Brabant in Belgium. He received Madame Marie Pasteur’s permission to rename the facility the Institut Pasteur de Bruxelles in 1903. In 1907, he was appointed professor of bacteriology in the Faculty of Medicine of the Free University of Brussels. In addition to his discovery of Bordetella pertussis, Bordet also described the mycoplasma of bovine pleuropneumonia, bacteriophage lysogeny. He discovered the spirochete of syphilis but did not publish this finding. These are in addition to his legion of discoveries in immunology that include specific agglutination (1895), conglutination (1909), the antigenicity of antibodies and complement and, most important, his investigations of complement reactions. Noting the similarity of specific lysis of cholera vibrios by Richard Pfeiffer to the phenomenon of hemolysis described by Hans Buchner, Bordet decided to use the red blood cell system to illustrate complement activity rather than lysis of pathogenic bacteria as a model system. He investigated both bacterial lysis and the lysis of red blood cells. The lytic action of serum was sensitive to heat but could be restored by the addition of unheated normal serum. Bordet’s experiment showed complement to be nonspecific and able to function only when target cells were sensitized. He showed the similarities of bacterial lysis and hemolysis. In 1900, Bordet showed that complement action was nonspecific by using the same source of complement for both hemolysis and bacterial lysis. He and Gengou described complement fixation, and pointed to its use in the diagnosis of infectious diseases. They showed that complement, specifically C1, is fixed by bacteria that resist lysis, providing the foundation for the highly sensitive complement fixation test to detect specific antibodies in serum. Von Wassermann applied the complement fixation technique to the diagnosis of syphilis, which was used worldwide. Bordet debated with Paul Ehrlich concerning his complex explanations of immune reactions. For example, Bordet believed toxin to

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be neutralized by antitoxin through adsorption, similar to a fabric interacting with a dye. He considered antitoxic sera to contain substance sensibilisatrice (i.e., antibody) which sensitized red blood cells or bacteria to the action of alexine, his term for complement. He postulated that this acts as a mordant does for a dyestuff. Thus, he disputed with Ehrlich about the nature and mechanism of action of antibodies and complement. This led to the publication in 1909– 1910 of competing volumes entitled Studies on Immunity, one by the “Bordet school” and the other by Ehrlich and his colleagues. He received the Nobel Prize in Medicine or Physiology in 1919 for his studies on complement and its reactions. During his long and productive career, Bordet wrote a popular text entitled Trait de l’Immunité dans les Maladies Infectieuses and trained a spate of gifted investigators, including the prominent American bacteriologist/ immunologist Frederick P. Gay of Columbia University. Ferrata, in 1907, recognized complement to be a multiple component system, a complex of protein substances of mixed globulin composition present in normal sera of many animal species. When electrolytes were removed from serum by dialysis against distilled water, the euglobulin fraction of serum proteins precipitated. A portion of complement activity was present in this fraction and a part in the supernatant. Neither was active alone, but activity was present when the two were combined. Ferrata demonstrated that euglobulin and pseudoglobulin fractions were each hemolytically inactive. However, hemolytic activity could be restored when they were combined. The component present in the euglobulin portion was found to combine with antigen–antibody complexes and not produce lysis. The soluble fraction failed to combine with antigen–antibody complexes but did combine with antigen–antibody complex plus euglobulin fraction. Therefore, in the early literature, the euglobulin fraction was called the midpiece of complement, while the other pseudoglobulin was designated as the endpiece. Current information reveals that what was referred to as the endpiece did not contain C1 but did contain all of the C2 and some other complement components. Bordet believed alexine (i.e., complement) to be a transient colloidal state of the serum. By contrast, Ehrlich hypothesized that complement represented a specific substance with the ability to induce lysis. Heidelberger at Columbia University designed investigations to determine whether complement was a distinct substance which could be measured with respect to weight and size. They showed that complement-containing sera added appreciable weight to specific precipitates formed with rabbit antipolysaccharide or antiprotein antibodies. Their classic experiments proved that complement was a real nitrogen-containing substance, or substances, with weight. Thus, the Ehrlich hypothesis rather than Bordet’s theory of complement was substantiated. This discovery, and Pillemer and Ecker’s independent purification of the first component, enabled other investigators to purify and analyze the complement system.

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The classical pathway of complement activation was described first by investigators using sheep red blood cells sensitized with specific antibody and lysed with guinea pig or human complement. Coca in 1914 described the third complement component and postulated an alternative pathway of complement activation by yeast cell walls, which should not involve C1 or C2. The lack of advanced biochemical methods delayed demonstration that the complement system is protein and comprises multiple components. In the late 1920s, Ferrata, followed by Coca and Gordon, identified four complement components. By 1941, Pillemer and associates confirmed the protein nature of complement. In the 1960s, Nelson identified a minimum of six components in guinea pig serum that were requisite for hemolytic activity. Each of these was subsequently purified and characterized by Müller-Eberhard and colleagues. During this same period, Ueno and subsequently Mayer (Figure 1.59) elucidated the classical pathway’s reaction sequence by combining partially purified components with antibody-sensitized sheep red blood cells. Since these early studies, multiple (i.e., more than 20) complement proteins have been identified. These include C1qrs C2, C3, C4, C5, C6, C7, C8, and C9. In addition to immune lysis, complement has many other functions and is important in the biological amplification mechanism that is significant in resistance against infectious disease agents. Complement’s mechanism of action in the various biological reactions in which it participates has occupied the attention of a host of investigators. In 1954, Pillemer et al. (Figure 1.60) suggested the existence of a non-antibody-dependent protein in the serum that is significant for early defense of the host against bacteria and viruses. This protein was named properdin. Pillemer et al. first described properdin as a serum factor that activates

Figure 1.59  Manfred Mayer, student of Heidelberger and professor at Johns Hopkins University, who described the role of complement in immune lysis.

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Ohio, when he identified a new protein, which he believed served as an alternative nonspecific defense mechanism to trigger complement action without antibody. His theory was hailed at first, but then opinion turned against him. In 1958, Nelson challenged Pillemer’s interpretation of these data and suggested that the properdin system was in fact the classical pathway activated by antibodies to zymosan. In later years, the properdin system came to be known as the “alternative pathway of complement activation.” When target cells stimulate the production of complement-fixing antibodies of the IgG or IgM class, complement is fixed by the classical pathway. By contrast, when cells stimulate the production of antibody classes that do not fix complement by the classic pathway (e.g., IgA), or if lysis is required prior to expression of an antibody response, the alternative pathway is activated.

Figure 1.60  Louis Pillemer, who described properdin and the alternative pathway of complement activation.

complement without antibody. It was found to be a serum protein physiochemically, and immunologically distinct from the immunoglobulins. It participates in a nonspecific manner in various types of immune reactions of normal serum. It acts in combination with certain inorganic ions and complement components that make up the so-called properdin system, which constitutes a part of the natural defense mechanism of the blood. In later years, the properdin system came to be known as the alternative pathway of complement activation.

Müller-Eberhard’s (Figure 1.61) subsequent purification of C3 proactivator and proposal that the C3 activator system is an alternative pathway of complement activation substantiated Pillemer’s original concept. Regrettably, Pillemer’s untimely death preceded the proof that his hypothesis was correct. His colleague and student Irwin Lepow prepared nine of his papers for posthumous publication. Lepow (Figure 1.62) and others published details of the characteristics of properdin in 1968, and in 1970 several laboratories correctly described its role as an alternative pathway to complement activation. Pillemer’s theories were vindicated, the truth being a synthesis of the opposing points of view. Dr. Lepow paid tribute to Pillemer in his presidential address to the American Association of Immunologists in 1980. A century of investigations has revealed that more than 20 soluble plasma and other body fluid proteins, together with an

Pillemer et al. proposed an alternative mechanism of complement activation which they termed the properdin pathway. Since antibody was not involved, they predicted that this alternative pathway was significant in nonspecific immunity. They conceived this mechanism to have significance in resistance against infectious diseases and neoplasms. The alternative pathway hypothesis was challenged in the late 1950s but came to be accepted in the 1960s once C2 and C4 genetically deficient sera were described. When target cells stimulate the production of complement-fixing antibody of the IgG or IgM class, the classical complement pathway is activated. By contrast, antibody classes that do not fix complement by the classic pathway (e.g., IgA), as well as selected nonantibody substances, may activate the alternative pathway. The separate complement proteins began to be isolated and identified immunologically and the biochemistry of their reactions determined. Further clarification of the alternative pathway evolved in the 1970s. In 1953, Pillemer reported that zymosan depleted C3 from serum without affecting C1, C2, and C4 levels. He also described a properdin pathway of complement activation with properdin as the activating factor. Louis Pillemer was a professor of biochemistry at Case Western Reserve in Cleveland,

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Figure 1.61  Hans J. Müller-Eberhard, who described later complement components.

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Figure 1.63  F. Magendie, who first observed anaphylaxis in rabbits in 1837.

Figure 1.62  Irwin Lepow, co-worker of Pillemer, who was also instrumental in describing the alternative pathway.

equivalent number of cell receptors and control proteins found on blood and other tissue cells, constitute the complement system. These proteins play a critical role in the phagocytosis of immune complexes, which activate the complement system. These molecules and their fragments resulting from the activation process are significant in the regulation of cellular immune responsiveness. Once complement proteins identify and combine with the target substance, serine proteases are activated. This leads ultimately to the assembly of C3 convertase, a protease on the surface of the target substance. The enzyme cleaves C3, yielding the C3b fragment that is bound to the target through a covalent linkage. C3b or iC3b bound to phagocytic cell surfaces become ligands for C3 receptors as well as binding sites for C5. The union of C5b with C6, C7, C8, and C9 generates the membrane attack complex (MAC), which may associate with the cell’s lipid bilayer membrane to produce lysis, which is critical in resistance against certain species of bacteria.

same antigen, produced severe symptoms, and in some cases death. These two physiologists from the University of Paris had been asked by the Prince of Monaco to study the toxic properties of the Physalia found in the South Seas. What had been intended to result in a state of protective immunity, much to their surprise, resulted in a state of hypersusceptibility of the tissues of the animals receiving the injections. The administration of this same extract into normal animals produced no adverse effect. In 1837, Magendie (Figure  1.63) noted the violent death of rabbits following repeated injections of egg albumin, and published his findings in 1839. Charles Richet (Figure 1.64) and Paul Portier (Figure 1.65) decided to consider that state of hypersusceptibility as the opposite of protection or prophylaxis. They used the prefix ana, derived from the Greek, which does not mean against. Although chosen in error, its

Thus, it is apparent that complement has captured the interest of an eclectic group of investigators from biochemists to internists. Indeed, the century-old science has emerged as the distinct and independent discipline of “complementology,” with its proponents often referring to themselves as “complementologists.” The future holds bright prospects that should further elucidate the significant role that the complement system plays in molecular and cellular biology, as well as in health and disease.

Anaphylaxis Charles Richet (1850–1935) and Paul Portier, in 1902, observed that the injection of toxic extracts from the tentacles of various sea anemones or eel serum into dogs, which had 3 weeks previously received a smaller injection of the

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Figure 1.64  Charles Richet, Nobel laureate for his discoveries related to anaphylaxis.

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Figure 1.67  Kimishige and Terako Ishizaka.

Figure 1.65  Paul Portier, colleague of Charles Richet in research on anaphylaxis.

use became well established to imply antibody-mediated hypersensitivity in man and other animals. Richet continued to study the phenomenon and was awarded the Nobel Prize for his work in 1913. A. Besredka wrote extensively on anaphylaxis. Henry Hallett Dale (1875–1968) (Figure  1.66), British investigator who made a wide range of scientific contributions including work on the chemistry of nerve impulse transmission, the discovery of histamine, and the development of an in vitro assay for anaphylaxis termed “the Schultz–Dale test for anaphylaxis.” He received a Nobel Prize in Medicine or Physiology in 1935. In the Schultz–Dale test, strong contraction of the isolated uterine horn muscle of a virgin guinea pig that has been either actively or passively sensitized occurs following the addition of specific antigen to the 37°C tissue bath in which it is suspended. Muscle contraction is caused

by the release of histamine and other pharmacological mediators of immediate hypersensitivity following antigen interaction with antibody fixed to tissue cells. Following the demonstration of the three major classes of immunoglobulin, IgG, IgA, and IgM, a patient with multiple myeloma was found to have significant quantities of antibody or immunoglobulin in the serum which did not cross-react with the antisera against the three known major immunoglobulin classes. Kimishige and Terako Ishizaka (Figure 1.67) described this newly discovered immunoglobulin as IgE. It was found to occur in minute quantities in all normal human sera examined, and to be responsible for the mediation of anaphylaxis in man. Although certain IgG antibodies are associated with anaphylactic reactions in guinea pigs, human anaphylaxis is dependent upon these IgE antibodies, which were formerly known as reagins, a group of antibodies with physical and biological characteristics different from those producing the classical serological reactions such as precipitation and agglutination. The IgE antibodies, which have the basic four-chain unit structure of immunoglobulin molecules, are bound through their Fc receptors to mast cells of the tissue or basophils of the blood. Following the combination of the antigen-binding receptors with antigen or allergen, the pharmacological mediators stored in granules in the cytoplasm of the cells are released, resulting in increased vascular permeability, smooth muscle contraction, and vasomotor shock. Kimishige Ishizaka (1925– ) and Terako Ishizaka, discovered IgE and have contributed to elucidation of its function.

Figure 1.66  Sir Henry H. Dale, Nobel laureate, is known for the in vitro anaphylaxis assay, the Shultz–Dale test.

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Among the many substances in clinical use that are known to induce hypersensitivity of the anaphylactic type, penicillin and related antibiotic substances are among the more notorious. This led to the search for many substitute drugs to circumvent allergic reactions.

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Charles Robert Richet (1850–1935), Parisian physician who became professor of physiology at the University of Paris. He was interested in the physiology of toxins, and with Portier discovered anaphylaxis, for which he was awarded the Nobel Prize in Medicine or Physiology in 1913. He and Portier discovered anaphylaxis in dogs exposed to the toxins of murine invertebrates to which they had been previously sensitized. Thus, an immune type reaction that was harmful rather than protective was demonstrated. Experimental anaphylaxis was later shown to be similar to certain types which lent clinical as well as theoretical significance to the discovery. L’Anaphylaxie, 1911. Paul Jules Portier (1866–1968), French physiologist who, with Richet, was among the first to describe anaphylaxis. Alexandre Besredka (1870–1940), Parisian immunologist who worked with Metchnikoff at the Institut Pasteur. He was born in Odessa. He contributed to studies of local immunity, anaphylaxis and antianaphylaxis. Anaphylaxie et Antianaphylaxie, 1918; Histoire d’une Idee: L’Oeuvre de Metchnikoff, 1921; Etudes sur l’Immunité dans les Maladies Infectieuses, 1928. Daniel Bovet (1907–1992), primarily a pharmacologist and physiologist, Bovet received the Nobel Prize in 1957 for his contributions to the understanding of the role histamine plays in allergic reactions and the development of antihistamines. Structure Chimique et Activite Pharmacodynamique des Médicaments du Systeme Nerveux Vegetatif, 1948; Curare and Curare-Like Agents, 1959.

Allergy and Atopy The Jekyll and Hyde nature of the body’s immune response mechanism was recognized early by Clemens von Pirquet (Figure  1.68), who introduced the term allergy to denote a state of altered reactivity. It was found that some types of allergy or hypersensitivity could be passively transferred to previously nonreactive individuals with serum, whereas others required lymphoid cells. Coca (Figure  1.69) and Cooke introduced the term atopy, which means out of place or strange disease, to characterize certain hypersensitivity states which were considered to be heritable and to be limited in occurrence to the human being. The atopic diseases include hay fever and allergy to foods and pollen, and constitute the so-called clinical allergies. Antibodies in the blood sera of atopic patients were described as being different from the precipitins and agglutinins involved in common serological reactions. The reagins, as these antibodies were called, are now known to belong to the IgE class of immunoglobulin. Two physicians, Prausnitz (Figure 1.70) and Küstner, transmitted sensitivity to fish from one of them to the other by the injection of serum. They were the first to demonstrate the passive transfer of specific reactivity with serum from atopic individuals to local skin sites in normal recipients, who subsequently demonstrate a wheal and flare response following

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Figure 1.68  Dr. Clemens Freiherr von Pirquet.

injection of specific antigen into the same site. In studies on the relationship of hypersensitivity to immunity, Arthus, in 1903, observed that repeated injections of protein antigen into the same skin site of rabbits led to a hemorrhagic and necrotic reaction. This local hypersensitivity reaction was subsequently demonstrated to be caused by immune complexes, classified by Coombs and Gell as type III hypersensitivity. This phenomenon, known since that time as the Arthus reaction, is proven by its specificity and dependence upon immune complexes to be immunologic in nature. By contrast, the Shwartzman phenomenon, which is remarkably similar to the Arthus reaction in many respects, is not immunologic. In

Figure 1.69  Dr. Arthur F. Coca.

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for tuberculosis. He held academic appointments at Vienna, Johns Hopkins, and Breslau, and returned to Vienna in 1911 as director of the University Children’s Clinic. Die Erumkrankheit (with Schick), 1905; Klinische Studien über Vakzination und Vakzinale Allergie, 1907; Allergy, 1911. Arthur Fernandez Coca (1875–1959), American allergist and immunologist. He was a major force in allergy and immunology. He named atopic antibodies and was a pioneer in the isolation of allergens. Together with Robert A. Cooke, Coca classified allergies in humans. Robert Anderson Cooke (1880–1960), American immunologist and allergist who was instrumental in the founding of several allergy societies. With Coca, he classified allergies in humans. Cooke also pioneered skin test methods and desensitization techniques. Figure 1.70  Dr. Carl Prausnitz.

1928, Gregory Shwartzman (Figure 1.71) found that a hemorrhagic and necrotic reaction could be produced at a local site 4 h following a provocative intravenous injection of a small amount of Salmonella typhi culture filtrate in rabbits treated 24 h previously by an intradermal injection of an agar culture washing of the microorganisms. This led to the localized Shwartzman reaction. If both injections were administered intravenously, the generalized Shwartzman reaction took place with features closely resembling disseminated intravascular coagulation (DIC). Clemens Freiherr von Pirquet (1874–1929), Viennese physician who coined the term allergy and described serum sickness and its pathogenesis. He also developed a skin test

Figure 1.71  Dr. Gregory Shwartzman.

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Carl Prausnitz-Giles (1876–1963), German physician from Breslau who conducted extensive research on allergies. He and Küstner successfully transferred food allergy with serum. This became the basis for the Prausnitz–Küstner test. He worked at the State Institute for Hygiene in Breslau and spent time at the Royal Institute for Public Health in London earlier in the century. In 1933, he left Germany and practiced medicine on the Isle of Wight. Nicolas Maurice Arthus (1862–1945), Paris physician. He studied venoms and their physiological effects, and was the first to describe local anaphylaxis, or the Arthus reaction, in 1903. Arthus investigated the local necrotic lesion resulting from a local antigen–antibody reaction in an immunized animal. De l’Anaphylaxie a l’Immunité, 1921. Gregory Shwartzman (1896–1965), Russian–American microbiologist who described systemic and local reactions that follow the injection of bacterial endotoxins. The systemic Shwartzman reaction, a nonimmunologic phenomenon, is related to disseminated intravascular coagulation. The local Shwartzman reaction in skin resembles the immunologically based Arthus reaction in appearance. Phenomenon of Local Tissue Reactivity and Its Immunological and Clinical Significance, 1937. Following the development of antitoxin for the treatment of diphtheria, clinicians noted an adverse side effect of the injection of the horse serum antitoxin. Within 8–12 days following the administration of a single large dose of diphtheria antitoxin, children began to develop signs and symptoms of serum sickness. Von Pirquet and Schick reported their observations in a book entitled Die Serumkrankheit (Serum Sickness). The signs and symptoms they observed were caused by the formation of immune complexes which fixed complement, attracted polymorphonuclear neutrophils, and led to inflammation in anatomical sites where the complexes were deposited, including the vessels, kidneys, and joints.

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As discussed later, Hans Zinsser, famous American bacteriologist and immunologist, in 1925 pointed to the differences between immediate and delayed-type hypersensitivity. Thus, these early investigators had observed two main features associated with allergy or hypersensitivity. They found that it was important to know whether the reaction occurred within seconds to minutes following the administration of antigen, in which case it was referred to as immediate hypersensitivity, or whether it occurred hours to days after the administration of antigen, in which case it was termed delayed-type hypersensitivity. Besides the significance of this temporal relationship with respect to antigen administration, they observed what was necessary to transfer the hypersensitivity passively, whether serum or specifically sensitized lymphoid cells. Frank J. Dixon and associates developed an experimental model for serum sickness in the 1950s. As antigen, they chose iodinated bovine serum albumin and injected it into rabbits. They followed the elimination of the antigen in three phases, one of which was immune elimination, and studied the development of experimentally induced pathological lesions in the vessels, kidneys, and joints of the animals correlating with the immune complex content of the blood serum. As a result of studies by these and numerous other investigators, immune complex disorders have been demonstrated to be dynamic processes implicated in the pathogenesis of a host of immunological diseases. The pathological alterations which they induce are governed by their molar ratios in the deposition sites. The British immunopathologists Gell and Coombs developed a simplified classification of immunopathological phenomena that fall within the realm of hypersensitivity (type I, anaphylaxis; type II, cytotoxicity; type III, immune complex disease; type IV, delayed-type hypersensitivity). Immune complex disorders of the serum sickness variety are called type III hypersensitivity in the Gell and Coombs classification. The Scripps Clinic and Research Foundation directed by Dixon became a center for immunology research. By applying modern techniques such as radiolabeling of serum proteins to a disease process which had been described almost half a century earlier, they were able to shed light on some of the more important basic mechanisms of immunologically mediated human diseases. Cochrane and associates at Scripps carried out fundamental studies on the uptake of immune complexes by polymorphonuclear neutrophils (PMNs) and the role of complement in this phenomenon. Other investigators have contributed much to our understanding of alterations in complement during the course of disease processes. Frank James Dixon (1920–2008), American physician and researcher noted for his fundamental contributions to immunopathology that include the role of immune complexes in the production of disease. He is also known for his work on antibody formation. Dixon was the founding director of the Research Institute of Scripps Clinic, La Jolla, California.

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Cellular Immunity and Hypersensitivity Metchnikoff was among the first investigators to appreciate the role of cells in the immune response. He attempted to explain immunity to infectious diseases on the basis of the capabilities of phagocytes to destroy infecting microorganisms in vivo by absorption phenomena. This was attributed to the action of two digestive ferments, one of which was released to the plasma and body fluids. The component referred to as cytase, comprising macrocytase and microcytase, by Metchnikoff was the same substance Ehrlich called das Komplement. Metchnikoff’s term fixateur meant the same as Ehrlich’s Amboceptor. Thus, this represented the first cellular theory of immunity which is quite different from our modern concept of the role that cells play in the immune response. In his extensive studies on tuberculosis, Robert Koch, director of the Institute for Infectious Diseases in Berlin, discovered that an extract which he termed “old tuberculin” was able to induce delayed hypersensitivity skin reactions in guinea pigs as well as in humans. Histological studies of these local sites of reactivity in the skin revealed accumulations of lymphocytes. Delayed skin reactivity was found to be manifest within 24 h of the inoculation and to reach maximum reactivity at about 48 h after injection. Albert Calmette (1863–1933) (Figure  1.72), French physician who was subdirector of the Institut Pasteur in Paris. In a popular book published in 1920, Bacillary Infection and Tuberculosis, he emphasized the necessity of separating tuberculin reactivity from anaphylaxis. Together with Guérin, he perfected bacillus Calmette–Guérin (BCG) vaccine and also investigated snake venom and plague serum. Hans Zinsser (1878–1940) (Figure 1.73), a leading American bacteriologist and immunologist who was a Columbia,

Figure 1.72  Albert Calmette (right) and C. Guérin (left).

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Henry Sherwood Lawrence (1916–2004), American immunologist. While studying type IV hypersensitivity and contact dermatitis, he discovered transfer factor. Cellular and Humoral Aspects of Delayed Hypersensitivity, 1959.

Figure 1.73  Hans Zinsser.

Stanford, and Harvard educator, whose work in immunology included hypersensitivity research, plague immunology, formulation of the unitarian theory of antibodies, and demonstration of differences between tuberculin and anaphylactic hypersensitivity. His famous text Microbiology (with Hiss), 1911, has been through two dozen editions since its first appearance. From the turn of the century until approximately the early 1960s, in vivo reactivity manifested as skin lesions was essentially the only means to test for cell-mediated immune reactivity. However, Rich and Lewis, in 1928, developed a technique for the demonstration of delayed-type hypersensitivity (DTH) in vitro, which involved the immobilization of cells migrating from the edges of small fragments of lymphoid tissue in culture in the presence of specific antigens. Of course, this was the predecessor of the migration inhibition factor (MIF) assay, which was widely employed in both clinical and research immunology laboratories. In the early 1940s, Landsteiner and Merrill W. Chase, and Battisto, performed extensive investigations into DTH reactions to simple chemical haptens and described the successful transfer of reactivity to previously nonreactive animals by suspensions of lymphoid cells. H.S. Lawrence, in 1949, described transfer factor, which was an extract of leukocytes from tuberculin positive-reacting patients that was released upon freezing and thawing the cells, spinning down the cellular debris and transferring the cell-free supernatant into tuberculin-negative individuals. These recipients became tuberculin positive. Thus, a cell-free extract proved capable of conferring specific delayed-type hypersensitive reactivity on individuals not previously contacted by the antigen 4. Transfer factor has been extensively studied biochemically, biologically and by other methods.

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A real breakthrough in cell-mediated immunity came with the demonstration that lymphocytes exposed to phytohemagglutinin or other plant mitogens could be successfully cultured in vitro over extended periods of time. This came about by serendipity. In blood-banking procedures where a plant extract called phytohemagglutinin (PHA) was added to agglutinate red cells, it was found that, upon centrifugation, the buffy-coat lymphocytes underwent blast transformation and developed the ability to survive over extended periods, a property not shared by lymphocytes untreated with phytohemagglutinin. Besides the plant lectin’s hemagglutinating property, it also had a mitogenic principle that induced blast transformation and division of lymphocytes in culture. These findings secured the lymphocyte as the central cell in the immune response. With this wealth of new information, lymphocytes were demonstrated to release a variety of soluble mediator substances termed lymphokines. Some of these were demonstrated to have effects on other cells such as MIF produced by lymphocytes, which actually prevents the migration of macrophages from the sites where they are needed. John R. David and Barry R. Bloom, working independently, showed that immune lymphoid cells activated by their corresponding antigen secrete a substance that inhibits macrophage migration, a feature of delayed hypersensitivity. They termed this soluble factor migration inhibitory factor (MIF). David found that MIF had a molecular weight greater than 10,000Da and was not preformed. Bloom and associates proved that lymphocytes synthesized MIF and macrophages were the target. Stimulation of lymphocytes was not antigenspecific, as mitogens or purified protein derivative (PPD) from tubercle bacilli could induce MIF synthesis and release. MIF was not species-specific. The description of MIF and its properties was the first demonstration that soluble factors regulate immune responses and play a significant role in intercellular communication. Billingham and Simonsen, working independently, discovered the graft-versus-host reaction (GVHR) in 1957. They demonstrated that the inoculation of immunocompetent lymphoid cells into an immunosuppressed host from whatever cause could lead to a reaction of the lymphoid cell graft against the host on the basis of allogeneic differences between the two. This graft-versus-host disease is very significant in bone marrow transplantation, or can occur following the administration of a unit a blood containing as few as 1.0 × 106 lymphocytes to a child with T-cell immunodeficiency. The Koch phenomenon is a delayed hypersensitivity reaction in the skin of a guinea pig after it has been infected with Mycobacterium tuberculosis. Robert Koch described the

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phenomenon in 1891 following the injection of either living or dead M. tuberculosis microorganisms into guinea pigs previously infected with the same microbes. He observed a severe necrotic reaction at the site of inoculation, which occasionally became generalized and induced death. The injection of killed M. tuberculosis microorganisms into healthy guinea pigs caused no ill effects. This is a demonstration of cellmediated immunity and is the basis for the tuberculin test.

Immunobiology and Cellular Immunology There was a renaissance of interest in cellular immunology from 1942 through 1952. In 1942, Coons perfected immunofluorescence to demonstrate antigens and antibodies in cells. That same year, Landsteiner and Chase reported the transfer of delayed-type hypersensitivity with lymphoid cells but not with serum.

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To speak of cellular immunity apart from humoral immunity could be considered unwarranted, because in actuality all of immunology is cellular. The renewed interest in cellular immunology is reminiscent of the revolution brought about by Virchow when he published his lectures on cellular pathology in 1858. Nevertheless, it is important for us to trace the main events relating cells of the lymphoid system to antibody production and cell-mediated immunity. Even in the 1920s, Murphy, of the Rockefeller Institute for Medical Research, wrote a monograph on the lymphocyte. In 1945, Harris and associates discussed the role of the lymphocyte in antibody formation, and in 1948, Astrid Fagraeus (Figure 1.74) wrote her doctoral thesis on the role of the plasma cell in antibody formation. While attempting to immunize chickens in which the bursa of Fabricius had been removed, Glick (Figure 1.76)

Figure 1.75  Milan Hasek.

Figure 1.74  Astrid Fagraeus.

Figure 1.76  Bruce Glick.

and associates (1956) noted that antibody production did not take place. This important discovery, which appeared in the journal Poultry Science, subsequently permitted the demonstration that the immune system of the chicken is divisible into a thymic-dependent T-cell system and a bursa-dependent B-lymphocyte limb. Glick correctly interpreted a laboratory mistake to show the role of the bursa of Fabricius in the production of antibody and the division of labor in lymphocyte populations. H. Wolfe, University of Wisconsin, and Robert Good (Figure  1.77), University of Minnesota, immediately realized the significance of this finding for childhood immunodeficiencies. Good and his colleagues in Minneapolis and J.F.A.P. Miller (Figure 1.78) in Melbourne went on to show the role of the thymus in the immune response, and various

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Figure 1.77  Robert Alan Good.

investigators began to search for bursa equivalents in many other animals. Interestingly, William Hewson’s (1739–1774) early concepts on the thymus were remarkably correct. J.F.A.P. Miller demonstrated the role of the thymus in immunity while in pursuit of Gross leukemia virus in neonatal mice. Dr. Good and associates helped establish the role of the thymus in the education of lymphocytes and made fundamental contributions to understanding the ontogeny and phylogeny of immunity. Hasek (Figure 1.75), studying parabiosis in chick embryos, made fundamental contributions to immunologic tolerance and transplantation biology. In 1959, Gowans (Figure  1.79) proved that lymphocytes actually recirculate. His insight that the lymphocytes recirculate via the thoracic duct made radical changes in the understanding of the role of lymphocytes. In 1966, Harris, Hummeler, and Harris clearly demonstrated that lymphocytes could form antibodies. In 1966 and 1967, Claman, Davies, and Mitchison showed that

Figure 1.78  J. F. A. P. Miller.

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Figure 1.79  J. L. Gowans.

T and B lymphocytes cooperate with one another in the production of an immune response. Henry N. Claman (1930– ) produced some of the first evidence that T and B cells act synergistically in the humoral response. Various phenomena, such as the switch from forming one class of immunoglobulin to another by B cells, were demonstrated to be dependent upon a signal from T cells, which could induce B cells to change from immunoglobulin IgM to IgG or IgA production. B cells stimulated by antigen in which no cell signal was given continued to produce IgM antibody. Such antigens were referred to as thymic-independent antigens, and others requiring T-cell participation as thymic-dependent antigens. In 1971, Gershon (Figure 1.80) and Kondo demonstrated that

Figure 1.80  R. Gershon.

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among the T-lymphocyte subsets were suppressor T cells. This was one of the first demonstrations of the suppressive role of the T cell. They were the subject of much investigation and were postulated to play an important role in immunoregulation and in various autoimmune disease states of man and animals. Thus, immune regulation was considered to depend in part upon a delicate balance between helper T lymphocytes and these T-suppressor cells. Later advances in molecular biology cast some doubt on the suppressor concept. Jerne (1974) proposed a network theory of immune regulation, and Cantor and Boyse (1975) described T-cell subclasses that were distinguished by their Ly antigens. They also investigated cell cooperation in the cell-mediated lympholysis (CML) reaction. Astrid Elsa Fagraeus-Wallbom (1913– ), Swedish investigator noted for her doctoral thesis which provided the first clear evidence that immunoglobulins are made in plasma cells. In 1962, she became chief of the Virus Department of the National Bacteriological Laboratory, and in 1965, professor of immunology at the Karolinska Institute in Stockholm. She also investigated cell membrane antigens and contributed to the field of clinical immunology. Antibody Production in Relation to the Development of Plasma Cells, Stockholm, 1948. Milan Hasek (1925–1985), Czechoslovakian scientist whose contributions to immunology include investigations of immunologic tolerance and the development of chick embryo parabiosis. Hasek also made fundamental contributions to transplantation biology. Bruce Glick, as a graduate student at Ohio State University, became interested in the bursa of the chicken and removed the organ from the cloacas of some test animals for study. Some of the bursectomized chicks happened to be used for a class demonstration of antibody formation and failed to produce antibody. Glick and associates sent an article to Science, which was refused, so they published their finding in Poultry Science. Harold Wolfe, at the University of Wisconsin, Madison, understood the importance of this fact, and the hunt began to identify the two big classes of lymphocytes, T and B cells, the latter being so called because they are “bursa derived.”

Atlas of Immunology, Third Edition

J.F.A.P. Miller (1931– ) (Figure 1.78), proved the role of the thymus in immunity while investigating gross leukemia in neonatal mice. James Gowans (1924– ), (Figure 1.79) British physician and investigator whose principal contribution to immunology was the demonstration that lymphocytes recirculate via the thoracic duct, which radically changed the understanding of the role lymphocytes play in immune reactions. He also investigated lymphocyte function. He served as director of the Medical Research Council (MRC) Cellular Immunobiology Unit, Oxford, 1963. Richard K. Gershon (1932–1983), (Figure 1.80) one of the first to demonstrate the suppressor role of the T cell. The suppressor T cell was described as a subpopulation of lymphocytes that diminish or suppress antibody formation by B cells or downregulate the ability of T lymphocytes to mount a cellular immune response. The inability to confirm the presence of receptor molecules on their surface has cast a cloud over the suppressor cell; however, functional suppressor-cell effects are indisputable. In 1975, Köhler and Milstein successfully fused splenic lymphocytes from mice forming antibody with tumor cells to produce what they called a hybridoma. The spleen cells conferred the antibody-forming capacity, while the tumor cells provided the capability for immortality or endless reproduction. This important technique permitted the formation of a homogeneous antibody population of a desired specificity by taking the spleen cells from animals specifically immunized with a certain antigen. At the 1980 International Congress of Immunology held in Paris, Henry Kaplan and Lennart Olsson of Stanford University reported a hybridoma formed with human cells. Zinkernagel and Doherty (Figure 1.81) (1975)

Henry Claman in Denver and A.J.S. Davies in London conducted studies with lethally irradiated mice, which proved that both bursa-derived and thymus-derived lymphocytes were needed to produce an immune response, T and B cooperation. Robert Alan Good (1922–2003), (Figure  1.77) American immunologist and pediatrician who has made major contributions to studies on the ontogeny and phylogeny of the immune response. Much of his work focused on immunodeficiency diseases and the role of the thymus and the bursa of Fabricius in immunity. He and his colleagues demonstrated the role of the thymus in the education of lymphocytes. The Thymus in Immunobiology, 1964; Phylogeny of Immunity, 1966.

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Figure 1.81  Peter Doherty and Rolf Zinkernagel.

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Phenomenon of Local Tissue Reactivity in 1937 that the injection of one dose of endotoxin into a tumor site followed 24 h later by an intravenous injection of endotoxin resulted in a local Shwartzman reaction, leading to hemorrhage and necrosis in the tumor.

Figure 1.82  Pamela Björkman.

proposed an altered-self concept and dual recognition by T cells. They proved major histocompatibility complex (MHC) restriction in the reaction of T cells with antigen. Further investigations of T cells have proceeded at an unprecedented pace. Pamela J. Björkman (Figure  1.82) was trained by Don Wiley in x-ray crystallography at Harvard. She determined the 3D structure of the human leukocyte antigen (HLA) molecule HLA-A2. Björkman described a prominent groove on the upper surface of the molecule which, surprisingly, contained antigen even after purification. The antigen and top edges of the groove were contiguous; from that vantage point, the T cell receptor is able to “see” chemical information from both the antigen and HLA-A2. Demonstration of this structure revealed how T lymphocytes could recognize antigen and HLA protein simultaneously, which is requisite to initiate killing.

Studies on transplantable tumors in inbred strains of mice are discussed elsewhere in this volume. In the 1950s, F. M. Burnet and Lewis Thomas (Figure 1.83) proposed a concept of immune surveillance to describe the interaction between tumor cells and the immune system. They postulated that somatic mutations of cells with the potential to develop into malignant tumors are recognized as alien by antigenic determinants not present on normal cells, and are destroyed by immunocytes that police the bodily tissues to eliminate cells not recognized as self by seek-and-destroy tactics. They theorized that failure of the immune system to carry out this function could lead to proliferation of these aberrant cells into a tumor mass too great to be eliminated by immunological means. Indirect evidence offered in support of such a concept included the increased frequency of tumors in children with immunodeficiency of the T-cell system, tumors that appear in individuals on prolonged immunosuppressive therapy, as in transplant patients, and the increase in tumor incidence with advancing age. One of the many paradoxes in cancer immunology is the phenomenon of immunological enhancement, in which antibodies favor the establishment of a tumor graft rather than hasten its rejection. Immunological enhancement was defined by Kaliss as the “successful establishment of a tumor

Tumor Immunity It has long been the dream of medical scientists to be able to resist cancer by immunological methods. William Coley, in 1891, observed that cancer patients who developed certain infections derived beneficial results. This led him to inject mixtures of bacterial toxins into cancer patients in an attempt to alter the pathogenesis of their malignant disease. His efforts met with success in 1893 when an inoperable tumor in a 16-year-old boy receiving bacterial toxin regressed and disappeared over several months of treatment. Coley applied his treatment to 250 other cancer victims whose survival ranged from 5 to 72 years. Perhaps because of the unavailability of sufficient immunological information to understand Coley’s experiments at the time, their significance was not appreciated until years later. Shwartzman reported in his classic monograph on The

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Figure 1.83  Lewis Thomas.

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homograft and its progressive growth (usually to death of the host) as a consequence of the tumor’s contact with specific antiserum in the host.” This definition was later altered to the successful establishment or prolonged survival (conversely the delayed rejection) of an allogeneic graft. In 1932, Casey observed enhancement of Brown–Pearce sarcoma in rabbits. He reported the presence of a principle termed XYZ factor in preserved preparations of Brown–Pearce sarcoma, which, upon injection into rabbits 2 weeks prior to transplantation of the tumor, resulted in an increased incidence of tumors, larger metastases, and a briefer period of survival following transplantation. Snell and associates studied tumor enhancement using inbred strains of mice of known genetic constitution. These investigators aimed at solving the mechanism and other aspects of immunological enhancement. Besides antibody, immune complexes comprising antigen and antibody were found to favor the establishment of a tumor by blocking or interfering with the immunological rejection mechanism. Karl and Ingegerd Hellström demonstrated immune complexes that acted as blocking factors in the serum of certain cancer patients. They subsequently discovered unblocking factors capable of eliminating complexes that interfere with the immune system of the tumor-bearing host. A renaissance of interest in Dr.  Coley’s treatment occurred following the demonstration that tumorbearing mice injected with bacillus Calmette–Guérin (BCG)attenuated tuberculosis vaccine showed regression of their tumors. After that, numerous clinical trials with BCG were carried out in selected tumor patients with considerable success. Malignant melanoma, breast cancer, mycosis fungoides, and acute lymphoid leukemia are among tumors treated with some success by BCG immunotherapy. Foley, in 1953, provided the first clear demonstration of specific antigenicity in a class of experimental tumors. He found that ligation and atrophy of methylcholanthrene-induced sarcoma in mice of the C3H-He pure line were followed by immunity. In 1957, Prehn extended Foley’s observation to provide conclusive evidence of an immune response to cancer-specific antigens of autochthonous tumors of putatively nonviral origin. In 1965, Phil Gold described carcinoembryonic antigen (CEA) in the sera of patients with cancer of the colon. At first there was great hope that the detection of this antigen in the blood sera of patients might aid in the diagnosis of cancer. Unfortunately, patients with other types of cancer as well as certain nonneoplastic diseases may experience derepression of α gene encoding its formation. It is still useful to monitor blood sera for the reappearance of CEA in patients who have undergone surgical excision of colonic cancer, to signal any recurrence or metastasis of the malignancy. Another cellcoded antigen that shows a more restricted association with one type of tumor than does CEA is α-fetoprotein observed in the sera of patients with hepatoma. Regrettably, however, this is of little consequence for the patient in whom no effective therapy can be applied. Burkitt lymphoma, a B-cell malignancy, which shows strong evidence of association with the

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Epstein–Barr virus, shows remission in some patients. This appears to have an immunologic basis.

Autoimmunity The historical development of autoimmunity has been presented in the past in widely scattered notes in the medical literature. In an attempt to assimilate an account of the metamorphosis of this fascinating chapter in the history of immunology, we present the following major advances in autoimmunity and their discoverers, who are no less fascinating than the subject itself. It should be pointed out that a comprehensive or exhaustive review of the literature of autoimmunity was not our intention. By contrast, we only outline the milestones in the development of autoimmunity, which has significance not only for selected disease mechanisms but also for many features of normal immune reactivity and immunoregulation. In 1900, Ehrlich and Morgenroth found that the injection of self-antigens failed to elicit an immune response in the autologous host. On discovering that goats immunized with their own erythrocytes failed to produce autoantibodies, Ehrlich formulated a concept of “horror autotoxicus” to explain the animal body’s failure to mount an immune response against itself. Nevertheless, he recognized also that autoimmunity might occur as an aberration and lead to disease. As Ehrlich and Morgenroth stated: “In the explanation of many disease phenomena, it will in the future be necessary to consider the possible failure of the internal regulation, as well as the action of directly injurious exogenous or endogenous substances.” Apparently this latter report was overlooked in the confusion of the World War I, and only the earlier concept of horror autotoxicus prevailed in succeeding years, leading future generations of immunologists to be told incorrectly that Ehrlich had not appreciated the possibility that autoimmunity could constitute a part of the etiopathogenesis of selected disease states. An investigator of rare genius and foresight, Ehrlich was already making contributions that still have relevance in cellular immunology and molecular genetics today, even before de Vries rediscovered Mendel’s basic principles of inheritance in 1901. Subsequently, Donath and Landsteiner showed that autoantibodies were responsible for paroxysmal cold hemaglobinuria in syphilis. Wassermann and colleagues reported a diagnostic serologic test for syphilis based on autoantibodies. Meanwhile, many claims were made that one type of disease process or another had an autoimmune basis. These have been reviewed extensively. For many years immunologists accepted Ehrlich’s dictum that the animal body would not form harmful immunologic reactions against itself. Half a century later, Burnet and Fenner expressed the concept as “self-tolerance” and suggested the presence of a thymic censor which was supposed to prevent the appearance of forbidden clones. Once early investigators began to use antigens from diverse sources, Metchnikoff and other pathfinders in immunology

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reported the development of cytotoxic antibodies in animals immunized with spermatozoa and tissue cells, thereby initiating the concept of autoallergy in which an animal mounts an autoimmune response against its own self-antigens. Early discoveries about eye diseases in which autoallergy was found to play a key role included phacoanaphylaxis, caused by inflammatory reactivity against protein antigens released from a traumatized lens capsule. Sympathetic ophthalmia was also found to occur as a result of autoallergic sensitization following trauma to one eye, leading to immunologic sensitization of the host against the uninjured companion eye. Although the use of adjuvant substances to potentiate the immune response to antigens dates from the time of Pasteur, Freund and co-workers established (1942) the efficiency of water-in-oil emulsions in potentiating the antibody response to a variety of antigens. The water-in-oil emulsion without added mycobacteria, known as incomplete adjuvant, stimulated sensitivity of the immediate (immunoglobulin-mediated) variety, which was dependent upon antibody titers in the serum, in contrast to sensitivity of the delayed or tuberculin-type, which was mediated by sensitized lymphoid cells. It was soon found that the incorporation of various normal tissues into Freund’s adjuvant, or similar adjuvant-like materials, could lead to the production of an autoimmune response in the animal body. In 1951, Voisin and colleagues produced aspermatogenesis experimentally by the injection of testicular tissue incorporated into Freund’s adjuvant. It was postulated that the use of an adjuvant material such as Freund’s adjuvant might cause stimulation of so-called forbidden clones, according to the clonal selection concept of acquired immunity, and that these forbidden clones might augment an immune response against self-antigens leading to pathological sequelae. Witebsky developed certain criteria later known as Witebsky’s postulates that were patterned after Koch’s postulates in bacteriology. Ernest Witebsky (1901–1969), German–American immu­ nologist and bacteriologist who made significant contributions to transfusion medicine and to concepts of autoimmune diseases. He was a direct descendent of the Ehrlich school of immunology, having worked at Heidelberg with Hans Sachs, Ehrlich’s principal assistant, in 1929. He went to Mt. Sinai Hospital in New York in 1934 and became professor at the University of Buffalo in 1936, where he remained until his death. A major portion of his work on autoimmunity was the demonstration with Noel R. Rose of experimental autoimmune thyroiditis. According to these criteria, an autoimmune response should be considered the cause of a human disease if:

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1. It is regularly associated with that disease; 2. Immunization of an experimental animal with antigen from the appropriate tissue causes the animal to make an immune response (i.e., form antibodies or develop a cell-mediated response);



3. Associated with this response, the animal develops pathological changes that are basically similar to those of the human; 4. The experimental disease can be transferred to nonimmunized animals by serum or by lymphoid cells.

Of course, in human diseases, it is difficult to gather all of these criteria to establish a particular disease process as having an autoimmune etiology or pathogenesis. Witebsky’s postulates reflect the thinking of that period concerning autoimmunity when a single cause was often believed to be responsible for an autoimmune disease. In comparison with Koch’s postulates, where a single microorganism was proven to be the etiologic agent of an infectious disease, Witebsky’s postulates were designed to follow the same pattern. Many investigators suspected that viruses were especially significant etiologic agents of autoimmune diseases. Indeed, viruses do represent one of several factors important in the etiology and pathogenesis of autoimmunity. It was also widely held that disease resulted from qualitative abnormalities resulting in autoimmune disease. In recent years, several investigators have attempted to present a unifying concept of autoimmunity since the demonstration that autoimmune reactions may be physiologic as well as pathologic. Contemporary immunologic research has demonstrated clearly that self-reactivity is entirely normal. Indeed, normal immune function and immune regulation are dependent upon appropriate self–self interactions. It is now recognized that quantitative rather than qualitative abnormalities, including the relative quantities of autoantibody or immune complexes formed or the degree of stem cell proliferation, may lead to disease. Recent research demonstrates also that autoimmune diseases have a multifactorial etiology, where genetics, environmental and hormonal factors, and defective immune regulation are now recognized to all act in concert to produce an autoimmune disease. In contrast to the horror autotoxicus concept of Ehrlich and Morgenroth and Burnet’s forbidden clone explanation, Boyden, Grabar and Kay, and Makinodan emphasized the physiologic regulatory functions of autoimmunity. In an attempt to explain how physiologic autoimmunity might be both physiologic and pathologic, and in an attempt to account for the various etiologic factors, Beutner and colleagues presented a “unified concept of autoimmunity.” They based this concept on the effects produced in vivo by autoantibodies or autoreactive T cells in the autologous host. Smith and Steinberg described a new classification for autoimmune diseases, in the light of current information.

Development of Immunobiology and Cellular Immunology Medawar (1944–1946) provided the first convincing proof that the rejection of grafts between individuals who are not related to one another (i.e., allografts or homografts) has

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immunity, which Nossal and Pike, and Green and co-workers proved to be correct.

Figure 1.84  The Walter and Eliza Hall Institute Group. Left to right: Professor Ian Mackay; Sir Macfarlane Burnet; Sir Gustav Nossal.

an immunological basis. During the same period, Owen described dizygotic cattle twins in which blood cells of one twin were tolerated immunologically by the other (i.e., they were chimeras). Burnet and Fenner at the Walter and Eliza Hall Institute (Figure 1.84) were beginning to take a view of antibody production different from that proposed by chemists adhering to the template theory of antibody production. The second edition of their classic monograph entitled The Production of Antibodies, published in 1949, contains an exposé of their developing concepts. Modifying their views through various explanations that included a self-marker hypothesis to explain antibody production, Burnet noted with interest that Jerne had proposed a selective theory of antibody formation in 1955. Whereas Jerne discussed various antibody populations, Burnet and Talmage independently emphasized replicating cells in a cell selection theory in 1957, leading to formulation of a new hypothesis which Burnet termed the “clonal selection theory of acquired immunity” in 1959. The template theory of antibody production, which had been popular with the chemists for so many years, could no longer explain these new biological revelations that included immunological tolerance, and it had never explained the secondary (or anamnestic) immune response. The coup de grâce to this hypothesis was the observation that mature antibodysynthesizing cells contained no antigen. Burnet proposed lymphoid cells genetically programmed to synthesize one type of antibody. Antigen would have no effect on most lymphoid cells but would selectively stimulate those cells already synthesizing the corresponding antibody at a low rate. The cell surface antibody would serve as receptor for antigen and proliferate into a clone of cells producing antibody of that specificity. Burnet introduced the “forbidden clone” concept to explain autoimmunity. Cells capable of forming antibody against a normal self-antibody were forbidden and eliminated during embryonic life. Since that time various modifications of the clonal selection hypothesis have been offered. The renaissance of cellular immunology developed following Burnet’s description of the clonal selection theory of acquired

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In 1948, Fagraeus established the role of the plasma cell in antibody formation. The fluorescent antibody technique was a major breakthrough for identification of antigens in tissues and subsequently for demonstrating antibody synthesis by individual cells. While attempting to immunize chickens in which the bursa of Fabricius had been removed, Glick and colleagues noted that antibody production did not take place. This was the first evidence of bursa dependence of antibody formation. Good and associates immediately realized the significance of this finding for immunodeficiencies of childhood. He and his associates in Minneapolis and Miller in England went on to show the role of the thymus in the immune response, and various investigators began to search for bursa equivalents in man and other animals. Thus, the immune system of many species was found to have distinct bursa-dependent, antibody-synthesizing and thymus-dependent, cell-mediated limbs. In 1959, Gowans proved that lymphocytes actually recirculate. In 1966, Harris and co-workers demonstrated clearly that lymphocytes could form antibodies. In 1966 and 1967, Claman and colleagues, Davies and colleagues, and Mitchison and associates showed that T and B lymphocytes cooperate with one another in the production of an immune response. Various phenomena, such as the switch from forming one class of immunoglobulin to another by B cells, were demonstrated to be dependent upon a signal from T cells activating B cells to change from immunoglobulin IgM to IgG or IgA production. B cells stimulated by antigen in which no T-cell signal was given continued to produce IgM antibody. Such antigens were referred to as thymusindependent antigens, and others requiring T cell participation as thymus-dependent antigens. Research focused next upon subpopulations of T lymphocytes. Mitchison and colleagues described a subset of T lymphocytes demonstrating helper activity (i.e., helper T cells), whose hyperactivity is well recognized to lead to manifestations of autoimmunity. In 1971, Gershon and Kondo described suppressor T cells that prevent development of autoimmune reactivity under normal circumstances. However, should their function become defective, autoimmunity may result. The concept of suppressor T cells has been brought into question because of the inability to characterize them by molecular biological analysis. However, the concept of immunosuppression, possibly through the action of cyto­ kines, is unquestioned. Subsequent studies concerned interactions among the various cell types of the immune system including interaction with antigen-presenting cells. Thus, elucidation of the T- and B-cell populations and subsets and their role in immunoregulation was requisite for improved understanding of autoimmune mechanisms. Especially critical to autoimmunity research was the discovery of the regulatory role of T cells in the immune response.

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Benacerraf and colleagues, Benacerraf and McDevitt, and McDevitt and Chinitz demonstrated the significant role played by gene products of the major histocompatibility complex in the specificity and regulation of T cell-dependent immune responses. For a review of genetic control of autoimmunity, the reader is referred to Rose and associates and Vaughan. In his network theory of immunity, Jerne suggested that the formation of antibodies against idiotypic specificities on antibody molecules followed by the formation of antianti-idiotypic antibodies constituted a significant additional immunoregulatory process for proper functioning of the immune system. His postulates have been proven valid by numerous investigators. Tonegawa and colleagues and Leder and associates identified and cloned the genes that encode variable and constant regions of immunoglobulin molecules, leading to increased understanding of the origin of diversity in antibody-combining sites. In 1975, Köhler and Milstein successfully fused splenic lymphocytes from mice forming antibody with tumor cells to produce what they called a hybridoma. The spleen cells conferred the antibody-producing capacity while the tumor cells provided the capability for endless reproduction. Monoclonal antibodies are the valuable homogeneous products of hybridomas with widespread application in diagnostic laboratory medicine. Human hybridomas formed by immortalization of lymphoid cells from patients with autoimmune disease, such as systemic lupus erythematosus (SLE), through fusion with a tumor cell line provide monoclonal antibodies that may be employed in the search for self-antigens eliciting autoimmune reactivity.

Autoimmune Manifestations of Disease The historical development of autoimmunity associated with the etiopathogenesis of selected disease processes is presented in the following paragraphs.

Autoimmune Hemolytic Anemia The association of autoantibodies with blood diseases dates from the observation by Donath and Landsteiner that paroxysmal cold hemaglobinuria patients develop a hemolytic antibody in the serum that adsorbs to their red cells at relatively low temperatures, leading to lysis of the cells by complement upon warming to 37ºC. Subsequently, Landsteiner proposed that this antibody was a consequence of autoimmunization with altered tissue components, possibly assisted by the spirochete of syphilis. Other early investigators such as Widal and colleagues performed studies suggestive of a role for autohemagglutinins in acquired hemolytic icterus, and Chauffard and Vincent pointed to the action of hemolysins in the production of acute hemolytic anemia with hemoglobinuria. Dameshek and Schwartz and Dameshek and colleagues (Figure  1.85)

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Figure 1.85  Dr. William Dameshek (left); Dr. H. Hugh Fudenberg (right).

described hemolytic antibodies in the blood sera of individuals with acute hemolytic anemia, pointing to the possibility that selected clinical hemolytic syndromes could be attributable to hemolysins in serum, rather than to intrinsically defective erythrocytes. They injected guinea pigs with rabbit antibody against guinea pig erythrocytes to produce experimental hemolytic syndromes, with associated spherocytosis, increased fragility of cells, and reticulocytosis, closely resembling comparable human disorders. Further investigation of immunologic aspects of human acquired hemolytic anemia was impeded by the lack of techniques for the detection of various types of antibodies against red cells, in particular those not demonstrable by agglutination in vitro. Coombs and colleagues’ introduction of the antiglobulin test for detection of red cells sensitized by incomplete rhesus isoantibodies represented the breakthrough needed for further progress. This technique was also found to be useful for detection of antibodies against red cells from selected idiopathic acquired hemolytic anemia cases. Thus, this test came into widespread use for the detection of incomplete (i.e., nonagglutinating) autoantibodies against erythrocytes. Coombs later credited Moreschi with development of a similar test after the turn of the century, even though this was unknown to Coombs and colleagues when they developed the antiglobulin technique. William Dameshek (1900–1969), noted Russian-American hematologist who was among the first to understand autoimmune hemolytic anemias. He spent many years as editor-inchief of the journal Blood. Robin R. A. Coombs (1921–2006), (Figure  1.86) British pathologist and immunologist who is best known for the Coombs’ test as a means for detecting immunoglobulin on the surface of a patient’s red blood cells. The test was developed in the 1940s to demonstrate autoantibodies on the surface of red blood cells that failed to cause agglutination of the cells. It is a test for autoimmune hemolytic anemia. He has also contributed much to serology, immunohematology, and immunopathology. The Serology of Conglutination and Its

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Relation to Disease, 1961; Clinical Aspects of Immunology (with Gell), 1963. Morton and Pickles developed other serological tests that included enzyme-treatment of erythrocytes to facilitate their agglutination by incomplete antibody. Other investigators described techniques to elute and recover antibody bound to red blood cell surfaces, thus permitting the study of their autoantibody specificity. Antibodies in the circulation and on the surface of erythrocytes were found to shorten red cell survival. Mollison showed that transfused normal 51Cr-labeled red cells, as well as the patient’s own erythrocytes, exhibited a remarkably briefer life span than the normal value of 110 days. Dacie suggested the serious nature of autoimmune acquired hemolytic anemia, pointing to the high mortality rate (>40%) in the warm antibody type, and emphasized the role of circulating autoantibodies against erythrocytes in the pathogenesis of the disease. After Coombs and colleagues introduced the antiglobulin test in 1945, autoantibodies were generally accepted to be etiologic factors in acquired hemolytic anemia. There was widespread introduction of the terms autoimmune and autoallergic into the medical literature. Subsequently, other blood diseases were shown to be associated with autoantibodies (e.g., antiplatelet antibodies in idiopathic thrombocytopenic purpura [ITP]). Certain types of neutropenia, red cell aplasia, and aplastic anemia have been linked also to autoantibodies and autoallergic cytotoxic reactions.

Atlas of Immunology, Third Edition

nephritis. Dixon and Wilson carried out fundamental investigations on the mechanism of immune complex-mediated injury in glomerulonephritis. As a result of studies by these and other investigators, immune complex disorders have been demonstrated to be dynamic processes implicated in the pathogenesis of a host of immunological diseases, including SLE and rheumatoid arthritis. In addition to their studies of immune complexes, Dixon and colleagues continued to examine immunoregulatory dysfunction of immune system cells in SLE. In an effort to unify the diverse descriptions of hypersensitivity phenomena, the British immunologists Coombs (Figure 1.86) and Gell developed a simplified classification of immunopathological phenomena that includes type I, anaphylaxis; type II, cytotoxicity; type III, immune complex disease; and type IV, delayed-type hypersensitivity. Immune complex disorders of the serum sickness variety belong in the type III hypersensitivity of the Coombs and Gell classification.

Systemic Lupus Erythematosus First recognized as a distinct cutaneous disease in the early 1800s, SLE was described by Kaposi as a multisystem disease. In 1895, Osler called the disease erythema multiforme exudativum, pointing to its visceral complications. The first

Ackroyd performed most of the basic work on drug-induced blood dyscrasias associated with autoimmunity. For example, the drug sedormid elicits antibodies reactive not only with the drug but also with platelets, erythrocytes, and even leukocytes bearing drug molecules, metabolites, or even drug– antidrug complexes.

Immune Complexes and Tissue Injury Following the development of horse antitoxin for specific therapy in the treatment for both diphtheria and tetanus early in this century, clinicians noted an adverse reaction to large doses of antitoxin which they termed serum sickness. In a series of elegant studies based upon this early model, Dixon and colleagues developed an experimental model for serum sickness in the 1950s to demonstrate the basic principles of mechanisms of immunopathologic injury mediated by immune complexes. As antigen, they utilized iodinated bovine serum albumin and injected it into rabbits. They followed the elimination of the antigen in three phases, one of which was immune elimination, and studied the development of experimentally induced pathological lesions in the vessels, kidneys, and joints of the animals, correlating this with the immune complex content of the blood serum. Cochrane studied the role of polymorphonuclear leukocytes in nephrotoxic

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Figure 1.86  The British Autoimmunity Group. Top: Professor Ivan Roitt and Professor Deborah Doniach. Bottom, left to right: Professor J. Irvine; Professor R.R.A. Coombs.

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immunological abnormality observed in SLE was a falsepositive serological test for syphilis. Pathologic lesions in the blood vessels and connective tissues of SLE cases were reported in the 1920s and 1930s. Gross, studying autopsy materials from SLE cases, discovered “hematoxylin-stained bodies.” In 1935, G. Baehr and associates reported the “wireloop” appearance of affected glomeruli in lupus. Klemperer emphasized the fibrinoid changes in lupus, but these were found also in other diseases of unknown pathogenesis. Thus, fibrinoid necrosis was widely used as a descriptive term concerning SLE and related disorders. Klemperer coined the term collagen disease to include rheumatic fever, rheumatoid arthritis, subacute and chronic glomerulonephritis, SLE, dermatomyositis, and scleroderma. It was his intention to point out the common pathogenic features of these conditions and to use the term collagen disease as a topographic rather than an etiopathogenetic description. Unfortunately, collagen disease came into widespread use by clinicians with incorrect connotations. The serologic breakthrough came with the development of autoantibody tests, beginning with the lupus erythematosus (LE) cell test by Hargreaves and colleagues in 1948. Although positive in only 50–70% of SLE patients and not specific for SLE, since it was also positive in some other connective tissue diseases such as rheumatoid arthritis, this test permitted the identification of previously occult mild cases and introduced the concept of autoantibodies in SLE. P. Miescher described antinuclear antibodies, thus establishing SLE as an immunological disease. Other prominent investigators of molecular aspects of autoimmune disease include N. Talal, A. Theofilopoulos, and A. D. Steinberg. Anti-DNA antibodies were described by several investigators. Other autoantibody tests developed subsequently included the fluorescent antinuclear antibody test (FANA), which became the most common single laboratory finding in SLE, since it is positive in 95–98% of SLE cases.

the pathogenesis of SLE was recognized to be influenced by genetic, hormonal, and infective mechanisms.

Rheumatoid Arthritis and Rheumatoid Factor Meyer observed coincidentally that selected human sera were able to agglutinate sheep red cells sensitized with rabbit amboceptor during the performance of routine complement fixation tests for syphilis. Waaler and Rose and associates reinvestigated the finding to determine whether it had relevance for rheumatoid arthritis, even though the original patient had suffered from chronic bronchitis. Limited agglutinating activity was present in most of the sera Waaler studied, yet this activity was accentuated in certain rheumatoid sera. Subsequent studies failed to demonstrate that rheumatoid factors in the sera of rheumatoid arthritis patients were qualitatively any different from those in the sera of patients with other diseases, or even of normal subjects. Noel Richard Rose (1927– ) (Figure  1.87), American immunologist and authority on autoimmune disease, who first discovered, with Witebsky, experimental autoimmune thyroiditis. Dr. Noel Rose’s discovery in the mid-1950s that rabbits could be immunized to their own thyroid protein overturned the established dogma of horror autotoxicus and showed unequivocally that autoimmunity could be the cause of disease. With Witebsky, he also demonstrated that patients with chronic thyroiditis develop similar antibodies to their own thyroid antigen, implicating autoimmunity as the cause of this human disease. The initial experiments were followed by others that elucidated the pathogenesis of autoimmune disease in animals and humans. In the 1970s, he described, for the first time, the critical role of genetics in conferring susceptibility to autoimmune disease and showed that the major histocompatibility complex includes the major susceptibility gene. In the 1980s, he showed how virus infections can serve as the trigger for autoimmune disease of the heart.

By 1957, several independent teams of investigators, including the United States group of W. C. Robbins and associates, described antibodies against DNA. Following introduction of the Farr ammonium sulfate technique to increase the sensitivity of the test for native (double-stranded) DNA antibodies in 1968, this method gained widespread clinical acceptance as a laboratory feature of SLE. Tan and Kunkel described the anti-Sm antibody which was found to be virtually diagnostic for SLE even though it was present in only 28–32% of SLE patients. Later investigations centered around the disordered suppressor T-cell function in SLE. Miller and Schwartz reported abnormal suppressor cell activity in 11 of 15 SLE patients as well as in 13 of 50 healthy first-degree relatives, including 12 females. Thus, observations in humans, as well as in experimental animal models of this disease, suggested that the immunologic defect in SLE was influenced by genetics. Hormones were believed also to have a significant role in the expression of immunologic parameters in SLE. Thus,

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Figure 1.87  Dr. Noel Richard Rose.

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These discoveries open the way to improved treatments and new prevention strategies for this group of diseases. Rose’s pioneering investigations initiated the modern era of research on autoimmune disease. Dr. Rose has served as professor of microbiology at SUNY, Buffalo, professor and chair of immunology and microbiology at Wayne State University, Detroit, and as professor and chair of immunology and infectious diseases at The Johns Hopkins University. The author of more than 600 articles and editor of numerous books and leading journals in the field, he is presently director of The Johns Hopkins Center for Autoimmune Disease Research and the WHO/PAHO Collaborating Center for Autoimmune Disorders. Dean demonstrated that a thermostable globulin from normal guinea pig serum could agglutinate red cells sensitized with dilute antiserum. This was attributable to a euglobulin unrelated to complement. He emphasized the significance of antigen access for this globulin to produce maximal agglutination or precipitation. This agglutinating globulin had the peculiar ability to enhance the action of an extremely dilute antibacterial serum. Agglutinating activating factor was later called rheumatoid factor, reflecting its association with rheumatoid arthritis. Subsequent studies demonstrated that sera from rheumatoid arthritis patients could agglutinate not only certain streptococci but other bacteria as well. Attempts to correlate reactivity against bacteria with the progress of the disease led to inconclusive and contradictory results. The demonstration that rheumatoid factor reacted best with denatured gammaglobulin led to the concept that it might be an autoantibody. Franklin and co-workers found both 19S rheumatoid factor and 7S gammaglobulin as a 22S complex when the sera of rheumatoid arthritis patients were studied by ultracentrifugation. Epstein and colleagues showed the ability of rheumatoid factor and gammaglobulin to give a positive precipitin reaction. The demonstration that autologous IgG was able to block the interaction of rheumatoid factor, and globulin coating-sensitized cells further substantiated the concept of rheumatoid factor as an autoantibody. IgM rheumatoid factors were found not to be species restrictive, indicating the sharing of active sites on the Fc fragment of human IgG with corresponding structures on immunoglobulin molecules of many other mammalian species. Milgrom and Witebsky immunized rabbits with aggregated autologous globulin and discovered that the rabbits produced antibodies with greater reactivity for human globulin than for rabbit globulin. Many of the studies on immunoglobulins, including rheumatoid factors, were conducted by Kunkel and coworkers at the Rockefeller University. Among these individuals were Franklin and co-workers, Fudenberg and co-workers, Edelman and colleagues, and a host of others.

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Henry George Kunkel (1916–1983), American physician and immunologist. The primary focus of his work was immunoglobulins. He characterized myeloma proteins as immunoglobulins and rheumatoid factor as an autoantibody. He also discovered IgA and idiotypy and contributed to immunoglobulin structure and genetics. Kunkel received the Lasker Award and the Gairdner Award. A graduate of Johns Hopkins Medical School, he served as professor of medicine at the Rockefeller Institute for Medical Research. It was difficult to accept that human IgG served as the antigen for stimulation of rheumatoid factor synthesis. Early investigators, such as Cecil and colleagues, attempted to show that microorganisms had stimulated formation of these factors. With the demonstration that IgG molecules possessed Gm groups capable of stimulating anti-immunoglobulin antibodies, it became more plausible to consider IgM rheumatoid factor as an antibody against native IgG, which was weakly antigenic in the autologous host. There still remained the difficulty of explaining antibodies against IgG in the serum of normal individuals, as well as those with a variety of diseases other than rheumatoid arthritis. The Fc region of denatured IgG molecules was found to be the primary antigen reactive with rheumatoid factor. Rheumatoid factors are natural humoral antibodies whose levels are markedly increased by the etiologic agent or agents of rheumatoid arthritis and, occasionally, the agents of other disease states. Despite a multitude of studies, the role of rheumatoid factor in the pathogenesis of rheumatoid arthritis remains an enigma. Further studies of rheumatoid arthritis revealed that antigen–antibody complexes became entrapped on the synovial surface and in fibrocartilage followed by activation of various biological amplification mechanisms, including complement, kinins, clotting, fibrinolysis, and phagocytosis. Lymphocytes and plasma cells in the subsynovium were found to produce rheumatoid factor and IgG molecules that were secreted into the joint space, perhaps perpetuating inflammation. The discovery of rheumatoid arthritis in agammaglobulinemic children and the fact that rheumatoid factor did not produce ill effects following infusion into healthy volunteers discouraged a pathogenic role for rheumatoid factor in rheumatoid arthritis. Nevertheless, immune complexes containing IgG rheumatoid factor and cryoglobulins were implicated in the pathogenesis of rheumatoid vasculitis. Regrettably, investigation of immune complexes was not able to account for joint inflammation or vasculitis. Abnormal cellular immune phenomena in rheumatoid arthritis were found to include anergy, depressed lymphoblastogenesis, depressed mixed leukocyte culture reactivity, and diminished antibody-dependent cell-mediated cytotoxicity of synovial fluid lymphocytes. Possible explanations for the uncontrolled synthesis of rheumatoid factor antibodies against native IgG have included chronic antigenic stimulation, enhanced T-helper cell function, or diminished T-suppressor function.

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Sjögren’s Syndrome This disorder was described as a triad of keratoconjunctivitis sicca, xerostomia, and rheumatoid arthritis. In one manifestation the sicca component predominates, whereas in the other the sicca occurs in conjunction with either rheumatoid arthritis or another disorder. Sjögren’s syndrome has been associated with the presence of specific antibodies and T-lymphocyte sensitization against salivary duct antigens. Patients were hypergammaglobulinemic and demonstrated serum antinuclear factor and antibody reactive by the autoimmune complement fixation test. Anderson and colleagues showed precipitating antibodies reactive with organ extracts in the sera of 10 of 29 patients with Sjögren’s syndrome. The presence of antinuclear antibodies and a positive test for rheumatoid factor suggested a resemblance of Sjögren’s syndrome to both SLE and rheumatoid arthritis. As evidenced by lymphoid infiltrate of the salivary glands, patients with Sjögren’s syndrome were shown to manifest many different kinds of lymphoproliferation, most as benign lymphoid infiltrate of the salivary and lacrimal glands, but also sometimes as lymphocytic infiltrate of the lungs, kidneys, and other organs. Occasional patients developed lymphoproliferation leading to lymphoma or Waldenström’s macroglobulinemia.

Autoimmune Thyroiditis Oswald described the globulin that R. Hutchison extracted from the thyroid as thyroglobulin. Subsequently, Hektoen and Shulhof and Hektoen and colleagues demonstrated that thyroglobulin antibodies were not species restricted. Rabbit antibodies specific for horse, dog, and human thyroglobulin all cross-reacted with bovine thyroglobulin. Rabbit antiporcine thyroglobulin cross-reacted with horse, beef, monkey, human, rat, and sheep, as well as swine thyroglobulin. In 1958, Witebsky and colleagues confirmed Hektoen’s observation using purified thyroglobulin. An antibody against thyroid cross-reacted with thyroid extracts of selected but not all species tested. Thus, thyroglobulin was proved to be organ-specific, yet less broadly cross-reactive than in the case of the lens proteins or the brain. Although Lerman could not elicit antibody against rabbit thyroid by the injection of rabbit thyroglobulin, he did observe that rabbit antibody against human thyroglobulin would precipitate rabbit thyroglobulin. Rose and Witebsky challenged rabbits with rabbit thyroid extract incorporated into Freund’s adjuvant. Upon finding that an antibody reactive with rabbit thyroid extract was produced, they proceeded to ascertain whether an autoantibody had been formed. Thyroidectomized or hemithyroidectomized rabbits were inoculated with autologous thyroid extracts incorporated into Freund’s complete adjuvant. Antibodies were produced not only against their own thyroid glands but against those of other rabbits and thyroids of other species. Histopathological examination revealed reduced colloid and infiltration by lymphoid, phagocytic and eosinophilic cells,

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further substantiating the autoimmune nature of rabbit antirabbit thyroid antibody. Following the demonstration by Witebsky and colleagues that ammonium sulfate fractionated rabbit thyroglobulin incorporated into Freund’s complete adjuvant could elicit antibodies in the autologous host that were reactive with rabbit thyroid extract, Beutner and associates showed by immunofluorescence that antibodies against rabbit thyroid would stain rabbit thyroid gland colloid containing thyroglobulin. Reactivity was shown to be thyroid specific. Although first considered to be anatomically isolated in the follicles of the thyroid gland, and not in contact with immunocompetent cells under normal conditions, relatively low levels of circulating thyroglobulin were subsequently demonstrated. T cells were found to be tolerant of low doses of thyroglobulin in the circulation, whereas B cells remained immunocompetent. In the absence of a signal from the T cell, B cells fail to mount an autoimmune response against thyroglobulin. Roitt and colleagues observed a positive precipitation reaction following interaction between serum of Hashimoto thyroiditis patients and human thyroglobulin in vitro. This represented the first proof that humans with thyroid disease contained circulating antibodies reactive with thyroglobulin. For further historical details of autoimmune thyroiditis, refer to Rabin.

Insulin-Dependent (Type 1) Diabetes Mellitus Inflammatory lesions termed insulitis in the pancreatic islets of diabetics at onset suggested that autoimmunity might have a central role in the pathogenesis of type 1 or insulin-dependent diabetes mellitus (IDDM). The immunologic attack appeared to be directed specifically against insulin-producing β cells, even though at least three other cell types were present in the pancreatic islets. Glucagon-containing α cells, somatostatin-containing δ cells and pancreatic polypeptidecontaining (PP) cells increased in relative proportion to the markedly reduced mass of β cells. Immunologic phenomena discovered in association with IDDM included evidence of cell-mediated immunity, killer cell activity and circulating antibody, reactive with the pancreatic islets. Considering the significance of the major histocompatibility complex in immunoregulation, it was interesting to note that certain HLA types, especially HLA-DR3 and -DR4, were found to be closely associated with IDDM. Thus, the presence of predisposing conditions associated with these alleles pointed to a significant role for autoimmunity in this disease. Therefore, it became clear that suppression of the immune response might represent a potential mode of therapy in IDDM. Antibodies specific against pancreatic islet cells were found in the circulation of insulin-dependent diabetics. Either environmental factors or loss of immunoregulatory control were credited with initiating autoimmunity against insulin-producing β cells. Several types of antibodies against both

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cytoplasmic and surface antigenic determinants of pancreatic islet cells have been linked to the onset of IDDM. Islet cell surface antibodies attracted special interest owing to their preferential β cytotoxicity. Historically, observations relating to cell-mediated autoimmune phenomena in the pathogenesis of diabetes preceded the recognition of humoral autoimmunity in this disease. Two-thirds of patients with short-duration juvenile-onset diabetes mellitus exhibited striking mononuclear cellular infiltrate, principally lymphocytes, in and around the islets of Langerhans at autopsy. In addition, many patients with type I diabetes mellitus had associated autoimmune disorders. Nerup and associates described inhibition of leukocyte migration in response to an extract of porcine insulin in diabetics by comparison with controls. Delayed-type hypersensitivity was demonstrated by intracutaneous injection of the insulin extract. Liver or kidney extracts and porcine insulin had no effect on lymphocyte migration. Migration inhibition had no association with a history of insulin therapy. Studies by Huang and Maclaren demonstrated that cell-mediated cytotoxicity could participate in the pathogenesis of type I diabetes. To establish the role of cell-mediated immunity in type I diabetes, Buschard and colleagues reported passive transfer of diabetes from humans to nude mice by injection of peripheral mononuclear cells derived from newly diagnosed patients with insulin-dependent diabetes. A valuable asset for experimental studies of type I diabetes was recognition of the spontaneously diabetic BB rat. Studies of the disease in this species have revealed the genetic basis for transmission of the syndrome, the insulin-dependent feature of physiologic abnormalities and the role of immune phenomena in pathogenesis. It is anticipated that future studies will reveal effector cells leading to β cell destruction and the antigenic targets against which the immune attack is directed.

Other Autoimmune Endocrinopathies: Autoimmunity of Adrenal and Gonads, Parathyroid and Pituitary This is reviewed by Doniach and colleagues. Following the successful introduction of antibiotic therapy for tuberculosis, most Addison’s disease cases were found to be due to autoimmune destruction of the adrenal cortex. Approximately 60% of Addisonian patients demonstrated antibodies reactive with adrenocortical cells in all three layers. An incidence of 88% adrenal antibodies in HLA-A1, B5, DR3 haplotype patients demonstrated the influence of genetic predisposition in development of this disease. Antibodies against steroid cells were shown to be cross-reactive with steroid-secreting cells in both the placenta and gonads. Milgrom and Witebsky and Witebsky and Milgrom differentiated clearly between antigens of the adrenal medulla and cortex in their investigations of experimental immune adrenalitis.

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Blizzard and colleagues first studied idiopathic hypoparathyroidism immunologically and found positive immunofluo­ rescence on parathyroid substrates with 38% of patient sera and with 6% of controls. Parathyroid gland atrophy and clinical associations placed this disease with the organ-specific autoimmune diseases. Antibodies against pituitary prolactin cells were first shown in polyendocrine disease. Subsequently, antibodies against some other types of pituicytes were discovered.

Pernicious Anemia and Autoimmunity Pernicious anemia together with its associated gastric atrophy were described between 1850 and 1870, and the therapeutic effect of liver and the extrinsic and intrinsic factor concept were developed between 1920 and 1930. Next came the identification of vitamin B12 as extrinsic factor and its structural analysis (1948–1955). The recognition of pernicious anemia as an autoimmune disease (1962–1965) was based upon the identification of autoantibodies against gastric parietal cells and intrinsic factor, and the association between gastric antibodies and defective absorption of vitamin B12. Other supportive evidence was the association of this disease with other autoimmune disorders, as well as the ability of prednisolone to facilitate regeneration of gastric mucosal atrophy. In the 1950s, continued use of hog intrinsic factor to treat pernicious anemia reduced its effectiveness with time. In 1958, Taylor and Morton found that rabbits injected with intrinsic factor developed anti-intrinsic factor activity, and Schwartz demonstrated development of an inhibitor of hog intrinsic factor activity in the serum of patients treated with this factor. Further interest in gastric autoimmunity stemmed from Tudhope and Wilson’s report that there was a different incidence of pernicious anemia in patients with thyroid disease compared with the general population. As Mackay and Whittingham pointed out, this linkage and the possibility that spontaneous hypothyroidism might be a consequence of autoimmune thyroiditis fueled speculation of an association between autoimmune reactivity against gastric cells and pernicious anemia. Irvine and colleagues found positive complement fixation reactions using sera from pernicious anemia patients and gastric mucosa as antigen. He also demonstrated serological crossreactivity between individuals with pernicious anemia and patients with Hashimoto thyroiditis, employing gastric and thyroid tissue antigens. Immunofluorescence was employed subsequently to demonstrate that the reactive antigen in pernicious anemia was that associated with parietal cells. Subsequent studies were directed toward antibodies against intrinsic factor reactive in vitro. Two different antibodies were shown to react with separate reactive sites on the molecule and were referred to as blocking and binding antibodies. Whereas parietal cell antibody was shown by

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immunofluorescence in 90–95% of pernicious anemia cases, only 50–55% of subjects showed antibody against intrinsic factor. It was then suggested that antibody against intrinsic factor in gastric juice rather than in serum might be a reliable indicator of gastric autoimmune reactivity. Mackay and Burnet set up criteria for the diagnosis of autoimmune diseases that included the following: 1 . Hypergammaglobulinemia 2. Serum antibodies 3. Lymphoid infiltration of the affected tissue 4. Responsiveness of the disease to corticosteroids 5. Association of the disease with clinical and serological features of other autoimmune diseases in that patient and/or blood relatives Pernicious anemia fulfilled several of these (i.e., antibody against both parietal cells and intrinsic factor, lymphoid infiltration of the stomach, regeneration of atrophic gastric mucosa in response to prednisolone therapy, and the demonstration of clinical and serological overlap between pernicious anemia and Hashimoto thyroiditis–thyrotoxicosis–myxedema complex of diseases). The central question in autoimmunity has always been whether autoantibodies are the cause or result of a given disease process. In the case of pernicious anemia, a positive correlation was demonstrated between the presence of gastric antibodies and impaired vitamin B12 absorption. For further details concerning the history of pernicious anemia, the reader is referred to the review article by Mackay and Whittingham.

Autoimmunity and the Liver Autoimmunity has been implicated in liver disease since 1908 when Fiessinger, cited by Paronetto and Popper, concluded from his serologic and experimental study that autoantibodies against liver could be induced following hepatic injury. The demonstration of an autoimmune component in chronic active hepatitis was shown in Melbourne in 1955 when Joske and King demonstrated that two “active chronic viral hepatitis” patients had positive LE cell tests. Mackay and co-workers reported five more cases, including predominantly females with hypergammaglobulinemia. Observing that some manifestations, such as arthralgia, suggestive of SLE were present, they termed the condition lupoid hepatitis, which they believed to be the result of autoimmunization. Complement fixation assays of the serum of patients with lupoid hepatitis, primary biliary cirrhosis (PBC), and SLE revealed reactivity with saline extracts of human tissues, especially liver and kidney. Further support for autoimmunity was provided by the use of immunofluorescence after 1960 to show positive antinuclear antibody in many patients with chronic active hepatitis whose LE cell tests were negative. Johnson and associates reported smooth muscle antibodies in chronic active hepatitis patients. Antibody titers appeared to

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be positively correlated with disease activity. These antibodies were also found in PBC as well as in selected other conditions, such as the low titers found in acute viral hepatitis. Walker and colleagues described antimitochondrial antibodies in PBC patients. Subsequently, these antibodies were found also in some chronic active hepatitis patients. The fluorescent antibody technique was found to be useful for detection of both smooth muscle and mitochondrial antibodies. Other antibodies found in liver disease included antinuclear and antiliver antibodies. Antinuclear and antismooth muscle antibodies were characteristically seen in chronic active hepatitis patients. Other identifiable antibodies were those against mitochondria, microsomes, cardiolipin, thyrogastric antigens, gammaglobulins, and erythrocytes. Immune complexes did not appear to contribute significantly to hepatocellular damage in chronic active hepatitis. By contrast, so-called antiliver antibodies demonstrated much variation from one case to another and little correlation with severity, progression, or even presence of disease. Since studies of humoral antibodies failed to explain pathogenetic mechanisms responsible for development of selected liver diseases, efforts were made to show a role for cellmediated immunity, which was suggested by the histologic appearance of lymphocytes, histiocytes, and large pyroninophylic cells in liver sections from patients with certain hepatic diseases. In vitro studies included lymphocyte stimulation by plant mitogens or hepatic tissue antigens. Soborg and Bendixen showed cell-mediated immunity by use of the leukocyte migration inhibition technique. Most of 34 untreated chronic active hepatitis patients showed positive in vitro correlates of cell-mediated immunity. Another study by Miller and colleagues showed cell-mediated immunity, as revealed by the lymphocyte migration inhibition technique, in 11 of 16 cases of chronic active hepatitis and seven of 12 cases of PBC. Bacon and co-workers found evidence of cellmediated immune reactivity using whole liver cell extracts as antigens in 24 of 32 patients with either chronic active hepatitis or PBC.

Autoimmune Neurologic Disorders Tissue injury produced by immune reactions was first described as a cause of disease in the nervous system. Occasional individuals who received the Pasteur vaccine for rabies virus, following its introduction in 1885, developed neurological signs and symptoms attributable to acute inflammation in the central nervous system (CNS). This was induced by an immune response against the nervous tissue in which the virus was cultured during attenuation. In 1933 and 1935, Rivers and colleagues and Rivers and Swentker reported development of acute disseminated encephalomyelitis in monkeys that had received repeated inoculations of CNS tissue extracts or emulsions. They observed a close similarity between this experimental disease and the encephalomyelitis found in occasional subjects following rabies vaccination. They suggested that the

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myelin injury might be attributable to an immune response to CNS antigens. Subsequently, Kabat and colleagues and Morgan reported independently that incorporation of either homologous or heterologous CNS tissue into Freund’s complete adjuvant could induce experimental allergic encephalomyelitis (EAE) in monkeys. Waksman and Adams pointed out that most histological studies of EAE had been made on animals with advanced disease, rendering unsuitable interpretation of the interrelationships among cells present in the lesions. To remedy this problem, they administered antigen by a technique known to provide clinical evidence of EAE in rabbits and guinea pigs 9–10 days following a single injection. The animals were killed at daily intervals beginning on the fifth day, which permitted the investigators to show that lymphocytes and small mononuclear cells were the first to infiltrate the area, apparently having been derived from circulating blood by emigration from adjacent vessels. They observed that many of the cells remained close to the vessel where they originated, either in meninges or in Virchow–Robin spaces. Yet many others migrated into the adjacent parenchyma and increased in number by mitosis. The infiltrating cells then underwent progressive metamorphosis into typical histiocytes, which Waksman and Adams suggested were directly responsible for demyelination in areas of infiltration. Thus, specific injury to the nervous system tissue was shown to be mediated by cells, an observation which was supported indirectly by failure to correlate the presence of circulating antibody with development of CNS lesions. Later studies demonstrated that the primary antigen was myelin basic protein (MBP), and pointed to the close resemblance between EAE and multiple sclerosis, the principal demyelinating disease of humans.

Myasthenia Gravis Although myasthenia gravis (MG) was probably first described by Willis in 1685, and approximately seven cases had been reported by the turn of the century, it was not until 1901 that Laquer and Weigert reported association of a thymic tumor with MG. Subsequently, thymectomy was found to improve the clinical status of some myasthenic patients with thymic tumor, even though it would be more than a half century before Good and associates and Miller described the cardinal role of the thymus in the immune response. Recent research has focused on whether the physiological defect could be presynaptic, with diminished quantities of acetylcholine, or postsynaptic, with acetylcholine receptor defects. This was resolved by Fambrough and associates, who demonstrated conclusively that the number of functional acetylcholine receptors is diminished in MG. Ito and colleagues demonstrated that this decrease in acetylcholine receptors could account for the physiological changes in the disease. Patrick and Lindstrom immunized rabbits with acetylcholine

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receptors purified from the electric organ of the electric eel, to develop an experimental model of MG that closely resembles the human disease. Serum from immunized animals was capable of transferring the disease passively to previously untreated recipients. Such antibodies have since been demonstrated in MG patients whose condition has been improved by plasma exchange or thoracic duct drainage.

Autoimmune Reactions of the Skin Increased understanding of bullous diseases, including pemphigus, began with Civatte’s description of acantholysis and with Lever’s delineation of pemphigus from pemphigoid syndromes. Beutner and Jordan found that serum from patients with pemphigus vulgaris contained an antibody reactive with intercellular substances of stratified squamous epithelium. Eight of 13 patients’ sera produced what has become known as a classic “chicken wire” reaction pattern when examined by indirect immunofluorescence microscopy. Normal skin adjacent to a blister of one pemphigus patient exhibited gammaglobulin in the intercellular space. Subsequent studies by these authors and others confirmed deposition of an IgG antibody in involved as well as in uninvolved areas of skin. Chorzelski and colleagues described antibodies in 23 of 35 patients with active pemphigus. They showed that antibody titers decreased as some patients responded to therapy. By contrast, exacerbations of the disease were noted in conjunction with the rising titer. The authors emphasized the significance of performing serial serologic tests in patient treatment. A subsequent report by Jordan and co-workers pointed to the diagnostic significance of direct immunofluorescence microscopy of skin biopsy specimens. Beutner and colleagues were able to identify the reactive principle in the serum of pemphigus patients as well as autoantibodies because in two patients the serum reacted with the patient’s own normal skin. The antibodies also demonstrated tissue specificity by fixing only to squamous epithelium whether from skin, oral mucosa, esophagus, anus, vagina, or cornea.

IMMUNOHEMATOLOGY Immunohematology is the study of blood group antigens and antibodies and their interactions in health and disease. Both the cellular elements and serum constituents of the blood have distinct profiles of antigens. There are multiple systems of blood cell groups, all of which may stimulate antibodies and interact with them. These may be associated with erythrocytes, leukocytes, or platelets. Another dream in early times was the possibility of performing blood transfusions (Figure  1.88). A well-documented early attempt was the effort to rejuvenate Pope Innocent VIII in 1492 with the blood of three young men, which ended disastrously for all four. Several Italians in the early part of the 1600s claimed that they had performed transfusions. Harvey’s

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experiments from sheep to dogs, which invariably failed. He revived a moribund obstetric patient with a transfusion of human blood in 1818, opening a new era. There continued to be great difficulties in the way of achieving human transfusion, for example with coagulation. Landsteiner’s elucidation of the blood groups in 1901 and the advent of sodium citrate as an anticoagulant in World War I finally made the matching of bloods comprehensible, and transfusions could become a medical possibility.

Figure 1.88  Early blood transfusions in dogs.

work on the circulation of the blood paved the way for a better understanding of the flow of blood in the body. Robert Boyle published a proposal in the Philosophical Transactions in 1667 for Richard Lower to experiment with transferring blood from one living animal to another, at Gresham College (Figure  1.89). In the same year at Montpellier, in southern France, a professor of philosophy, Jean Denis, began a series of transfusions of animal blood into humans. The amounts of blood given were small, and he was probably not able to introduce enough blood to produce death, although he reported predictable adverse effects. In November, Richard Lower claimed to have transfused 9 oz of lamb’s blood into a young man, reported by Samuel Pepys. In December, Denis again attempted to give calf blood to a manic depressive patient, without fatal results; indeed, observers thought him much improved. A third transfusion was planned and the man died in mysterious circumstances. Denis accused the man’s wife of poisoning him and was himself accused of causing the death but was acquitted. However, further experiments with transfusion came to an end for 150 years.

An article in the Berliner klinische Wochenschrift in 1900 by Ehrlich and Morgenroth, which described blood groups in goats based on antigens of their red cells, led Karl Landsteiner, a Viennese pathologist, successfully to identify the human ABO blood groups. He took samples of his own blood, and from his colleagues Sturli and Ardhein, Dr. Pletschnig, and his assistant Zaritsch. The small tables Landsteiner used to illustrate his reasoning bear the names of these colleagues. In 1902, Sturli and Alfred von Descastello, under Landsteiner’s direction, designated one more group, which was actually not named AB until 10 years later when von Dungern and Hirszfeld (Figure  1.90), studying the genetic inheritance of blood types, designated the fourth type and gave Landsteiner’s C group the designation O. It was for this discovery, rather than his elegant studies on immunochemical specificity, that he won the Nobel Prize in Medicine 30 years later. After 5 years of training in chemistry under the tutelage of Emil Fischer, Eugen von Bamberger, and Arthur Hantzsch, and the beginning of his work on the specificity of serological reactions using chemically modified antigens, Dr. Landsteiner served in Max von Grüber’s Institute for 2 years. After von Grüber’s departure for Munich, he became an assistant to Anton Weichselbaum, also of the Vienna University Faculty of Medicine, in 1897. In 1908, he was appointed to the staff of the Imperial Wilhelminen Hospital

In 1800, Paul Scheel made an extensive study of the literature of attempted transfusions. His work was reviewed by James Blundell, an English obstetrician who tried animal

Figure 1.89  Transfusion of a patient with animal blood (from Scultatus, courtesy of the National Library of Medicine).

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Figure 1.90  Ludwik Hirszfeld, who studied the genetics of blood groups along with Emil von Dungern.

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Figure 1.91  Alexander Wiener, codiscoverer of the Rh blood group system.

as head of the Department of Pathology. His years in Vienna were his most fruitful, with more than 170 papers. After a brief sojourn in Holland after World War I, he was invited to join the Rockefeller Institute for Medical Research by Simon Flexner in 1923. He continued there for the rest of his life, completing 346 papers during his enormously productive career. Several of the investigators who trained under his direction, including Merrill W. Chase, gained scientific acclaim in their own right in subsequent years. Among those who worked with him in the field of immunohematology were Alexander Wiener (Figure 1.91) and Philip Levine (Figure 1.92). Distin­ guished investigators who worked with Dr. Landsteiner in other fields of immunology are mentioned in other sections of this book. Landsteiner and Levine discovered the M and N blood group antigens by injecting human erythrocytes into rabbits. The antisera which they raised were able to divide human blood into three groups, M, N, and MN, based on the antigenic content. They showed that these antigens were under the genetic control of codominant alleles. Landsteiner and Wiener described the rhesus (Rh) factor in 1940. At first thought to be a simple system involving a single antigen, it was shown to be genetically, immunologically, and clinically complex. In studies on M-like factors on the erythrocytes of rhesus monkeys, antisera raised by injecting rabbits with rhesus red cells cross-reacted with human erythrocytes containing M antigen. It was subsequently demonstrated that the red cells of about 85% of the human population reacted with antisera against rhesus red cells. Thus, those individuals who shared an antigen with the rhesus monkey red cells were termed Rho positive and those who did not were termed Rho negative. It was subsequently demonstrated that multiple pregnancies with Rh

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Figure 1.92  Philip Levine, colleague of Landsteiner and codiscoverer of the MNS blood group system.

(D) positive fetuses in Rh negative mothers leads to stimulation of maternal anti-D antibodies of the IgG class which cross the placenta and cause lysis of fetal red cells. Ronald Finn (Figure 1.93), studying Kleihauer’s slides stained for fetal hemoglobin, suggested that freeing a mother’s blood of fetal cells would prevent Rh sensitization, thereby preventing erythroblastosis fetalis in subsequent pregnancies. Subsequently, anti-D antibody was used for this purpose. Unless the mother is treated with antibody against the D antigen after parturition, hemolytic disease of the newborn (erythroblastosis fetalis) may result in subsequent births. Race and Sanger (Figure  1.94) offered an alternative genetic concept of the Rh blood groups with a threeallele theory rather than a single locus with a large series of alleles proposed by Wiener. Race and Sanger developed a system of nomenclature (i.e., CDE/cde) different from that of Wiener. Besides those mentioned above, other red blood cell antigens discovered in the intervening years included Kell, Diego, P, Duffy, the I blood group system, and soluble

Figure 1.93  Dr. Ronald Finn.

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Karl Landsteiner (1868–1943), discoverer of the ABO and other blood group systems including MN and the Rh factor, developed artificial haptens to investigate antibody specificity, and received the Nobel Prize in Medicine or Physiology in 1930 for discovery of the human blood groups.

Figure 1.94  Dr. Ruth Sanger and Dr. Robert Race.

antigens such as the Lewis and Lutheran antigens that are in the secretions and are adsorbed to the red cell surface. Although most red cell groups are inherited as autosomal characteristics, the Xga blood group system is sex linked. Historically, new red blood cell antigens were discovered as a result of transfusion incompatibility reactions that could not be explained on the basis of existing or known antigens. Until the late 1950s immunohematology concerned itself almost exclusively with the immunology of the red cell. Since that time, however, tissue-typing techniques have demonstrated that HLAs in humans are present on leukocytes but are absent on erythrocytes. Since most blood group studies employed red cells, this might explain the delay in their discovery. Since these are significant for organ and bone marrow transplantation, improvements in techniques for tissue typing using patient and donor lymphocytes progressed at a rapid pace. Pioneers in this field of human histocompatibility testing included Bernard Amos, a student of Peter Gorer, Kissmeyer-Nielsen, Van Rood, Ceppelini, Fritz Bach, Paul Terasaki, and a host of others. Genes for the major histocompatibility complex (MHC) in man were localized to the short arm of chromosome 6. These include HLA-A, HLA-B, HLA-C, HLA-D, and HLA-DR. The transplantation of bone marrow and solid organ grafts such as the kidney is facilitated by HLA typing. Carlo Alberto Moreschi (1876–1921) graduated as doctor of medicine at Pavia in 1900. He was assistant to Pfeiffer, Ehrlich, and Ascoli and in 1920 was named Ordinarius in clinical medicine at Sassari and Messina Universities. He pursued studies on anaphylaxis, bacteriolysis, and complement fixation reactions. Starting from the specific inhibition reaction perfected by Landsteiner and colleagues (1902) to study haptens, Moreschi reported in 1907 a new method to identify serological antibodies, which drew praise from Landsteiner in 1909. Coombs, Mourant, and Race, in the 1940s, developed the antihuman globulin (Coombs) test based on the same principle as Moreschi’s earlier report.

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Landsteiner was born in Vienna on June 14, 1868. The son of a journalist, he attended the University of Vienna from 1885 to 1891, when he graduated as MD. He maintained a strong interest in chemistry and took special instruction in organic chemistry under Emil Fischer and Eugen von Bamberger. In 1896, he became assistant at the Institute of Hygiene directed by Max von Grüber where he was introduced to immunology and serology, which occupied his research interests for the remainder of his career. Two years later, he transferred to pathology. He coauthored nine papers with Donath, including a report on paroxysmal hemoglobinuria. Landsteiner was appointed research assistant at the Vienna Pathological Institute in 1898 where he remained until 1908 when he was appointed chief of pathology at the Royal Imperial Wilhelminen Hospital in Vienna and adjunct professor in the Medical Faculty of the University of Vienna. Following World War I the newly formed Republic of Austria was chaotic and experienced inflation and fuel and food shortages. Fleeing disruptive working conditions, Landsteiner left for The Hague in the Netherlands in 1919, where he accepted a position in the Catholic Hospital. He was lured to the Rockefeller Institute for Medical Research in New York City at the invitation of the institute’s director, Dr. Simon Flexner, in 1923, where he was made a full member and given a modest laboratory. He became an American citizen in 1929 and died in New York City on June 26, 1943. Landsteiner discovered the human blood groups in 1900 when he found that the blood serum of some individuals could cause the agglutination of red cells from others. This led him to characterize red blood cells according to their antigens, leading subsequently to a description of the ABO blood groups. The development of citrates as anticoagulants in 1914 by Richard Lewisohn, together with Landsteiner’s blood grouping, led to the widespread practice of blood transfusion. In 1927, Landsteiner and Phillip Levine discovered the additional blood group antigens M and N, which are not of significance in transfusion since human blood serum does not contain isoagglutinins (antibodies) against them, differentiating MN from the ABO groups. In 1940, Landsteiner, Levine, and Wiener discovered the Rh blood group system when they found that antibodies raised in rabbits and guinea pigs immunized with blood from a rhesus monkey were able to agglutinate the red blood cells of many humans, who were termed Rh positive, whereas those not possessing it were termed Rh negative. This factor was shown to be important in hemolytic disease of the newborn. Landsteiner investigated poliomyelitis between 1908 and 1922. He discovered that a rhesus monkey became paralyzed following the injection of brain and spinal cord from a polio

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victim. After finding no bacteria in the monkey’s nervous system, Landsteiner concluded that a virus must be the causative agent. He perfected a technique to diagnose poliomyelitis. Landsteiner discovered that an extract of ox hearts could replace the antigen derived from livers of babies with congenital syphilis, for use in the Wassermann test for syphilis. With his excellent background in chemistry, medicine, and pathology, Landsteiner continued his serology and immunology work at the Rockefeller Institute. He examined the effect of position on attached radicals. He discovered that immunochemical specificity was altered by the ortho, meta, or para position on aromatic rings of stereoisomers. He found that position was more important than the nature of the radical. Landsteiner further showed that partial antigens, termed haptens, were unable to elicit antibody formation, but could react with those formed in response to a complete antigen comprising a hapten bound to a carrier molecule. He demonstrated carrier-specific and hapten-specific antibodies. His extensive investigations on immunochemical specificity were first summarized in his book entitled Die Specifizitat der Serologischen Reactionen published in 1933. The English translation was published in 1936, and a revised edition appeared following his death in 1945 entitled The Specificity of Serological Reactions. He found that haptens could combine with pre-existing antibodies to produce allergic desensitization. Landsteiner’s work laid a foundation for much of modern immunology, including the construction of synthetic vaccines as a representative application of his research. Philip Levine (1900–1987), Russian–American immunohematologist. With Landsteiner, he conducted pioneering research on blood group antigens, including discovery of the MNP system. His work contributed much to transfusion medicine and transplantation immunobiology.

Figure 1.95  St. Cosmas and St. Damian transplanting the leg of a Moor onto the stump of a young man who had lost his leg.

been amputated. A number of famous artists, including Fra Angelico and Fra Lippi, illustrated some aspects of this popular legend. John Hunter (Figure  1.96), who was given the title “father of experimental surgery,” was intrigued by the possibility of transplantation, and was successful in replacing a premolar tooth some hours after it had been knocked

Landsteiner’s rule (historical) Landsteiner (1900) discovered that human red blood cells could be separated into four groups based on their antigenic characteristics. These were designated as blood groups O, A, B, and AB. He found naturally occurring isohemagglutinins in the sera of individuals specific for the (ABO) blood group antigen which they did not possess (i.e., anti-A and anti-B isohemagglutinins in group O subjects; anti-B in group A individuals; and anti-A in group B persons; neither anti-A nor anti-B in group AB subjects).

Immunogenetics From early times, the practice of medicine was plagued with other problems which had to wait for solution until there was an understanding of the immune mechanisms of the human body. In time of war, in the days of hand-to-hand combat, the loss of a limb was the lot of many soldiers. The patron saints of the surgeons were St. Cosmas and St. Damian (Figure  1.95) who were said to have transplanted the leg of a Moor onto the stump of a young man whose leg had

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Figure 1.96  John Hunter, English surgeon and father of experimental surgery, who transplanted a tooth into the comb of a rooster.

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Figure 1.98  Gaspare Tagliacozzi of Bologna, who wrote a famous book on skin grafting. Figure 1.97  Early depiction of grafts of skin from the forehead, neck, and cheek, used to restore mutilations of the nose, ear, or lip.

out. He thought he was successful in transplanting a human tooth into the comb of a cock, a specimen still on view at the Hunterian Museum. Early understanding of the principles of skin grafting by the Indian physician Sushruta can be found in a Sanskrit text from India dated about 450 BC. Grafts of skin from the forehead, neck, and cheek were used to restore mutilations of the nose, ear, or lip (Figure 1.97). Apparently the physicians of the Alexandrian school understood how to repair such defects with skin flaps also, which is documented in the De Medicina of Celsus. During the 13th and 14th centuries there was a resurgence of surgery practiced in the ancient medical school of Salerno, and in the 15th century mention was made of two skilled practitioners of plastic surgery, Branca de Branca and his son Antonio, who practiced in Catania in Sicily. The next great step in transplantation was the work of Gaspare Tagliacozzi (Figure 1.98) of Bologna, who published De Curtorum Chirurgia per Insitionem, showing in detail his methods and procedure and illustrated with pictures of his instruments and methods of bandaging. He drew analogies from the agricultural practices of grafting in explaining his technique. He understood that xenografts were impossible and allografts were highly unlikely to unite, if only because of the awkwardness of keeping the two parts in close contact so that the graft could take. In spite of the excellence of Tagliacozzi’s book, the technique of skin grafting did not progress further until the 19th century when interest revived. A number of surgeons tried making free grafts in animals and also attempts were made in humans, although, without the refinements of aseptic technique and anesthesia and since the principles of immunology were not yet known, not much progress was made.

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Alexis Carrel (1873–1944) (Figure 1.99) was a French surgeon who received the Nobel Prize in Medicine or Physiology for successfully joining blood vessels by end-to-end anastomosis with triangulation sutures, thereby permitting the rapid reestablishment of blood circulation to a transplanted organ. Alexis Carrel was born in 1873 in Sainte-Foy near Lyon, France. In 1900 he graduated as doctor of medicine at Lyon, and departed for America in 1904 to work in the Physiology Department at the University of Chicago. In 1906, he joined the Rockefeller Institute for Medical Research in New York City as an associate member, achieving full membership in 1912.

Figure 1.99  Alexis Carrel.

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Becoming interested in tissue culture devised by Yale University researcher Ross G. Harrison, Carrel sent his colleague, M. T. Burrows, to learn the technique from Harrison. Their aim was to grow cells from warm-blooded animals, although this had already been accomplished in 1908 by Margaret Reid in Berlin. Carrel and Burrows claimed to have devised a method that employed a simple surgical technique, freshly sterilized glassware, and instruments. They transformed the simple technology of tissue culture into an elaborate laboratory ritual. Carrel and Burrows worked in two separate suites of rooms consisting of animal preparation rooms, scrub room, and culture/operating room fitted with built-in sprays used to cause dust to settle by spraying with water prior to commencing work. He required technicians, including Charles Lindbergh of subsequent aviation fame, to dress somewhat theatrically in black, full-length gowns fitted with hoods. Carrel stated that “the technique [of tissue culture] is delicate and in untrained hands, the experimental errors are of such magnitude as to render the results worthless.” This caused other investigators to shy away from research involving tissue culture because of its apparent complexity, the cost of laboratory equipment, and space. Critics termed this Carrel’s “mumbo jumbo” that stalled medical progress for years. He developed the technique of end-to-end anastomosis of blood vessels in 1902. This technical advance permitted him to transplant organs successfully 6 years later. Continuing the work on organ transplantation begun by Emerich Ullmann in Vienna, Alexis Carrel became the first researcher in America to win the Nobel Prize in Medicine or Physiology by devising a skillful technique of approximating the ends of blood vessels to be anastomosed through triangulation sutures. He also successfully perfected surgical techniques to suture small vessels. In 1905, together with his colleague, Charles Guthrie, Carrel successfully performed a kidney autotransplant in a dog even though the animal died after the kidney failed.

Atlas of Immunology, Third Edition

The Vichy French Government assisted him during World War II and he negotiated with the Germans. In August 1944, following the liberation, Carrel was accused of collaborating with the enemy, but died before he could be arrested. When Professor M. R. Irwin of the University of Wisconsin in Madison coined the term immunogenetics in 1933 to describe an uncertain association between immunology and genetics, no one could have predicted that revelations of histocompatibility genes and antigens and of immune response (Ir) genes would lead to the award of a Nobel Prize in Medicine in 1980 to George Snell, Baruj Benacerraf, and Jean Dausset for immunogenetics research. The studies of Jensen and Loeb, working independently with inbred strains of mice, at the turn of the century, demonstrated that the genetic similarity between a tumor transplant and the host into which it was inoculated governed the success or failure of the tumor allograft. These findings were confirmed and expanded by Tyzzer in 1909. Little (1914) suggested that dominant genes govern susceptibility to tumor allografts. This line of research led to the development of more inbred strains of mice which became a valuable tool in biomedical research. Several investigators sought antibodies to tumor-specific antigens in animals making an immune response against transplanted tumors, but without success. It was Haldane in 1933 who pointed out that a tumor retains many of the alloantigens of the tissue from which it arises. He further postulated that these alloantigens, rather than tumor-specific antigens, stimulate an immune response in a transplant recipient lacking them. For such a hypothesis to have credibility, it would be necessary to demonstrate blood group antigenic differences among various strains of mice. Regrettably, such information was not available. Peter Gorer (Figure  1.100), father of modern histocompatibility testing, described four blood group antigens in mice, which he

He demonstrated that blood vessels could be maintained in the cold for “prolonged periods” before use for transplantation. Carrel and Burrows used Harrison’s technique to grow sarcoma cells successfully in culture in 1910. Together with Tuffier, Carrel performed a number of successful experimental valvotomies. In World War I, he and Dakin developed a treatment for wounds that was used extensively. A positive accomplishment in his tissue culture research was the Carrel flask which reduced bacterial contamination that had been a principal cause of failure of tissue cultures before the discovery of antibiotics in the 1940s. He also claimed to have developed an immortal cell line of chick embryo heart cultures that was begun in 1912 and supposedly maintained by him and a colleague, A. H. Ebeling, until 1942. How this was accomplished remains an enigma, as cells are now known to have a finite longevity. In 1938, Carrel retired from the Rockefeller Institute and returned to France, where he set up an Institute for the Study of Human Problems in Paris.

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Figure 1.100  Peter A. Gorer.

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designated I, II, III, and IV (1936–1938). To examine whether an association existed between genes controlling blood groups and those predicted to govern tumor susceptibility, Gorer inoculated A strain carcinoma into A and C57BI mice, their hybrids, and back-crosses. He concluded that tumor susceptibility was governed by two or three genes, and that one of these was the same as the blood group gene encoding antigen II. This finding led him to the belief that tumor regression in these animals had an immunological basis. The blood sera from animals rejecting the tumor allotransplant contained hemagglutinins specific for red blood cells derived from A strain mice. Thus, antibodies against blood group antigen I represented strong evidence that the tumor tissue expresses antigen II in common with normal tissues. Thus, Gorer’s research showed that genes governing susceptibility to tumor allotransplants were the same as those encoding alloantigens. His demonstration that alloantibodies were produced by mice rejecting tumor transplants proved that this process had an immunological basis. Gorer’s tissue transplantation concept proposed that both normal and tumor tissues contain genetically determined isoantigens, and that the host makes an immune response against those isoantigens in grafted tissue which the host does not possess. Peter Alfred Gorer (1907–1961) was a British pathologist who was professor at Guy’s Hospital Medical School, London, where he made major discoveries in transplantation genetics. With Snell, he discovered the H-2 murine histocompatibility complex. Most of his work was in transplantation genetics. He identified antigen II and described its association with tumor rejection. The Gorer Symposium. Oxford: Blackwell, 1985. From 1944 to 1946, Medawar and associates provided the first convincing proof that the rejection of grafts between individuals who are not related to one another (i.e., allografts or homografts) has an immunological basis. He showed that allogeneic skin grafts heal-in and appear healthy. After several days, however, host lymphoid cells may infiltrate the skin graft and undergo blastogenesis. Twelve days later the allograft may appear necrotic, representing first-set rejection. A second graft of the same donor specificity is rejected at an accelerated rate, i.e. within 6–10 days. This is termed second-set rejection, which represents host sensitivity to transplantation antigens of the graft. During this same period, Ray Owen (Figure 1.101) described dizygotic cattle twins in which blood cells of one twin were tolerated immunologically by the other (i.e., they were chimeras). Ray David Owen (1915– ) is an American geneticist who described erythrocyte mosaicism in dizygotic cattle twins. This discovery of reciprocal erythrocyte tolerance contributed to the concept of immunological tolerance. This observation that cattle twins that shared a common fetal circulation were chimeras and could not reject transplants of each other’s tissues later in life provided the groundwork for Burnet’s ideas about tolerance and Medawar’s work in transplantation.

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Figure 1.101  Sir Peter B. Medawar.

Medawar, Billingham, and Brent went on to conduct grafting experiments in which tissues and cells from one strain of mouse were successfully transplanted in recipients of a different strain that had been administered cells bearing donor antigen prior to birth. Their classic paper on acquired immunological tolerance, which was published in Nature in 1953, demonstrated that sensitization could be transferred passively with specifically sensitized lymphoid cells. Peter Brian Medawar (1915–1987) (Figure  1.102) was a British transplantation biologist who received his PhD at Oxford, 1935, where he served as lecturer in zoology. He was subsequently a professor of zoology at Birmingham (1947)

Figure 1.102  Ray Owen.

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and at University College, London, 1951. He became director of the Medical Research Council, 1962 and of the Clinical Research Center at Northwick Park, 1971. Together with Billingham and Brent, he made seminal discoveries in transplantation and immunobiology and described immunological tolerance and its importance for tissue transplantation. He shared the 1960 Nobel Prize in Medicine or Physiology with Sir Frank Macfarlane Burnet. Although serologically identifiable antibodies are produced in animals rejecting allografts, the demonstration of round cell infiltration (i.e., lymphocytes) in the graft bed suggested to Mitchison that graft rejection was dependent upon lymphoid cells rather than serum antibodies. By taking lymphoid cells from animals rejecting a tumor and injecting them into a recipient never before exposed to the graft, he showed that sensitization against graft antigens could be transferred with specifically immune lymphoid cells but not with serum. Mitchison termed this procedure adoptive immunization. Those investigators who could not accept the idea that lymphoid cells rather than humoral antibodies were responsible for graft rejection postulated that cytophilic antibodies in the serum adsorb to lymphoid cells and confer specific reactivity on them. This subject was explored by Boyden and Sorkin in the 1960s. Meanwhile, Burnet and Fenner, at the Walter and Eliza Hall Institute, were beginning to take a view of antibody production different from that proposed by chemists adhering to the template theory of antibody synthesis. Their first monograph, The Production of Antibodies, was published in 1941, and the second edition appeared in 1949. Modifying their views through various explanations that included a self-marker hypothesis to explain antibody production, Burnet noted with interest that Jerne had proposed a selective theory of antibody formation in 1955. Whereas Jerne discussed various antibody populations, Burnet applied this natural selection concept, which was the first selective or genetically based theory of immunity since the time of Ehrlich, to a new hypothesis, which he termed the clonal selection theory of acquired immunity, in 1959. The template theory of antibody production, which had been popular with the chemists for so many years could no longer explain these new biological revelations that included immunological tolerance, and it had never explained the secondary (or anamnestic) immune response. Burnet proposed precursor cells with a limited range of specificity for interaction with antigens. Upon stimulation, the precursor cell would proliferate into a clone of cells producing antibody of that specificity. Since that time, various modifications of the clonal selection hypothesis have been offered. Dreyer and Bennett offered a germ line theory. Smithies proposed a half-gene concept to explain antibody diversity, and Tonegawa won the Nobel Prize for his elegant studies and explanation of the generation of diversity in antibody synthesis. Snell (Figure 1.103) in 1948 introduced the term histocompatibility antigens to designate the antigenic specificities that confer tissue compatibility. He stated that histocompatibility

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Figure 1.103  George D. Snell in his laboratory.

genes (or H genes) determine these antigens. Snell went on to develop the sophisticated genetic methods required to unravel the mystery of histocompatibility loci. He developed coisogenic (or congenic) strains of mice showing only one H difference. Snell’s development of the congenic lines of mice was the most significant event in transplantation biology since the introduction of inbred strains. His careful development of the congenic lines made the mouse the prototype animal in transplantation genetics and immunology. He went on to discover the existence of major (strong) and minor (weak) H genes in mice. The major histocompatibility locus of the mouse was designated H-2 by Snell and co-workers. With the demonstration that more than one gene is present at the H-2 locus, it was called the major histocompatibility complex (MHC) in the mouse. Snell was subsequently awarded the Nobel Prize for his work. Following the success of Snell’s histogenetic methods, Gorer in England proceeded to characterize antibodies to H-2 products by serological methods. He and his co-workers employed hemagglutination and cytotoxicity tests. Among the group of investigators gathered around Gorer at Guy’s Hospital Medical School in London during the 1950s were Bernard Amos, Z. B. Mikulska, O’Gorman, Gustavo Hoecker, and Nathan Kaliss, who later made important contributions in immunological enhancement of tumor allografts. George Davis Snell (1903–1996), American geneticist who shared the 1980 Nobel Prize with Jean Dausset and Baruj Benacerraf, “For their work on genetically determined structures of the cell surface that regulate immunologic reactions.” Snell’s major contributions were in the field of mouse genetics, including discovery of the H-2 locus (together with Gorer) and the development of congenic mice. He made many seminal contributions to transplantation genetics and received the Gairdner Award in 1976. Histocompatibility (with Dausset and Nathenson), 1976.

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Although conceded by all to have attracted a group of brilliant investigators and to represent exquisite scientific methodology and results, the H-2 system was considered by many to be an esoteric topic in biology and medicine. Studies designed to decipher the H-2 system were aimed at clarifying the two-locus model (i.e., H-2K and H-2D) and elucidating the nature and function of I-regions, among others, in the major histocompatibility complex of the mouse. With the discovery of histocompatibility antigens of human leukocytes in 1958 by Dausset and Rapaport, all of the valuable data garnered by the mouse H-2 researchers took on a new significance. The remarkable similarity between the H-2 of the mouse and the HLA of man, and the numerous crossreactions between the two, provided researchers on human histocompatibility testing with a head start in their attempt to define the immunogenetics of human HLA. A major difference between human and mouse histocompatibility was the need to study large groups of unrelated human subjects, in striking contrast to the inbred strains of mice where numerous genetically identical individuals of a single strain could be tested. Using the leukoagglutination technique, Dausset described the first HLA antigen, designated Mac, which was subsequently identified on platelets by complement fixation. The fortunate demonstration that multiparous women produce antibodies against lymphocytes provided a major source of antibodies for tissue-typing trays used in clinical histocompatibility testing. Dausset’s work encouraged Rose Payne to undertake studies of human histocompatibility and immunogenetics in the United States. With advances in DNA technology, molecular typing is supplanting serological assays.

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Figure 1.105  Dr. Rose Payne.

Jean Baptiste Gabriel Dausset (1916–2009) (Figure 1.104), French physician and investigator. He pioneered research on the HLA system and the immunogenetics of histocompatibility. For this work, he shared a Nobel Prize with Benacerraf and Snell in 1980. He made numerous discoveries in immunogenetics and transplantation biology. Immunohematologie Biologique et Clinique, 1956; HLA and Disease (with Svejaard), 1977.

Rose Payne (1909–1999) (Figure  1.105), earned her PhD in bacteriology at the University of Washington, Seattle. She spent her professional career at Stanford where she co-authored an important paper on Evans syndrome, a combination of autoimmune hemolytic anemia and thrombocytopenic purpura. She became an authority on red cell and platelet autoantibodies, and in 1957 found that chill-fever transfusion reactions were attributable to patient alloantibodies to donor leukocytes. She also discovered an association between previous pregnancies and the presence of leukoagglutinins, which prompted her to begin an extensive program of serum collection searching for potential typing sera. She and Emanuel Hackel showed that the leukocyte antigens were inherited. Payne and Walter Bodmer identified two alleles from a single locus: LA-1 and LA-2 (HLA-A1 and HLAA2). Subsequently they reported a third allele at the LA locus (LA-3 or HLA-A3), and additional new antigens. Further definition of HLA antibodies was achieved through International Histocompatibility Workshops.

Figure 1.104  Professor Jean Dausset.

Baruj Benacerraf (1920– ) (Figure 1.106), American immunologist born in Caracas, Venezuela. Beginning his scientific career studying hypersensitivity mechanisms in Elvin Kabat’s laboratory at Columbia, he moved to Paris to investigate reticuloendothelial function in relation to immunity. On returning to the United States, he joined the pathology faculty at New York University where he resumed experiments on hypersensitivity mechanisms. He investigated cellular hypersensitivity, immune complex disease, anaphylactic hypersensitivity, tumor-specific immunity, and the structure of antibodies, in relation to their specificity. He mentored a host of gifted fellows and students at NYU. He initiated immunogenetics studies that led to the observation that random-bred animals immunized with antigens with restricted heterogeneity, such as hapten conjugates of poly-L-lysine, distribute themselves

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Figure 1.106  Professor Baruj Benacerraf.

into two groups, responders and nonresponders. Benacerraf determined that responsiveness to these antigens is controlled by dominant autosomal genes termed immune response (Ir) genes, located in the major histocompatibility complex of mammals. His studies led to understanding of the manner in which these genes exercise their function and determine immune responsiveness. His subsequent work at the National Institutes of Health and at Harvard concerned the role of immune response genes in the regulation of specific immunity and the control of immune suppression, among other investigations. His multiple contributions include the carrier effect in delayed hypersensitivity, lymphocyte subsets, major histocompatibility complex (MHC), and Ir immunogenetics, for which he shared the Nobel Prize in 1980 with Jean Dausset and George Snell. Benacerraf and colleagues showed that many of the genes within the MHC control the immune response to various immunogens. Using synthetic polypeptides as antigens, Benacerraf, McDevitt, and coworkers demonstrated that immune response Ir genes control an animal’s response to a given antigen. These genes were localized in the I region of the MHC. (Benacerraf B, Unanue ER. Textbook of Immunology. Baltimore, MD: Williams & Wilkins, 1979.) In 1964, Bernard Amos invited a group of HLA researchers to gather for the first workshop on histocompatibility testing in Durham, North Carolina. Although concerned that their methods were contradictory, the international group met again in 1965 in Leiden at the invitation of J. J. van Rood (Figure 1.108). Using improved techniques they demonstrated that different human tissue types do indeed exist. Ceppelini organized the third workshop held in Turin, Italy, in 1967, where genetic studies using Italian families demonstrated classic Mendelian inheritance of tissue specificities. The fourth workshop organized by Paul Terasaki (Figure 1.107)

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Atlas of Immunology, Third Edition

Figure 1.107  Paul Terasaki.

was held in Los Angeles in 1970, where the major histocompatibility system of man was clearly established as a result of serological investigations of 300 families. Paul Terasaki (1929– ), American immunologist, who began his career in transplantation immunology as a postdoctoral research fellow under Peter Medawar in London in 1957. On returning to University of California, Los Angeles, in 1959, he perfected the microcytotoxicity test which was important in identification of the HLA system. He established the usefulness of HLA in cross-matching and detection of presensitization in organ grafting. The principal theme of his research has concerned evaluation of the role of HLA matching in transplantation. His more recent work has been in establishing the role of HLA antibodies in chronic rejection. Terasaki has played a significant international role in tissue typing as director of tissue typing at UCLA and of the Regional Organ Procurement Agency of Southern California. He is one of the world’s leading researchers in HLA and transplantation. The company he founded, One Lambda, Inc., has made key contributions to HLA technology.

Figure 1.108  Dr. J.J. van Rood.

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J. J. van Rood, of the Netherlands, wrote his MD thesis on leukocyte grouping and techniques. He reported the first two allele systems, which he called 4a and 4b. The 4a and 4b antigens were difficult to define, public antigens shared by many molecules. By testing leukocyte alloantibodies obtained from the sera of a large number of women during pregnancy against leukocytes from 100 unrelated donors, a systematic approach was developed to decipher the complexity of the HLA system. Van Rood and associates described nine HLA antigens and a diallelic system not linked to HLA (the group-5 system). Balner and van Rood showed that leukocyte antigens were transplantation antigens. Dr. van Rood’s contributions to HLA research are legion. Fifty-four human populations studied over the vast reaches of the earth constituted the subject of the fifth workshop sponsored by Dausset in France in 1972. They found that the same genetic laws govern tissue types among divergent populations from remote areas as the ones they had described previously in national or ethnic populations. Kissmeyer-Nielsen sponsored the sixth histocompatibility or transplantation workshop in 1975, where six HLA-D alleles were described and the HLA-C locus was demonstrated. Besides genes coding for histocompatibility or transplantation antigens, the major histocompatibility complex in man (i.e., HLA) contains genes that govern specific immunological responsiveness, T and B lymphocyte cooperation and complement components. A seventh workshop was held in Oxford in September 1977, the main theme being the serological investigation and genetics of the Ia determinants and serological studies of specificities of HLA-A, -B and -C loci. Subsequent international workshops have addressed equally important topics in HLA. The eleventh was held in Japan. In addition to the use of clinical histocompatibility testing to predict the success of organ and bone marrrow grafts, many human diseases were found to be associated with certain HLA antigenic specificities. Notable among these associations was the finding that some B-27 positive individuals suffer from ankylosing spondylitis. HLA testing has also found wide application in resolving cases of disputed paternity. E. Donnall Thomas (1920– ) and Joseph E. Murray (1919– ) (Figure 1.109), recipients of the 1990 Nobel Prize for Medicine or Physiology for their work during the 1950s and 1960s on reducing the risk of organ rejection by the body’s immune system. Murray performed the first successful organ transplant in the world, which was a kidney from one identical twin to another, at the Peter Bent Brigham Hospital in 1954. Two years later, Thomas was the first to perform a successful transplant of bone marrow, which he achieved by administering a drug that prevented rejection. The two doctors have made significant discoveries that “have enabled the development of organ and cell transplantation into a method for the treatment of human disease,” said the Nobel Assembly in its citation for the prize. In subsequent years other organ and tissue transplants were pioneered. Dr. James D. Hardy, of the University of Mississippi Medical Center, successfully performed the first

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Figure 1.109  E. Donnall Thomas (left) and Joseph Murray (right).

human lung transplant in 1963. The following year he transplanted a chimpanzee heart, which functioned successfully for 90 minutes, into the chest of a dying man with advanced heart failure. Dr. Christian Barnard of South Africa went on to transplant a healthy human heart into a dentist with heart failure. Dr. Thomas Starzl of Pittsburgh pioneered liver transplantation in 1963. Dr. Paul Lacy and colleagues of Washington University in St. Louis perfected islet cell transplantation in the BB rat model of diabetes mellitus. Rolf Zinkernagel (1944– ) and Peter Doherty (1940– ), recipients of the 1996 Nobel Prize for Medicine or Physiology for their demonstration of MHC restriction. In an investigation of how T lymphocytes protect mice against lymphocytic choriomeningitis virus (LCMV) infection, they found that T cells from mice infected by the virus killed only infected target cells expressing the same major histocompatibility complex (MHC) class I antigens but not those expressing a different MHC allele. In their study, murine cytotoxic T cells (CTLs) would only lyse virus-infected target cells if the effector and target cells were H-2 compatible. This significant finding had broad implications, demonstrating that T cells did not recognize the virus directly but only in conjunction with MHC molecules. The finding that repeated blood transfusions prior to grafting actually improved the survival of kidney allotransplants, possibly through tolerance induction, led to deliberate transfusions prior to grafting. This assumed lesser importance with the introduction of cyclosporine for immunosuppressive therapy. The best transplant results guaranteeing long-term graft survival were with HLA-A, -B, -DR matching in addition to cyclosporine and subsequent immunosuppressive drug therapy. The Bretscher–Cohn theory maintains that self/not-self discrimination occurs at any stage of lymphoreticular development. The concept is based on three principles: engagement of the lymphocyte receptor by antigen provides signal (1), and signal (1) alone is a tolerogenic signal for the lymphocyte; provision of signal (1) in conjunction with signal (2),

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a costimulatory signal, results in lymphocyte induction; and delivery of signal (2) requires associative recognition of two distinct epitopes on the antigen molecule. The requirement for associative recognition blocks the development of autoimmunity in an immune system where diversity is generated randomly throughout an individual’s lifetime.

Immunization against Infectious Diseases Nearly a century after Edward Jenner discovered a vaccination against smallpox, Pasteur and associates used attenuated microorganisms to induce a state of protective immunity against chicken cholera. Subsequently, killed bacterial vaccines were developed to induce immunity against other diseases, such as anthrax. There was a great ferment among medical investigators during the latter half of the 19th and early 20th centuries to confer immunoprophylaxis through the use of vaccines, to protect the population against all sorts of infectious diseases. In addition to vaccines, antitoxin and toxoids were developed for diseases induced by exotoxins, that is, diphtheria and tetanus. Ehrlich’s venture into the development of a chemotherapeutic agent for the treatment of syphilis, which he termed Salvarsan, an arsenical compound, signaled the beginning of diminished emphasis on the preparation of new bacterial vaccines. With the discovery of the sulfonamides in the mid-1930s by Gerhard Gomagk, and the subsequent use of penicillin and other antibiotic agents, interest in vaccines was confined principally to those directed against virus diseases, which could not be treated successfully by chemotherapy or antibiotics. As bacterial strains resistant to antibiotic agents appeared, there was renewed interest in bacterial vaccines. Typhoid and pertussis vaccines proved highly effective, and an attenuated strain of bovine tubercle bacillus called bacillus Calmette–Guérin (BCG) produced limited success in immunization against tuberculosis during the first third of the 20th century. In later years this vaccine found another use as adjunct therapy in reawakening the subdued immune response in cancer victims, apparently by producing a generalized activation of the cells involved in the immune response. Nevertheless, the major accomplishments in immunoprophylaxis were associated with the development of viral and rickettsial vaccines. Pasteur was the first to treat a case of human rabies successfully by repeated injections of a vaccine prepared from the spinal cords of rabbits challenged with the virus. Since that time, rabies vaccines prepared from hen and duck embryos infected with the virus have been used with some success. But in the 1960s, Martin Kaplan and Hilary Koprowski (Figure  1.110) prepared a highly potent vaccine from virus grown in normal human embryonic cell cultures. It had the advantage of requiring fewer injections than the Pasteur treatment, and eliminated the possibility of allergy to duct embryo antigens. The chick embryo and the mouse have proved highly beneficial for the development of viral and rickettsial vaccines. One of the more important of these

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Figure 1.110  Hilary Koprowski.

was the production of a highly effective yellow fever vaccine from the attenuated 17D strain of the virus, which led to the award of a Nobel Prize to Max Theiler, its discoverer. John Enders (Figure 1.111), F. C. Robbins, and T. H. Weller of Harvard Medical School found in 1949 that polio viruses could be successfully propagated in human embryonic and adult nonnervous tissue cultures. They were awarded the Nobel Prize in 1954 for this work. Dr. John F. Enders (1897–1985), American microbiologist, shared the 1954 Nobel Prize in Medicine with T.H. Weller and F.C. Robbins for discovering that many viruses (specifically poliomyelitis virus) can be grown in tissue culture and thereby studied and isolated, making possible the production of vaccines. Jonas Salk (Figure  1.112), at the University of Pittsburgh, prepared a vaccine including all three types of polio virus,

Figure 1.111  John F. Enders.

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of the Merck Institute contributed to the research that led to hepatitis B vaccine.

Figure 1.112  Jonas Salk.

inactivated by formaldehyde, which proved safe and effective and was used in 650,000 children in 1954. Employing the principles of local immunization established by Besredka at the Pasteur Institute during the early part of the 20th century, Albert Sabin (Figure 1.113) developed a live attenuated oral vaccine for poliomyelitis, which produced local immunity in the intestine. Later, Enders and associates isolated the measles virus, grew it in tissue culture, and developed highly effective vaccines. This was followed by the isolation of rubella virus in 1962 by Weller and Parkman, and a successful vaccine was developed. A live mumps vaccine was introduced for general use in 1967, and is generally given with measles and rubella at age 15 months. Other vaccines developed include one against arboviruses, which infect domestic animals, meningococcal polysaccharide vaccines, adenovirus, herpes virus and some others, such as a rickettsial vaccine for typhus. Later a vaccine was developed for B viral hepatitis that is carried in the blood and other bodily fluids of some 200 million humans. A team led by Wolf Szmuness of the New York Blood Center in 1980 concluded a large field trial on a preparation developed from work begun in 1963, when the Australia antigen was first recognized by Baruch Blumberg in Philadelphia. Saul Krugman of New York University and Maurice Hilleman

Figure 1.113  Albert Sabin.

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Some attempts at vaccination have been disappointing. Although strains A, B, and C, as well as A2, which causes Asian influenza, have been known for many years, the ability of influenza viruses to vary their antigenic patterns makes immunization difficult. Once a variant strain with a new antigenic structure develops against which the host does not have antibodies, the disease results. Much attention has been devoted to the development of protozoal vaccines. Vaccines for malaria have received top priority. There is great hope for the production of effective vaccines against other protozoan infections, which could be effectively used in the underdeveloped countries of the world. Immunoprophylaxis is not without hazard. For example, the use of live virus vaccines can prove fatal in children with T-cell immunodeficiencies or in adults who have been deliberately immunosuppressed by drugs. Smallpox vaccination was sometimes followed by encephalitis or progressive vaccinia, both of which could lead to death. Yellow fever vaccination can lead to encephalitis. Live measles vaccine may produce convulsions. There is always the chance that live polio virus vaccine can lead to paralytic poliomyelitis. A live virus might penetrate the placenta, leading to infection of the fetus. Viruses contaminating tissue culture materials may cause problems. SV40 virus was found in live polio virus vaccine, and fowl leukosis viruses were discovered in yellow fever vaccine preparations. Viruses oncogenic for one species could hybridize with a human virus, thereby passing on its oncogenic potential. In recent years, conjugate vaccines have proven especially valuable in the protection of very young children against Haemophilus influenzae and related infections. Max Theiler (1899–1972) (Figure  1.114), South African virologist who received the Nobel Prize in 1951 “for his development of vaccines against yellow fever.”

Figure 1.114  Max Thelier.

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Interferon While studying the phenomenon of viral interference (i.e., the resistance shown by cells infected by one virus to simultaneous infection by another), Alick Isaacs and Jean Lindenmann discovered a substance which they named interferon. In these experiments, chick cells infected with influenza viruses were resistant to infection with other viruses. Healthy chick cells treated with an extract prepared from the growth medium were resistant to infection when challenged with a live virus preparation. Thus, it was clear that the virus-infected cells had produced a substance which interfered with the infection of other cells by viruses. The substance was soon shown to be cell or species specific and not virus specific. This was all the more reason to pin great hopes on a substance that might aid the animal in resisting a cadre of viruses associated with various types of infectious diseases. Although impressive at first, interest began to wane because living cells produced only minute amounts, and interferon was technically difficult to purify and the process expensive. Kari Cantell, a Finnish virologist, produced interferon from leukocytes collected from 500–800 pints of blood collected each day from donors at the Finnish Red Cross. After infecting leukocytes with Sendai virus, he incubated the cells and then extracted interferon. His interferon was used in clinical trials throughout the world; it not only protected individuals against potentially devastating virus diseases, but it also proved effective in the treatment of some forms of cancer, apparently through the activation of natural killer (NK) cells. Recombinant DNA technology and cDNA cloning techniques have revolutionized our understanding of complex molecules, such as interleukin-2 (IL-2) and other lympho­ kines, produced in picomolar quantities by immune system cells but made available for analysis in milligram quantities by the marvels of genetic engineering technology and the polymerase chain reaction.

Congenital Immunodeficiencies In 1952, Bruton described hereditary sex-linked agammaglobulinemia in young boys who had only trace amounts of immunoglobulins in the blood, but their T-cell immune function remained intact. This represented a classic defect of B cells. By contrast, DiGeorge syndrome, characterized by defective T-cell immunity, was described later and attributed to failure of the thymus to develop. On the other hand, the severe combined form (Swiss type) of immunodeficiency represented a failure of both B- and T-cell limbs of the immune response. Robert Good, Donnall Thomas, and others instituted widespread clinical application of bone marrow transplants for treatment of these unfortunate children with defective immunity. Richard Hong at Wisconsin successfully treated selected T-cell and combined immunodeficient children with fetal tissue thymus transplants.

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Adenosine deaminase-deficient patients have been successfully treated by “gene therapy” at the National Institutes of Health (United States) as a consequence of phenomenal advances in genetic engineering and gene transfection technology.

Acquired Immune Deficiency Syndrome In mid-1981, five young homosexual males in Los Angeles contracted Pneumocystis carinii pneumonia, and two died. This heralded the beginning of what became known as the AIDS (acquired immune deficiency syndrome) epidemic. An intensive research program was launched that led to rapid advances, with the discovery in 1984 of a retrovirus as the etiologic agent of the disease. Luc Montagnier and associates, at the Institut Pasteur in Paris, discovered LAV (lymphadenopathy-associated virus), which was subsequently named HIV (human immunodeficiency virus), the causative agent of AIDS. This was followed by the development of an antibody test to protect the blood supply. In spite of phenomenal advances in unraveling the molecular biology of AIDS, development of an effective vaccine has proved elusive thus far. The infection, which was first described in the United States but was subsequently found in countries around the world, is characterized by profound immunosuppression as the virus targets CD4 lymphocytes. This renders patients susceptible to a litany of opportunistic microbial agents, secondary neoplasms, and neurologic manifestations. Although first described in homosexual and bisexual males, it also afflicts intravenous drug abusers, and recipients of HIV-infected blood and blood products. In Africa it is a heterosexual disease. Controlling the AIDS epidemic is one of the most challenging public health problems in the world today.

Assays of Antigens and Antibodies Just as the eclectic science of immunology intersects essentially all of the basic biological sciences, it makes use of many biochemical techniques such as chromatography and protein fractionation. It also employs the newer methods of molecular genetics such as gene sequencing and related techniques. Advances in technology have armed the immunologist with the powerful tools of polymerase chain reaction (PCR) technology, immunophenotyping by flow cytometry, hybridomas and monoclonal antibodies, DNA typing, enzyme-linked immunosorbent assay (ELISA), and radiolabeling of immune system molecules. In addition, the time-honored methods of precipitation, agglutination, complement fixation, and related techniques have long been used by the immunologist. Since its inception, immunologic science has not only maintained a unique nomenclature but also special techniques that have elucidated some of nature’s most jealously guarded secrets through scientific investigation. Inbred mice, of known genetic constitution, and more recently, transgenic animals including knockout mice, offer new avenues for elucidating some of immunology’s most

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perplexing conundrums. Great tomes are currently available that describe the myriad immunological techniques now available. Although Charrin and Roger observed that immune serum could produce clumping of bacteria in suspension, Durham in 1896 discovered bacterial agglutination by immune serum and applied the method as a diagnostic tool. Widal applied the principle discovered by von Grüber and Durham that the blood serum of typhoid patients would agglutinate typhoid bacilli to develop a clinically useful test for typhoid fever in which blood sera of patients were tested against known bacteria in an agglutination assay. Kraus introduced the precipitin test the following year. He discovered precipitation in a mixture of immune serum and culture filtrates of Vibrio cholerae. Both of these methods found immediate widespread use in diagnostic bacteriology. When Landsteiner described the human blood groups in 1900, he found natural isohemagglutinins in the serum. The next year (1901), Bordet and Gengou discovered the complement fixation test and applied it to measure antigen–antibody reactions. The complement fixation test found widespread use in the diagnosis of infectious diseases. When used to diagnose syphilis, the test became known as the Wassermann reaction. After the demonstration by Denys and Leclef in 1895 that immunization has an enhancing effect upon phagocytosis, Wright and Douglas (1903) discovered antibodies in the blood, called opsonins, that attached to bacteria or other particles and assisted their phagocytosis. This constituted the basis of an opsonocytophagic index that correlated the ability of patient leukocytes to phagocytize bacteria in vitro with the patient’s level of immunity against the infectious disease agent. Although these studies had limited significance, they did yield some valuable information concerning opsonins in immunity. Agglutination, precipitation, and complement fixation were the tools of serology, the study of antigen–antibody interactions in vitro. Dean and Webb (1928) developed an optimal proportions procedure for the quantitation of precipitating antibodies. In the 1930s, Heidelberger and Kendall developed precise methods for the quantitation of antibody, and they discovered and characterized nonprecipitating antibodies, which were functionally univalent or incomplete. Pappenheimer and Robinson in the 1930s described a variant of precipitation called flocculation, which was observed in studies on horse antisera against bacterial toxins and later in the human reaction to thyroglobulin. In 1934, Marrack proposed a lattice theory of secondary aggregates of antigen and antibody in his monograph The Chemistry of Antigens and Antibodies. A significant breakthrough in methodology came in 1937 when Tiselius proposed the electrophoretic method of separating serum proteins and the assignment of antibody activity to the globulin fraction. In 1929, Kabat and Tiselius demonstrated by electrophoresis and ultracentrifugation that 19S antibody is found early and 7S antibody late in the immune response.

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August von Wassermann (1866–1925), German physician who, with Neisser and Bruck, described the first serological test for syphilis, the Wassermann reaction. Handbuch der Pathogenen Mikroorganismen (with Kolle), 1903. Gaston Ramon (1886–1963), French immunologist who perfected the flocculation assay for diphtheria toxin. Bela Schick (1877–1967), Austro-Hungarian pediatrician whose work with von Pirquet resulted in the discovery and description of serum sickness. He developed the test for diphtheria that bears his name. Die Serumkrankheit (with Pirquet), 1905. Michael Heidelberger and associates in 1933 reported the use of colored materials in quantitative studies of the precipitation reaction. They attached a salt of benzidine to egg albumin through a diazo linkage. Spectrophotometric methods were employed in the studies. Marrack (1934) showed that dyes could be introduced into antibody molecules without altering their immunological specificity. In 1941, Coons (Figure 1.115) and associates at Harvard Medical School first labeled antibody molecules with fluorescent dyes. Coons’ group later developed an improved optical filter system, a procedure for the preparation of isocyanate and a technique for the conjugation of antibody globulin with fluorescence dye. Tissue sections or smears of material containing antigen were covered with specific antibody labeled with a fluorescent compound, and observed in ultraviolet light for fluorescence. Although nonfluorescent dyes were first employed, they were inferior to the fluorescence variety because of the greater ease of detecting a minute amount of light against a dark background compared with detecting a small amount of color in a bright microscopic field. This fluorescent antibody

Figure 1.115  Albert H. Coons.

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method found wide application in diagnostic medicine as well as in research. Albert Hewett Coons (1912–1978), American immunologist and bacteriologist who was an early leader in immunohistochemistry with the development of fluorescent antibodies. Coons, a professor at Harvard, received the Lasker Award in 1959, the Ehrlich Prize in 1961, and the Behring Prize in 1966. The antiglobulin technique described in 1908 by Moreschi was rediscovered and perfected in 1945 by Coombs. This has been a useful tool in the detection of incomplete (i.e., more correctly termed nonagglutinating) antibodies, such as those found in certain types of autoimmune hemolytic anemia and hemolytic disease of the newborn. It detects antibodies attached to cell surfaces that are unable to bridge the gap between cells to produce agglutination in saline. The use of a medium that reduces the zeta potential surrounding cells and the use of rabbit antihuman immunoglobulin permitted Coombs to develop the test. Jules Freund (1890–1960), Hungarian physician who later worked in the United States. He made many contributions to immunology, including work on antibody formation, studies on allergic encephalomyelitis, and the development of Freund’s adjuvant. He received the Lasker Award in 1959.

Figure 1.116  Orjan Ouchterlony.

Jacques Oudin (1908–1986), French immunologist who was director of analytical immunology at the Institut Pasteur, Paris. His accomplishments include discovery of idiotypy and the agar single diffusion method antigen–antibody assay.

Singer first developed methods for the detection and localization of antigen or antigen–antibody complexes by electron

hypogammaglobulinemia patients. This valuable technique found broad applications in both clinical diagnosis and research.

Orjan Thomas Gunnersson Ouchterlony (1914–2004) (Figure  1.116), Swedish bacteriologist who developed the antibody detection test that bears his name. Two-dimensional double diffusion with subsequent precipitation patterns is the basis of the assay. Handbook of Immunodiffusion and Immunoelectrophoresis, 1968. In 1946, Oudin discovered that bands of precipitation occurred in tubes containing antiserum and antigen incorporated into agar. This represented diffusion in a single dimension. The technique was expanded by Ouchterlony and independently by Elek, who used agar in Petri plates to allow antigen and antibody to diffuse from wells cut in the agar in two dimensions. With this technique, relative similarities or differences in antigens reacting with a common antiserum could be detected. Laser nephelometry proved beneficial in quantifying the precipitin reaction. In 1953, Grabar (Figure 1.117) and Williams developed the immunoelectrophoresis technique in which a serum sample or other antigen preparation was electrophoresed to separate its components prior to interaction with antibody to produce precipitation lines in gel. This method proved that certain immunoglobulin classes were lacking in the sera of

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Figure 1.117  Pierre Grabar (1898–1986), French-educated immunologist, born in Kiev, who served as chef de service at the Institut Pasteur and as director of the National Center for Scientific Research, Paris. He is best known for his work with Williams in the development of immunoelectrophoresis. He studied antigen– antibody interactions and developed the “carrier” theory of antibody function. He was instrumental in reviving European immunology in the era after World War II.

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engineering account for the windfall of progress in immunological research.

Figure 1.118  Rosalyn Yalow.

microscopy. He conjugated ferritin to antibody molecules, rendering them highly electron scattering, thus permitting their detection as a row of gray points along a cell membrane where antigens were located. Rosalyn Sussman Yalow (1921– ) (Figure 1.118), American investigator who shared the 1977 Nobel Prize with Guillemin and Schally for her endocrinology research and perfection of the radioimmunoassay (RIA) technique. With Berson, Yalow made an important discovery of the role that antibodies play in insulin-resistant diabetes. Her technique provided a test to estimate nanogram or picogram quantities of various types of hormones and biologically active molecules, thereby advancing basic and clinical research. RIA revolutionized the treatment of hormonal disorders. It made possible the diagnosis of medical conditions associated with minute changes in hormones and launched the new field of neuroendocrinology.

Techniques for the study of cell-mediated immunity are mentioned elsewhere in this book. Following the demonstration that ficoll-hypaque could be used to isolate lymphocytes as well as neutrophils, techniques were employed to separate T and B cells from each other. The affinity of B cells for nylon wool permitted their separation from T cells, which pass through as the B cells adhere. This was later replaced by magnetic beads. Different subpopulations of macrophages have also been separated by ultracentrifugal methods. Flow cytometry has largely replaced more primitive methods for T-cell subset analysis and B- and T-cell sorting. This method permits the rapid and accurate separation of cells with different antigenic markers and functional capabilities into distinct populations for further studies. The enzyme linked immunosorbent assay (ELISA) replaced many of the radioisotypic immunoassays of serum antibodies developed in the modern era. Georges J. F. Köhler (1946– ) (Figure  1.119), German immunologist who shared the Nobel Prize in Medicine or Physiology in 1984 with César Milstein for their work on the production of monoclonal antibodies by hybridizing mutant myeloma cells with antibody-producing B cells (hybridoma technique). Monoclonal antibodies have broad applications in both basic and clinical research as well as in diagnostic assays. Köhler was born in Munich and educated at the University of Freiburg, Germany, where he received a diploma in biology in 1971 and a PhD in 1974. His research for the doctorate was conducted at the Institute for Immunology in

The immunoperoxidase technique was introduced in the 1970s for immunohistopathologic evaluation of cells and tissues that formerly relied on fluorescence antibody staining. This technique offered several advantages that include high sensitivity, the use of conventional light microscopy rather than special fluorescence microscopy, the detection of antigenic markers on cells fixed in paraffin sections for many years, and permanent sections which obviate the need for photographs required for immunofluorescence. This technique found wide application in diagnostic pathology. Without the significant advances in the techniques of biochemistry made in the past 30 years, the great strides made in immunological research would not have been possible. Highly sophisticated hybridoma technology and monoclonal antibodies, DNA sequencing, flow cytometry, chromatographic and ultracentrifugal techniques, amino acid analysis and synthesis, x-ray diffraction studies, immunoenzymology, recombinant DNA technology, and advances in genetic

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Figure 1.119  Georges J. F. Köhler.

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nearby Basel. Visiting the Basel Institute in 1973 to present a seminar, Milstein invited Köhler to join him at Cambridge as a postdoctoral fellow in 1974. Köhler accepted, and later returned to Basel in 1976 as a member of the institute. The pivotal advance made by Köhler and Milstein had neither been planned nor was it an accident. Michael Potter, in 1962, had devised technology to produce myelomas in mice, and described their maintenance. Milstein and Cotton had already used rat and mouse myelomas to investigate whether or not different chromosomes encode variable and constant regions of immunoglobulins. Their results revealed that both variable and constant regions were always of one species, even though heavy and light chain hybrids did occur. Köhler began his postdoctoral fellowship in Milstein’s laboratory at Cambridge University in 1974. His dissertation research had shown that 1,000 different murine immunoglobulins were able to react with a single epitope. He embarked upon a project with Milstein on antibody gene mutations. Encountering difficulties with the research, Köhler sought ways to synthesize myeloma antibodies of known specificity. He conceived the idea of producing hybridomas at the end of 1974, while lying in bed halfway between wakefulness and sleep. On learning his concept the next morning, Milstein encouraged Köhler to proceed. Initial failures were traced to the use of toxic reagents, but eventually the experiments succeeded. They fused antibody-forming spleen cells with mutant myeloma cells to create hybridomas, to study the genetic basis of antibody diversity. The mutant myeloma cells conferred immortality on the fused cell line, whereas the spleen cells conferred antibody specificity. Thus, the hybrid cell or hybridoma cell line was endowed with the ability for long-term survival in culture, producing an unlimited quantity of monoclonal antibodies. The success of the technique depended upon a selective method to recover only fused cells. Therefore, they employed a mutant myeloma cell line that was deficient in the enzyme hypoxanthine phosphoribosyltransferase. Cells deficient in this enzyme would perish in a medium containing hypoxanthine, aminopterine, and thymidine (HAT). However, hybrid cells would survive and could be isolated, since the antibodysynthesizing cell incorporated into the hybridoma would furnish the enzyme. Thus, the immortal hybridoma clone would produce abundant quantities of monoclonal antibodies with specificity for a specific antigenic determinant. Milstein wrote the report and received most of the initial honors and credit. Yet both were awarded the Nobel Prize in Medicine for 1984, together with N. K. Jerne. Monoclonal antibodies have broad applications in both basic science and clinical research. They have been developed against a multitude of antigens, including tissue markers that are of critical importance in surgical pathologic diagnosis of tumors, to cite but one example. The thousands of articles and dozens of monographs on monoclonal antibodies published in the last quarter-century reflect their widespread use and significance.

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Figure 1.120  César Milstein (1927–2002), an Argentinian molecular biologist who, with Georges Köhler, perfected the technique of monoclonal antibody production from hybridomas, for which they received the Nobel Prize in Medicine or Physiology (1984) which was shared with Niels K. Jerne. This technology revolutionized immunological research.

César Milstein (Figure 1.120) was born on October 8, 1927, in Bahia Blanca, Argentina. He attended the Collegio Nacional, Bahia Blanca, from 1939 to 1944, earning his bachelor’s degree. He matriculated at the University of Buenos Aires in 1945 and graduated in 1952. He continued studies for the doctorate, which he received in 1957, and continued as a staff member in the Institute of Microbiology until 1963. During a leave of absence, Milstein worked in the Department of Biochemistry at Cambridge University in England, receiving a second doctorate there in 1960. Returning to Cambridge in 1963, he joined the staff of the Medical Research Council Laboratory of Molecular Biology as a member, later directing its Protein Chemistry Division together with F. Sanger. Milstein and associates established the complete sequence of the light-chain portion of an immunoglobulin molecule. He determined the nucleotide sequence of a large segment of the light-chain messenger RNA, discovering that there is only one type of RNA for both domains within that chain. The separate domains of light and heavy chains are designated constant and variable. Milstein reasoned that the constant domain genes may be separate in the germ line but that they must come together in antibody-synthesizing cells. Milstein and Köhler proceeded to develop a method for preparing monoclonal antibodies from hybridomas in 1975, for which they subsequently shared the Nobel Prize in Medicine or Physiology. They fused mutant myeloma cells, which contributed immortality, with spleen cells, which produced antibodies against a designated antigen, contributing specificity to produce a hybridoma (hybrid cells) that could be cultured perpetually, producing antibodies

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against any antigen used to immunize the spleen cells. By selecting clones, Milstein and Köhler developed cell lines that produced monoclonal antibodies of a single antigen-binding specificity. This revolutionary technique permitted the development of permanent cultures from a single clone that could be maintained indefinitely, providing an unlimited amount of specific monoclonal antibody. The technique developed with mouse cells was later extended to perfect human hybridomas, and could even be used to synthesize purified monoclonal antibodies against impure antigens.

The Founding of the American Association of Immunologists The ferment in immunological research in European laboratories in the early 1900s attracted American and Canadian physicians to Germany, France, and England, where they became acquainted with late-breaking scientific discoveries. In addition to Ehrlich, von Behring, Koch, and Mechnikov on the continent, Sir Almroth E. Wright, director of the Inoculation Department of St. Mary’s Hospital, London, was a favorite mentor. In his Praed Street Laboratories, Sir Almroth taught his students the “new immunotherapy” that employed preventive vaccines and mass inoculation. As von Behring proved the therapeutic efficacy of diphtheria antitoxin, Wright, in 1898–1900, established the efficacy and feasibility of typhoid vaccine and the practicability of mass inoculation of British forces in the South African War and in India. In 1903, Wright and a colleague, Stewart R. Douglas, discovered antibodies in the blood, termed opsonins, that attach to bacteria or other particles and facilitate their phagocytosis. They developed an opsonocytophagic index that correlated the ability of a patient’s leukocytes to phagocytize bacteria in vitro with the level of immunity against an infectious disease agent. Leonard Colebrook joined Wright and Douglas in 1907. Recognizing that it would take many years to gain sufficient fundamental knowledge to understand immune processes, Wright predicted that “the physician of the future will be an immunisator.” Among the North American disciples attracted to Wright’s laboratory were Drs. Martin J. Synnott (Figure  1.121) of Montclair, New Jersey; Gerald Bertram Webb (Figure 1.122) of Colorado Springs, Colorado; F. J. Clemenger of Asheville, North Carolina; R. Matson of Portland, Oregon; Benjamin H. Matthews of Denver, Colorado; and F. M. Pottenger of Monrovia, California. On returning to the United States following studies on immunity at the Praed Street Laboratory, Synnott suggested that the American and Canadian students of Wright form a “society of vaccine therapists.” Dr. Gerald Bertram Webb, who had also been a pupil of Wright’s, expressed the belief that laboratory researchers should be included together with therapists to form an interdisciplinary society with a philosophy sufficiently flexible to accommodate changes in direction as immunological knowledge

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Figure 1.121  Martin J. Synnott, MD, who in 1912 had the idea for the formation of a Society of Vaccine Therapists. Courtesy of the New York Academy of Medicine Library.

increased. He believed that the proposed objective of the society and the criteria and qualifications for membership were too narrowly focused and too restricted. Dr. Webb suggested that physicians who had studied under other founders of immunology besides Sir Almroth, including Metchnikoff, von Behring, Ehrlich, and Koch should also be invited to membership.

Figure 1.122  Gerald Bertram Webb, MD, who coined the name The American Association of Immunologists and served two terms as the association’s first president. Courtesy of Mrs. Marka Webb Stewart.

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Figure 1.123  Victor Clarence Vaughan, MD, championed the cause of the laboratory researcher as vital to the interdisciplinary goals of the association. Courtesy of the University of Michigan Library.

Dr. Victor Clarence Vaughan (Figure  1.123), of Ann Arbor, Michigan, champion of laboratory researchers interested in the biochemistry of serology and vaccine therapy, expressed the view that progress in medicine was not possible without advances in physical, chemical, and biological sciences. Webb’s wise counsel prevailed, and his suggestion was adopted by the few charter members at a National Tuberculosis Association Meeting in May, 1913, in Washington, DC. The society was not chartered formally until June 1913, at a meeting of the American Medical Association in Minneapolis. Drs. Webb, Synnott, and A. Parker Hitchens and apparently the 52 enrolled charter members met on June 19 and founded the American Association of Immunologists (AAI), the name proposed by Dr. Gerald B. Webb. Other officers included Drs. George W. Ross as vice-president, Martin J. Synnott as secretary, and Willard J. Stone as treasurer. The council, which comprised Dr. A. Parker Hitchens (Figure  1.124) as chairman and Drs. Oscar Berghausen, Campbell Laidlaw, Henry L. Ulrich, and J. E. Robinson, was charged with governance of the AAI. The elected officers served as ex officio members. The council and officers proposed three principal objectives for the association, which were subsequently adopted by the charter members. The first annual meeting was held on June 22, 1914, in Hotel Chelsea in Atlantic City, New Jersey. During this meeting, Dr. A. Parker Hitchens presented a draft of the association’s constitution and bylaws, which was adopted unanimously. The constitution and bylaws were subsequently enacted on April 6, 1917. Seven applicants were elected to charter membership. Sir Almroth Wright and Captain S. R. Douglas were elected to honorary membership. Sir Alexander Fleming,

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Figure 1.124  A. Parker Hitchens, MD, first chairman of the American Association of Immunologists (AAI) council, “father” of the association’s constitution and bylaws, and the force behind an association journal. Courtesy of the New York Academy of Medicine Library.

Dr. Leonard Colebrook, and Dr. John Freeman were named corresponding members. The first scientific meeting of the AAI consisted of three sessions. Dr. Webb’s presidential address was entitled “History of Immunity.” In principle, the 1917 constitution and bylaws still serve as the basis for governance of the association. They were modified or expanded in 1936 to make past presidents honorary members of the council. Later the membership, rather than the council, elected new members. The AAI, by vote of council, accepted membership in the Federation of American Societies for Experimental Biology (FASEB) on April 1, 1942. At that time the council also voted unanimously to include the editor-in-chief and four associate editors of the Journal of Immunology as ex officio members of the AAI Council. Although traditionally officers had served only 1 year, during World War II Dr. J. J. Bronfenbrenner served as president from 1942 until 1946, when Dr. Michael Heidelberger was elected to succeed him. He sought changes in the governance policies of the association. In 1947 the constitution and bylaws were revised and were formally adopted in 1950. The changes applied to the classes of membership (i.e., active, emeritus, and honorary), together with specific qualifications for active, criteria for transfer to emeritus, and the professional achievements associated with honorary membership. Active and honorary members, nominated by the council, were to be elected by the membership-at-large. Besides the officers, four councillors, one elected each year, with 4-year terms of service, would be elected. The council would consist of the four elected officers and five councillors, one of whom

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would be the editor-in-chief of the Journal of Immunology. Past presidents would be invited to attend council meetings but would not vote. It was not until 1965 that a central office for AAI affairs was established in Beaumont House at FASEB headquarters in Bethesda, Maryland. Twelve qualified members per year were accepted between 1914 and 1938, resulting in a total of 275 active members. Only 28 new members were added between 1939 and 1945 because of World War II. Approximately 35 new active members were added each year from 1946 through 1963, resulting in 626 new members on the association’s 50th anniversary. In 1972, specific criteria for membership eligibility were established. These were modified in 1975, 1977, and 1978. Trainee members were first admitted in 1983. Other categories of membership included honorary, emeritus, and sustaining associate (corporate) members. At the time of the AAI’s 75th anniversary meeting in 1988, membership had risen to 5,142, and by 1998, the association’s 85th anniversary, total membership was 5,774. The AAI was invited to meet with FASEB first in 1939, and became a Corporate Member Society in 1942. Until 1948, the secretary of the AAI was responsible for preparing and programming the annual scientific sessions. Thereafter, the council enlisted the assistance of two members to assist the secretary in program activities to improve the quality of sessions at the annual meeting. By 1958, FASEB had begun to hold intersociety symposia involving all member societies. Symposia were included in the annual meeting for the first time in 1948. Members were permitted to submit abstracts of 10-min volunteered papers to be presented at the annual meeting as the highlight of the program. By 1967, the program committee suggested that distinguished foreign, as well as nonmember, immunologists be invited to participate in symposia. Poster presentations were begun in 1975 and represented a major departure from the volunteered oral presentations. Since that time, posters have gained considerable popularity over oral presentations, which, nevertheless, provided valuable experience for younger investigators. By 1986, poster presentations developed into poster workshop/ discussion groups led by senior investigators. The number of mini-symposia was increased in 1979, and volunteered papers were integrated into them. The time allotted for the presentation of brief papers was increased to 15 min. Slide sessions were eliminated, and all abstracts not chosen for mini-symposia were programmed into poster sessions, which constitute more than three-quarters of volunteered papers. The format of mini-symposia and poster sessions has been modified in various ways during the past 20 years. The AAI has also met apart from the traditional FASEB annual meeting, combining efforts with other FASEB societies or immunology groups with related interests.

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The AAI Establishes an Official Journal In 1915, Dr. A. Parker Hitchens announced to the AAI council, on which he had served as chairman, that a movement was afoot outside of the AAI to launch a Journal of Immunity with Dr. Arthur F. Coca of New York City as editor. To avoid working at cross-purposes, the AAI council authorized Dr. Hitchens to contact Dr. Coca and the New York Society of Serology and Hematology, of which Coca served as president, concerning the establishment of the journal. Representatives of the two organizations met in 1915, together with a representative of Williams and Wilkins Company, to work out details for founding the Journal of Immunology. Dr. Coca was chosen unanimously to become managing editor, supported by an editorial board selected by a committee comprising two members from each of the two societies, with the managing editor serving ex officio. It was decided that the journal would publish scientific research articles on immunity, serology, and bacterial therapy. It would appear bimonthly with each volume consisting of six numbers and containing from 500 to 525 total pages. By 1930 the association’s financial position improved and the journal showed a profit for the first time. In 1940 the council decided to make the publication international by inviting Latin-American immunologists to serve on the editorial board. They appointed Dr. A. Sordelli of Buenos Aires and Dr. M. Ruiz Castaneda of Mexico City as the first two non-U.S. immunologists to serve on the board. After 30 years of service, Dr. Arthur F. Coca resigned as managing editor and was succeeded by Dr. Geoffrey Edsall, who brought considerable fiscal responsibility to the journal and worked with the council to arrange for the publisher to credit the AAI with journal pages saved per year. Dr. John Y. Sugg succeeded him in 1954. By 1963, the total pages published increased to 1,400 as the quality improved with the acceptance of meritorious articles representing the forefront of immunologic research. Two thousand pages were published in 1964. This number more than doubled to 4,650 by 1978, and in 1986 the journal published 9,400 pages. Financial arrangements with the publisher continued to improve in favor of the association. With the proliferation of immunological research, the journal developed subsections of immunochemistry, cellular immunology, immunopathology, immunogenetics, and microbial and viral immunology, each with its own subeditor. In 1968, Dr. Harry Rose succeeded Dr. Sugg. In 1971, Dr. Joseph D. Feldman became the fifth editor-in-chief. He proposed a new format for an 8 1/2- by 11-inch page size that would diminish the cost of published papers. This was implemented in 1975, and students were offered the journal at reduced rates. The AAI was given copyright to the Journal of Immunology in 1972, which strengthened its role in working with the publisher to regulate journal income and to set the ratio for net income distribution. Subsequently, in 1982, the editorial board arranged for a cost-plus arrangement

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with the publisher. The council established a journal reserve fund in 1983, and charged the editorial board with reorganization of scientific and business management functions. Editorial matters were transferred to the AAI Central Office at FASEB headquarters in Bethesda, and scientific functions were separated from financial responsibilities. In 1984, the editorial board became the publications committee, which gave it stature as a standing committee of the association. In 1985, the journal began publication twice monthly. The publications committee became responsible to the council for the quality of the Journal of Immunology. With the transfer of the journal’s editorial office from San Diego to Bethesda in early 1987, Dr. Ethan M. Shevach succeeded Dr. Joseph D. Feldman as editor-in-chief with scientific responsibility for the journal, and Dr. Joseph Saunders, executive officer, became managing editor. Subsequent chief editors included Drs. Peter Lipsky and Frank Fitch. In the intervening decade, the Journal of Immunology continued to increase in size and prestige, publishing scholarly articles not only from Americans but from the international community as well.

The AAI Supports Immunology Education The AAI has played a vital role in immunology education, sponsoring an advanced course which was first authorized by the council in 1964 at the suggestion of the AAI secretary/treasurer, Dr. Sheldon Dray. It began as a summer course, with 2–3 weeks of intensive training at the professional level. Its purpose was to facilitate dissemination of immunological information to individuals, who were themselves teachers and would pass on this knowledge to their students. The first course was held in the summer of 1966 at Lake Forest College in Illinois. It was designed for university instructors and investigators desiring to improve their knowledge of basic immunology. In succeeding years, both PhDs and MDs enrolled. In 1976, the council adopted the recommendation of an expert committee that two courses be organized each year at different locations on the east and west coasts. One would be on basic immunology and the other on clinical immunology. By 1981, the annual course became self-sustaining, and in 1982, the council and the Education Committee elevated the course level to include topics in advanced basic and clinical immunology. The Education Committee selected course instructors who taught the faculty members, PhD and MD postdoctoral fellows, graduate students, and industrial staff scientists who registered. In 1984, the course was moved to Lindenwood College, St. Charles, Missouri, under the capable leadership of Drs. Carl W. Pearce and Judith A. Kapp. In 1980, the Education Committee recommended that a course be established for high-school science teachers. Subsequently, the council approved a grant to develop an instructional monograph on advances in immunology for use by high-school and college biology teachers and students.

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The AAI Addresses Clinical Immunology In 1973, the council established an ad hoc committee on hospital-based clinical immunology that was designed to address problems and increase the standards of diagnostic immunology. In 1976, the AAI council established a Committee on Clinical Immunology and Immunopathology. Whereas the council decided against involvement in certification activities, it consented to cooperate with groups engaged in certification to ensure fair and sound procedures. In 1979, the council determined that AAI representation on the certification boards of the American Academy of Microbiology, the American Board of Pathology, and the American Board of Allergy and Immunology would be desirable. The council appointed representatives recommended by the Clinical Immunology and Immunopathology Committee. In 1983, clinical diagnostic laboratory immunologists formed a new international society named the Clinical Immunology Society (CIS). The AAI council consented to support and cosponsor a meeting of the CIS in the fall of 1986. Earlier that year, representatives of the CIS met with the AAI council to define further the affiliation and interaction between the two organizations. The AAI council recognized the CIS as an autonomous society formally affiliated with the AAI, and designated the CIS to serve as its clinical regent. The AAI council determined that the affiliation would include exchange of council/board members on each organization’s council to facilitate future meetings and other activities of mutual interest. At further meetings, representatives from the two groups identified areas of mutual interest and planned future interactions. The two societies decided to hold joint meetings and to exchange not only representatives to society councils but also selected committees, such as those for programs and publications. They decided that an ad hoc committee comprising representatives from each society would meet annually to facilitate future interactions between the AAI and the CIS. Dr. Bernhard Cinader of the Canadian Immunology Society approached the council in 1967 with a proposal to establish an International Congress of Immunology that would be held quadrennially. The following year, the council established an ad hoc steering committee that led to formation of the International Union of Immunological Societies (IUIS), which met first in 1969 in Bruges, Belgium. It was then decided that the first congress would be held in Washington, DC, in August 1971. Since that time, International Congresses of Immunology have been held around the world, most recently in Montreal in 2004. On its 75th anniversary, the AAI honored Professor Michael Heidelberger, of Columbia University, on his 100th birthday. Among the distinguished members present were Professor Elvin Kabat, Heidelberger’s first PhD graduate in immunochemistry, and Professor Merrill W. Chase, long-term associate of Karl Landsteiner at the Rockefeller Institute for Medical Research (Figure 1.125).

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of immunology; and to contribute to the advancement of immunology in all its aspects. There are currently 54 member societies of the IUIS. International Congresses of Immunology are held every 3 years under the auspices of the IUIS. The IUIS also contributes to the staging of regular congresses and conferences by each of the four Regional Federations and to various educational activities in immunology.

Landmarks in the History of Immunology Figure 1.125  Professor Elvin A. Kabat (left) and Professor Merrill W. Chase (right) at the 75th annual meeting of the AAI in Las Vegas, Nevada, 1988.

Awaiting commencement of the celebratory proceeding were Professor Baruj Benacerraf, Professor David W. Talmage, Professor Julius M. Cruse, who introduced Professor Heidelberger, Dr. Heidelberger, and Mrs. D. Shreffler and Professor D. Shreffler, AAI President (Figure 1.126). Following the founding of the AAI, national immunological societies were established in many other countries. The IUIS was established in 1969 by representatives from 10 societies meeting in Bruges, Belgium, to facilitate communication and cooperation among immunologists of all nations. The IUIS is an umbrella organization for many of the regional and national societies of immunology throughout the world. The objectives of IUIS are to organize international cooperation in immunology and to promote communication among the various branches of immunology and allied subjects; to encourage, within each scientifically independent territory, cooperation among the societies that represent the interests

Figure 1.126  From right to left: Professor Baruj Benacerraf, Professor David Talmage, Professor Julius M. Cruse, Professor Michael Heidelberger, Mrs. D. C. Shreffler (AAI President Donald Shreffler not shown) at the symposium honoring Professor Michael Heidelberger on his 100th birthday at the 75th annual meeting of the American Association of Immunologists in Las Vegas, Nevada, 1988.

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1798

Vaccination Edward Jenner published Inquiry... on vaccination

1830–1850 Modern microscopes 1839

Francois Magendie, Lectures on the Blood, described secondary injection of egg albumin causing death, first description of anaphylaxis

1835–1836 Bassi described pathogenic microorganism of silkworm 1840

F. G. J. Henle’s essay on miasms and contagions, etiological relation of bacteria to disease

1850

Anthrax in sheep blood

1858

Fermentation due to microbe Natural selection

1860

H. H. Salter, On Asthma, “best work in 19th century,” G. M. (Garrison-Morton)

1864

Spontaneous generation disproved

1865

J. A. Willemin, tuberculosis inoculable in rabbits

1867

Aseptic surgery

1870

T. Klebs observed bacteria in wounds

1871

DNA in trout sperm

1876

Robert Koch, description of anthrax Anthrax transmitted from culture

1878

Koch’s masterpiece on the etiology of traumatic infections, the role of bacteria in infection, and the specificity of infection

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1879–1881 Attenuated vaccine



Complement Richard Pfeiffer recorded the occurrence of bacteriolysis in cholera vibrio incubated with specific antibody and fresh guinea pig serum, the “Pfeiffer” phenomenon first demonstrated in vivo Jules Bordet described properties of the sera of immunized animals. He found both a heat-stable and a heat-labile component, antibody and alexine (complement: Ehrlich) J. Denys and J. Leclef, immunization greatly increased phagocytosis Precipitins

1880

Pasteur’s first paper on attenuation of infective organisms, studying fowl cholera; followed by work on anthrax, rabies, and swine erysipelas

1883

Phagocytic theory

1895

1884

Elie Metchnikoff described phagocytosis, studied the phenomenon in starfish larvae and Daphnia



1885

1886

Paul Ehrlich’s book on the need of the organism for oxygen, first reference to his side-chain theory, Das SauerstoffBedurfniss des Organismus



D. E. Salmon and Theobald Smith found that dead virus could produce immunity against the living virus Louis Pasteur described method of preventing rabies



1896

1897 1887

F. B. Löffler, a first history of bacteriology, incomplete

1888

George Nuttall, demonstration of the bactericidal power of the blood of certain animals Emile Roux and A. E. J. Yersin found that a bacterium-free filtrate of the diphtheria bacillus culture contained the exotoxin Killed vaccines First antigen and antibody

1889

1890



Hans Buchner demonstrated that the bactericidal power of defibrinated serum was in cell-free serum and was lost on heating the serum to 55°C for 1 h A. Charrin and J. Roger observed clumping of bacterial suspension by immune serum Emil von Behring and Shibasaburo Kitasato published papers describing the use of antitoxins against diphtheria and tetanus in therapy, passive transfer of immunity Koch announced the preparation of “tuberculin” prematurely at the 10th International Congress of Medicine in Berlin Hypersensitivity Antitoxins

1891

Ehrlich studied the plant toxins ricin and abrin and raised antibodies to them

1892

Filterability of virus



1898 1900



1901

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Max von Grüber and Henry Durham discovered agglutination of the typhoid bacillus, by the serum of typhoid patient Fernand Widal used the same reaction in reverse, testing sera of patients against known bacteria to identify typhoid, the Grüber–Widal test Ehrlich developed a method for standardizing the antitoxin used in treatment of diphtheria, one of the founding discoveries of immunochemistry R. Kraus showed that bacterial culture filtrate could produce an antibody which formed precipitate when added to the filtrate used for injection. The precipitin reaction could be demonstrated with a variety of protein and complex polysaccharides Bordet published paper on bacterial hemolysis, bringing it to the attention of many investigators Side-chain theory Intracellular growth of virus Karl Landsteiner mentioned the agglutination of red blood cells of healthy human blood from another individual, perhaps due to inborn differences, in a footnote Blood groups P. Ehrlich and J. Morgenroth studied blood of six goats, finding that there are clumping reactions between some, not published until 1901, the horror autotoxicus theory J. Bordet and O. Gengou developed the complement fixation test, basis of many laboratory tests Emil von Behring awarded Nobel Prize in Medicine for diphtheria, tetanus antitoxin sera Max Neisser and R. Lubowski demonstrated complement deviation

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Complement fixation Yellow fever transmission

1908

Paul Ehrlich, Elie Metchnikoff, Nobel Prize for work on immunity

1902

Jan Danysz studied the Danysz phenomenon, different results from mixing antitoxin–toxin in equal parts or the antitoxin added in two doses, resulting in continued toxicity August von Wassermann, studies on hemolysin, cytotoxin, and precipitin Paul Portier and Charles Richet described anaphylaxis in dogs using Portuguese manof-war and anemone poisons P. T. Uhlenhuth, tissue-specific antigens, recognition of bird egg albumin Anaphylaxis

1909

Inbred mice

1910

John Auer and Paul Lewis; S. J. Meltzer identified the physiological reaction in anaphylaxis, recognized that asthma was a phenomenon of anaphylaxis L. Hirzfeld and E. von Dungern, blood groups were genetically determined Sir Henry Dale isolated histamine from ergot, demonstrated allergic contraction of muscle William H. Schultz developed Schultz–Dale test for anaphylaxis

Nicholas Arthus studied local necrotic lesion resulting from a local antigen–antibody reaction in an immunized animal, the Arthus reaction (type III hypersensitivity) Opsonins Allergy

1911

1903





1912

1904





1905

1906

Almroth E. Wright and Stewart R. Douglas studied opsonization reactions (coined from opsono: I prepare food for). A bridge between the cellular and humoral theories George Nuttall’s Blood Immunity and Blood Relationships published. Precipitin reactions used to demonstrate blood relationships among various animals F. Obermayer and C. P. Pick described altered protein antigens, first hapten study Fred Neufeld, bacteriotropins named and described Svante Arrhenius delivered a series of lectures in Berkeley, later to be published in the book Immunochemistry, term coined by him

1913

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Alexis Carrell, Nobel Prize for organ grafting Charles Richet, Nobel Prize for work on anaphylaxis Viruses cultured Ludvig Hektoen proved that x-rays suppressed the antibody response American Association of Immunologists founded

1914

Frederick Twort announced the transmissible lysis of bacteria by viruses, first mention of bacteriophage

1915

G. Sanarelli observed what was later called Shwartzman reaction Bacterophage discovered



Clemens von Pirquet and Bela Schick published text on serum sickness of children receiving serum therapy. Pirquet coined allergy to indicate an altered reactivity of the body, ergcia reactivity and allos altered Robert Koch, Nobel Prize for investigations in tuberculosis

1916

Landsteiner began study on haptens, carriers, and antibody specificity

1919

Jules Bordet, Nobel Prize for theories of immunity, complement

1920

Wassermann developed the test for syphilis which bears his name Richard Otto, the “Theobald Smith phenomenon” described: anaphylactic death of guinea pigs



Felix D’Herelle wrote Le Bacteriophage, filterable substance capable of bacteriolysis Albert Calmette and Camile Guérin used BCG vaccine in experimental vaccination of newborns, later in mass vaccinations to control tuberculosis

1921 1907

John Freeman, Leonard Noon, treated hay fever with injection of pollen extract Tumor viruses

A. Besredka and E. Steinhardt gave term anti-anaphylaxis to the desensitization of animals sensitized to an antigen



Carl W. Prausnitz and Heinz Küstner studied reagin; allergy to fish was passively transmitted, Prausnitz–Küstner test Bacterial lysogeny

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Alexander Fleming described lysozyme Michael Heildelberger and O. T. Avery, studies of antibody binding using capsular polysaccharides of pneumococci Clarence Little established that the homograft reaction was due to genetic differences between donor and recipient Sir Thomas Lewis wrote on the role of a histamine-like substance in the anaphylaxis complex Carl S. Williamson of the Mayo Clinic described the pathology of transplant rejection Hans Zinsser identified immediate and delayed hypersensitivity

1936 1938

1939

1924

Besredka, local immunity, oral immunizations BCG vaccine

1928

Gregory Shwartzman, local skin reactivity to B. typhosus filtrate, the Shwartzman phenomenon L. Dienes and E. W. Schoenheit wrote on delayed hypersensitivity to simple protein antigens injected into tubercles, forerunner of the adjuvant Alexander Fleming, penicillin described William Taliaferro’s monograph on immunity to parasitic infections

1929

1930 1931 1933 1934

1935

Friedrich Breinl and Felix Haurowitz, the template theory of antibody formation Direct template theory Thomsen’s description of the panagglutination reaction named for him in human erythrocytes Virus culture on embryo Virus size measured Landsteiner’s first edition of The Specificity of Serological Reactions John Marrack proposed the “lattice” theory of antibody–antigen secondary aggregates of antibody–antigen Tobacco mosaic virus crystallized Viral interference

1935–1936 Michael Heidelberger and Forrest E. Kendall isolated pure antibodies, performed quantitative precipitin reactions P. D. McMaster and S. S. Hudack, antibody produced by cells of lymph nodes

1940

1941

1942

1944 1945

1935–1939 Peter Gorer detected antigenic differences in mouse erythrocytes using immune sera

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Gorer identified the first histocompatibility antigens MHC antigens Specificity of serologic reactions Viral nucleoproteins Arne Tiselius and Elvin Kabat demonstrated that antibodies were gammaglobulins in Sweden using analytical ultracentrifuge; 19S antibody found early, 7S late in immune response Philip Levine and R. E. Stetson studied hemolytic disease of newborn, case of Mary Seno γ-globulins Karl Landsteiner and Alexander Wiener, Rh antigens Linus Pauling proposed a theory of antibody formation, the template idea again, the necessity of stereophysical complementarity of reaction Variable folding theory George Hirst discovered virus hemagglutination; also independently discovered by McClelland and Hare F. M. Burnet, Freeman, Jackson, and Lush proposed an early theory of antibody formation Jules Freund, Freund’s complete adjuvant which enhanced antibody response to antigen and directed response to development of delayed hypersensitivity A. H. Coons and H. H. Creech used fluorescein labeling, immunofluorescence as a tool for research Lloyd D. Felton observed immunological tolerance in mice injected with minute amounts of polysaccharides Karl Landsteiner and Merrill Chase demonstrated cellular transfer of sensitivity in guinea pigs Immunofluorescence Delayed-type hypersensitivity transferred with cells Bacterial transformation with DNA Peter Medawar proved that the mechanism of tissue transplant rejection was immunological Acquired immunological tolerance R. R. A. Coombs, R. R. Race and A. E. Mourant developed the antiglobulin test for incomplete Rh antibody

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Ray Owen, Wisconsin, bovine twins were chimeras T. N. Harris, E. Grimm, E. Mertens, W. E. Ehrich, the role of the lymphocyte in antibody formation Cattle chimeras Recombination in phage

1955 1956

1946

Jacques Oudin, precipitin reaction in gels

1948

Orjan Ouchterlony and Stephen Elek independently, double diffusion of antigen and antibody in gels Antibodies in plasma cells

1949 1950

A. H. Coons and Melvin H. Kaplan on the fluorescent antibody technique

1958

1951

Max Theiler, Nobel Prize for development of yellow fever vaccine



1952

Ogdon Bruton described agammaglobinemia in humans James R. Riley and Geoffrey B. West found histamine in mast cells Transduction in phage Infective agent was DNA

1959

Pierre Grabar and C. S. Williams, immunoelectrophoretic analysis P. Medawar, R. E. Billingham, and L. Brent on “actively acquired tolerance” of foreign cells, proof for Burnet and Fenner’s theory of antibody production L. Pillemer and others, properdin and its role in immune phenomena John F. Enders, F. C. Robbins, T.H. Weller, poliomyelitis virus grown in tissue cultures, Nobel Prize Chimeric tolerance The double helix

1960

1953



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Astrid Fagraeus, thesis on the correlation of antibody formation with the plasma cell series Elvin Kabat, W. T. J. Morgan, and others worked out the structure of ABO blood group antigens Burnet and Frank Fenner introduced the “self-marker” hypothesis in The Production of Antibodies P. S. Hench and others at Mayo found that adrenocorticotropic hormone inhibited allergic reaction Adaptive enzyme theory Polio virus in culture

1957





Rodney Porter, splitting IgG into Fab and Fc fragments Niels K. Jerne developed the natural selection theory of antibody production Ernest Witebsky and Noel Rose, induction of autoimmune thyroiditis in animals Discovery of human immunodeficiencies Roitt and Doniach, et al., autoantibodies in thyroid disease, Hashimoto thyroiditis B. Glick, T. S. Chang, and R. G. Jaap discussed the bursa of Fabricius in antibody production, B cells Alick Isaacs and Jean Lindenmann, discovery of interferon L. D. McLean, S. Zak, R. Varco, and R. Good, the role of the thymus in antibody production Hugh Fudenberg and H. G. Kunkel, macroglobulins with antibody activity, cold agglutinins, rheumatoid factor Daniel Bovet, Nobel Prize for antihistamine research Cell selection theories Interferon Jean Dausset, Rapaport, histocompatibility antigens on leukocytes R. S. Farr, quantitative measure of primary interaction between bovine serum albumin and antibody George Snell, the histocompatibility antigens of the mouse Solomon Berson and Rosalyn Yalow, radioimmunoassay Burnet’s clonal selection theory of antibody production Gerald Edelman, Alfred Nisonoff, and Rodney Porter studied the structure of antibody molecule using pepsin, papain J. L. Gowans, lymphocyte recirculation Nowell, lymphocyte transformation (phytohemagglutinin) Sir F. Macfarlane Burnet, Peter B. Medawar, Nobel Prize for work on acquired immunological tolerance Messenger RNA

1961

J. F. A. P. Miller, Robert Good, thymus dependence of immune responses

1962

G. B. Mackaness adoptively transferred cellular immunity against Listeria monocytogenes with cells but not with serum

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1964

A. E. Reif and J. M. V. Allen, the AKR thymic antigen and its distribution in leukemia and nervous tissues, the theta antigen

1966

John R. David and Barry R. Bloom described the first cytokine, i.e., the lymphokine, migration inhibition factor (MIF) T and B cells Genetic code completed Spleen cell cultures

1977

Rosalyn S. Yalow, radioimmunoassay

1978

Immune response gene rearrangement

1980

Prize

for

1966–1967 H. N. Claman, A. Davies, N. A. Mitchison, T–B cell cooperation



Jean Dausset, Baruj Benacerraf, George Snell, Nobel Prize for immunogenetics research, MHC W. Szmuness, hepatitis vaccine clinical trials Henry Kaplan, Lennart Olsson, human hybridomas Transgenic mice

1967

1983

T-cell receptors

1984

Human immunodeficiency virus (HIV-1) discovered to be the etiologic agent of acquired immune deficiency syndrome (AIDS) by Luc Montagnier, F. Barre–Sinoussi, and J. C. Chermann, Institut Pasteur, Paris T-cell receptor genes







Kimishige Ishizaka and T. Ishizaka, IgE as reaginic antibody Johansson and Bennich study an atypical myeloma globulin, E myeloma

1968

Immune response genes linked to MHC

1970

Site-specific restriction enzymes Reverse transcriptase

1971

Richard K. Gershon and Kazumnari Kondo, T-cell suppression

1972

Rodney R. Porter, Gerald M. Edelman, Nobel Prize for immunoglobulin structure Recombined DNA



1996



Niels Jerne, network theory of immune regulation Recombined DNA replicated Recombinant DNA

1974

T-cell restriction

1975

H. Cantor and E. A. Boyse, T-cell subclasses distinguished by their Ly antigens, cell cooperation in the cell-mediated lympholysis reaction Georges Köhler and César Milstein, monoclonal antibodies from hybridomas R. M. Zinkernagel and P. C. Doherty, the altered self-concept dual recognition by T cells Monoclonal antibodies

1987

1973



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Nobel

Pamela Bjorkman and associates determined the structure of the human class I histocompatibility antigen and its antigen binding and T-cell recognition regions Susumu Tonegawa received the Nobel Prize in Medicine or Physiology for his demonstration of how antibody diversity is generated Peter Doherty and Rolf Zinkernagel received the Nobel Prize in Medicine or Physiology for their demonstration of MHC restriction of cytotoxic T-cell recognition of viral antigens on infected cells

What better way to end our discussion of milestones in the history of immunology than to use them as a window to the future, fulfilling our hopes and aspirations for successfully combating devastating microbial and neoplastic diseases not with toxic chemotherapeutic agents, but with substances manufactured within the animal body, devised by nature to aid our survival.

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2

Molecules, Cells, and Tissues of the Immune Response

The generation of an immune response of either the innate or acquired variety requires the interaction of specific molecules, cells, and tissues. This chapter provides an overview of these structures with brief descriptions, enhanced by schematic representations and light and electron micrographs of those elements whose interactions yield a highly tailored immune response that is critical to survival of the species. Many of the molecules of immunity are described in subsequent chapters. Adhesion molecules that are important in bringing cells together in the generation of immune responses, of directing cellular traffic through vessels or interaction of cells with matrix are presented here. All lymphocytes in the body are derived from stem cells in the bone marrow. Those cells destined to become T cells migrate to the thymus where they undergo maturation and education prior to their residence in the peripheral lymphoid tissues. B cells undergo maturation in the bone marrow following their release. Both B and T cells occupy specific areas in the peripheral lymphoid tissues. Depictions of the thymus, lymph nodes, spleen, and other lymphoid organs are presented to give the reader a visual concept of immune system structure and development. The various cells involved in antigen presentation and development of an immune response are followed by a description of cells involved in effector immune functions. Understanding the molecules, cells, and tissues described in the pages that follow prepares the reader to appreciate the novel and fascinating interactions of these molecules and cells in the body tissues and organs which permit the generation of a highly specific immune response. Immunity may perform many vital functions; for example, the elimination of invading microbes, the activation of amplification mechanisms such as the complement pathway, or the development of protective antibodies or cytotoxic T cells that prevent the development of potentially fatal infectious diseases. By contrast, the immune system may generate responses that lead to hypersensitivity or tissue injury and disease. In either case, the process is fascinating and commands the attention and respect of the reader for Nature’s incomparable versatility. Immune:  Natural or acquired resistance to a disease. Either a subclinical infection with the causative agent or deliberate immunization with antigenic substances prepared from it may render a host immune. Because of immunological memory, the immune state is heightened upon second exposure of individuals to an immunogen. A subject may become immune as a consequence of having experienced and recovered from an infectious disease.

CAM (cell adhesion molecules)  (Figure  2.1) are cellselective proteins that promote adhesion of cells to one another and are calcium independent. They are believed to help direct migration of cells during embryogenesis. The majority of lymphocytes and monocytes express this antigen, which is not found on other cells. The “humanized” antibodies specific for this epitope are termed Campath-1H. See CD52 in Chapter 11. Cell adhesion molecules (CAMs)  on the cell surface facilitate the binding of cells to each other in tissues as well as in cell-to-cell interaction. They also facilitate cell-to-matrix adhesion and extravasation. Most are grouped into protein families that include the integrins, selectins, mucin-like proteins, and the immunoglobulin superfamily. Immune cell motility:  Migration of immune cells is a principal host defense mechanism for the recruitment of leukocytes to inflammatory sites in the development of cellmediated immunity. The induction of migratory responses follows the interaction of signal molecules with plasma membrane receptors, initiating cytoskeletal reorganization and changes in cell shape. Motile responses may be random, chemokinetic, chemotactic, or haptotactic. Random migration of unstimulated motility in chemokinetic migration, i.e., stimulated random movement of cells without a stimulus gradient, are motile responses that are not consistently directional. By contrast, responses that are directional include those that are chemotactic and haptotactic. They take place when cells are subjected to a signal gradient, and the cells migrate toward an increasing concentration of the stimulus. The various motile responses may participate in the mobilization of immune cells to sites of inflammation. The immune system  includes the molecules, cells, tissues, and organs that are associated with adaptive immunity such as the host defense mechanisms, mainly against infectious agents. Immune system anatomy:  The lymphocyte is the cell responsible for immune response specificity. The human mature lymphoid system is comprised of 2 × 1012 lymphocytes together with various accessory cells that include epithelial cells, monocytes/macrophages, and other antigen-presenting cells. Accessory cells are a requisite for both maturation and effective functioning of lymphocytes. The thymus is the site of maturation of T cells and the bone marrow is the maturation site of B cells. These two tissues comprise the primary 77

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78

Mac-1

Fibrinogen

Monocyte shed

P-Selectin

Endothelial Cells TNF ??

LFA-1

IFN

CD31

IL-4

Mac-1

L-Selectin Ligand

VLA-4

Lymphocyte

VLA-4

CD31

CD44

ICAM-1

ICAM-3

E-Selectin

L-Selectin

CD31

CD44

Monocyte

CD31

CD31

shed

P-Selectin Ligand P-Selectin

LFA-1

s.L-Selectin

E.Selectin Ligand

s.P-Selectin

VCAM-1

CD31

VLA-4 CD44 P-Selectin Ligand L-Selectin

L-Selectin CD31

Fibronectin

ICAM-1 LFA-1

s.VCAM-1

s.ICAM-1 E-Selectin Ligand

s.E-Selectin Mac-1

Neutrophil

CD31 ICAM-1 LFA-1

Endothelial Cells

ICAM-1 LFA-1

VCAM-1

ICAM-2

VLA-4

Atlas of Immunology, Third Edition

Figure 2.1  Cellular adhesion molecules.

lymphoid organs. The secondary lymphoid organs consist of the cervical lymph nodes, ancillary lymph nodes, spleen, mesenteric lymph nodes, and inguinal lymph nodes. Mature lymphocytes migrate from the central lymphoid organs by way of the blood vessels to the secondary or peripheral tissues and organs, where they respond to antigen. Peripheral

K10141.indb 78

lymphoid tissues comprise the spleen, lymph nodes, and mucosa-associated lymphoid tissue (MALT) which is associated with the respiratory, genitourinary, and gastrointestinal tracts, making up 50% of the lymphoid cells of the body. The mucosa-associated lymphoid system consists of the adenoids, tonsils, and mucosa-associated lymphoid cells

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Molecules, Cells, and Tissues of the Immune Response

79

of the respiratory, genitourinary, gastrointestinal tracts, and Peyer’s patches in the gut.

In vivo  is a term used to describe investigations performed (“in life”) in an intact living organism.

Immunity  refers to a state of acquired or innate resistance or protection from a pathogenic microorganism or its products or from the effect of toxic substances such as snake or insect venom.

Immunocompetent  is an adjective that describes a mature functional lymphocyte that is able to recognize a specific antigen and mediate an immune response. It also may refer to the immune status of a human or other animal to indicate that the individual is capable of responding immunologically to an immunogenic challenge.

Cluster of differentiation (CD):  The designation of antigens on the surface of leukocytes by their reactions with clusters of monoclonal antibodies. The antigens are designated as clusters of differentiation (CDs). CD (cluster of differentiation):  See cluster of differentiation. Molecular weights for the CD designations in this book are given for reduced conditions. CD antigens  are a cluster of differentiation antigens identified by monoclonal antibodies. The CD designation refers to a cluster of monoclonal antibodies, all of which have identical cellular reaction patterns and identify the same molecular species. Anti-CD refers to antiidiotype and should not be employed to name CD monoclonal antibodies. The CD designation was subsequently used to describe the recognized molecule, but it had to be clarified by using the terms antigen or molecule. CD nomenclature is used by most investigators to designate leukocyte surface molecules. Provisional clusters are designated as CDw. CD antigen is a molecule of the cell membrane that is employed to differentiate human leukocyte subpopulations based upon their interaction with monoclonal antibody. The monoclonal antibodies that interact with the same membrane molecule are grouped into a common cluster of differentiation or CD. CD molecules  are cell surface molecules found on immune system cells that are designated “cluster of differentiation” or CD followed by a number such as CD33.

Selectins  are a group of cell adhesion molecules (CAMs) that are glycoproteins and play an important role in the relationship of circulating cells to the endothelium. The members of this surface molecule family have three separate structural motifs. They have a single N-terminal (extracellular) lectin motif preceding a single epidermal growth factor repeat and various short consensus repeat homology units. They are involved in lymphocyte migration. These carbohydrate-binding proteins facilitate adhesion of leukocytes to endothelial cells. There is a single-chain transmembrane glycoprotein in each of the selectin molecules with a similar modular structure that includes an extracellular calcium-dependent lectin domain. The three separate groups of selectins include L-selectin (CD62L), expressed on leukocytes; P-selectin (CD62P), expressed on platelets and activated endothelium; and E-selectin (CD62E), expressed on activated endothelium. Under shear forces their characteristic structural motif is comprised of an N-terminal lectin domain, a domain with homology to epidermal growth factor (EGF), and various complement regulatory protein repeat sequences. See also E-selectin, L-selectin, P-selectin, and CD62. Immunocyte  literally immune cell, is a term sometimes used by pathologists to describe plasma cells in stained tissue sections, e.g., in the papillary or reticular dermis in erythema multiforme. Mucins  are heavily glycosylated serine- and threonine-rich proteins that serve as ligands for selectins.

CMI  is the abbreviation for cell-mediated immunity. Immunoblast:  Lymphoblast. Immunochemistry  is that branch of immunology concerned with the properties of antigens, antibodies, complement, T cell receptors, MHC molecules, and all the molecules and receptors that participate in immune interactions in vivo and in vitro. Immunochemistry aims to identify active sites in immune responses and define the forces that govern antigen– antibody interaction. It is also concerned with the design of new molecules such as catalytic antibodies and other biological catalysts. Also called molecular immunology. In vitro  is a term used to describe investigations with living cells or cellular components performed outside the body of an intact organism (“in glass”) performed in tissue culture plates or test tubes.

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Immunocytochemistry  refers to the visual recognition of target molecules, tissues, and cells through the specific reaction of antibody with antigen by using antibodies labeled with indicator molecules. By tagging an antibody with a fluorochrome, color-producing enzyme, or metallic particle, the target molecules can be identified. MEL-14  is a selectin on the surface of lymphocytes significant in lymphocyte interaction with endothelial cells of peripheral lymph nodes. Selectins are important for adhesion despite shear forces associated with circulating blood. MEL-14 is lost from the surface of both granulocytes and T lymphocytes following their activation. MEL-14 combines with phosphorylated oligosaccharides. MEL-14 antibody  identifies a gp90 receptor that permits lymphocyte binding to peripheral lymph node high

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endothelial venules. Immature double-negative thymocytes comprise cells that vary from high to low in MEL-14 content. The gp90 MEL-14 epitope is a glycoprotein on murine lymph node lymphocyte surfaces. MEL-14 antibody prevents these lymphocytes from binding to postcapillary venules. The gp90 MEL-14 is apparently a lymphocyte homing receptor that directs these cells to lymph nodes in preference to lymphoid tissue associated with the gut. Immunologic (or immunological)  is an adjective referring to those aspects of a subject that fall under the purview of the scientific discipline of immunology. An immunological reaction  is an in vivo or in vitro response of lymphoid cells to an antigen they have never previously encountered or to an antigen for which they are already primed or sensitized. An immunological reaction may consist of antibody formation, cell-mediated immunity, or immunological tolerance. The humoral antibody and cell-mediated immune reactions may mediate either protective immunity or hypersensitivity, depending on various conditions. A microenvironment  is an organized, local interaction among cells that provides an interactive, dynamic, structural, or functional compartmentalization. The microenvironment may facilitate or regulate cell and molecular interactions through biologically active molecules. Microenvironments may exert their influence at the organ, tissue, cellular, or molecular levels. In the immune system, they include the thymic cortex and the thymic medulla, which are distinct; the microenvironment of lymphoid nodules; and a microenvironment of B cells in a lymphoid follicle, among others. Microfilaments  are cellular organelles that comprise a network of fibers of about 60 Å in diameter present beneath the membranes of round cells, occupying protrusions of the cells, or extending down microprojections such as microvilli. They are found as highly organized and prominent bundles of filaments, concentrated in regions of surface activity during motile processes or endocytosis. Microfilaments consist mainly of actin, a globular 42-kDa protein. In media of appropriate ionic strength, actin polymerizes in a double array to form microfilaments, which are critical for cell movement, phagocytosis, fusion of phagosome and lysosome, and other important functions of cells belonging to the immune and other systems. An immunologically activated cell  is the term for an immunologically competent cell following its interaction with antigen. This response may be expressed either as lymphocyte transformation, immunological memory, cell-mediated immunity, immunologic tolerance, or antibody synthesis. Bystander effects  are indirect, nonantigen-specific phenomena that result in polyclonal responses. In contrast to antigen-specific interactions, bystander effects are the result of cellular interactions that take place without antigen

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recognition or under conditions where antigen and receptors for antigen are not involved. Bystander effects are phenomena linked to the specific immune response in that they do not happen on their own but only in connection with a specific response. Cells not directly involved in the antigenspecific response are transstimulated or “carried along” in the response. Innocent bystander  refers to a cell that is fatally injured during an immune response specific for a different cell type. Bystander lysis  refers to tissue cell lysis that is nonspecific. The tissue cells are not the specific targets during an immune response but are killed as innocent bystanders because of their close proximity to the site where nonspecific factors are released near the actual target of the immune response. Bystander lysis may occur by the Fas/FasL pathway depending on the polarity and kinetics of FasL surface expression and downregulation after TCR engagement. This cytotoxicity pathway may give rise to bystander lysis of Fas+ target cells. An immunologically competent cell  is a lymphocyte, such as a B cell or T cell, that can recognize and respond to a specific antigen. An immunologist  is a person who makes a special study of immunology. Immunology  is that branch of biomedical science concerned with the response of the organism to immunogenic (antigenic) challenge, the recognition of self from nonself, and all the biological (in vivo), serological (in vitro), physical, and chemical aspects of immune phenomena. Immunophysiology  refers to the physiologic basis of immunologic processes. LCAM  is the abbreviation for leukocyte cell adhesion molecule. A neural cell adhesion molecule-L1 (NCAM-L1)  is a member of the Ig gene superfamily. Although originally identified in the nervous system, NCAM-L1 is also expressed in hematopoietic and epithelial cells. It may function in cell– cell and cell–matrix interactions. NCAM-L1 can support homophilic NCAM-L1–NCAM-L1 and integrin cell-binding. It can also bind with high affinity to the neural proteoglycan eurocan. NCAM-L1 promotes neurite outgrowth by functioning in neurite extension. Activated leukocyte cell adhesion molecule (ALCAM/ CD166)  is a member of the immunoglobulin (Ig) gene superfamily. It is expressed by activated leukocytes and lymphocyte antigen CD6. The extracellular region of ALCAM contains five Ig-like domains. The N-terminal Ig domain binds specifically to CD6. ALCAM-CD6 interactions have been implicated in T cell development and regulation of

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Attachment (Rolling)

CD11b CD11a

CD62L

Adhesion

CD15s GlyCAM CD54 CD62P CD26Z

L-B

CD31

Fibronectin

Diapedesis

Laminin

Collagen

Extracellular matrix

Figure 2.2  Adhesion molecules.

T cell function. ALCAM may also play a role in progression of human melanoma. Ligand  refers to a molecule or part of a molecule that binds or forms a complex with another molecule such as a cell surface receptor. A ligand is any molecule that a receptor recognizes. Cell surface receptors and ligands:  Activation of caspases via ligand binding to cell surface receptors involves the TNF family of receptors and ligands. These receptors contain an 80-amino acid death domain (DD) that through homophilic interactions recruits adaptor proteins to form a signaling complex on the cytosolic surface of the receptor. The signaling induced by the ligand binding to the receptor appears to involve trimerization. Based on x-ray crystallography, the trimeric ligand has three equal faces; a receptor monomer interacts at each of the three junctions formed by the three faces. Thus, each receptor polypeptide contacts two ligands. The bringing together of three receptors, thereby orienting the intracellular DDs, appears to be the critical feature for signaling by these receptors. The adaptor proteins recruited to the aligned receptor DDs recruit either caspases or other signaling proteins. The exact mechanism by which recruitment of caspases-8 to the DD-induced complex causes activation of caspases-8 is not clear. Promiscuous binding:  A docking site that accepts several different ligands with related affinity manifests promiscuous binding. Cross-linking:  A process resulting from the joining of multiple identical molecules by the union of multivalent ligands such as antibodies. Cross-linking may occur with both soluble and cell-surface structures. Intracellular signaling pathway:  The mechanism whereby ligand binding to its cell surface receptor ultimately activates

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new gene expression patterns in the cell nucleus. Interaction between ligand and receptor activates a sequence of interactions among proteins, including enzymes and adaptors, which lead to activation of transcription factors with access to the nucleus where they change transcription patterns of genes governing cellular proliferation, differentiation, and effector functions. Homing receptors  are molecules on a cell surface that direct traffic of that cell to a precise location in other tissues or organs. For example, lymphocytes bear surface receptors that facilitate their attachment to high endothelial cells of postcapillary venules in lymph nodes. Adhesion molecules present on lymphocyte surfaces enable lymphocytes to recirculate and home to specific tissues. Homing receptors bind to ligands termed addressins found on endothelial cells in affected vessels. L-selectin on naïve lymphocytes binds to GlyCAM-1 molecules on high endothelial venules. Adhesion molecules  (Figure 2.2) are extracellular matrix proteins that attract leukocytes from the circulation. For example, T and B lymphocytes possess lymph node homing receptors on their membranes that facilitate passage through high endothelial venules. Neutrophils migrate to areas of inflammation in response to endothelial leukocyte adhesion molecule-1 (ELAM-1) stimulated by TNF and IL-1 on the endothelium of vessels. B and T lymphocytes that pass through high endothelial venules have lymph node homing receptors. Adhesion molecules mediate cell adhesion to their surroundings and to neighboring cells. In the immune system, adhesion molecules are critical to most aspects of leukocyte function, including lymphocyte recirculation through lymphoid organs, leukocyte recruitment into inflammatory sites, antigen-specific recognition, and wound healing. The five principal structural families of adhesion molecules are (1) integrins, (2) immunoglobulin superfamily (IgSF) proteins, (3) selectins, (4) mucins, and (5) cadherins.

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Neuropilin  is a cell-surface protein that is a receptor for the collapsin/semaphorin family of neuronal guidance proteins. Adhesion molecule assays:  Cell adhesion molecules are cell-surface proteins involved in the binding of cells to each other, to endothelial cells, or to the extracellular matrix. Specific signals produced in response to wounding and infection control the expression and activation of the adhesion molecule. The interactions and responses initiated by the binding of these adhesion molecules to their receptors/ ligands play important roles in the mediation of the inflammatory and immune reaction. The immediate response to a vessel wall injury is the adhesion of platelets to the injury site and the growth, by further aggregation of platelets, of a mass which tends to obstruct (often incompletely) the lumen of the damaged vessel. This platelet mass is called a hemostatic plug. The exposed basement membranes at the sites of injury are the substrate for platelet adhesion, but deeper tissue components may have a similar effect. Far from being static, the hemostatic plug has a continuous tendency to break up with new masses reformed immediately at the original site. Integrins  are a family of cell membrane glycoproteins that are heterodimers comprised of α and β chain subunits. They serve as extracellular matrix glycoprotein receptors. They identify the RGD sequence of the β subunit, which consists of the arginine-glycine-aspartic acid tripeptide that occasionally also includes serine. The RGD sequence serves as a receptor recognition signal. Extracellular matrix glycoproteins, for which integrins serve as receptors, include fibronectin, C3, and lymphocyte function-associated antigen 1 (LFA-1), among other proteins. Differences in the β  chain serve as the basis for division of integrins into three categories. Each category has distinctive α chains. The β chain provides specificity. The same 95-kDa β chain is found in one category of integrins that includes LFA-1, p150,95, and complement receptor 3 (CR3). The same 130kDa β chain is shared among VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, and integrins found in chickens. A 110-kDa β chain is shared in common by another category that includes the vitronectin receptor and platelet glycoprotein IIb/IIIa. There are four repeats of 40 amino acid residues in the β chain extracellular domains. There are 45 amino acid residues in the β chain intracellular domains. The principal function of integrins is to link the cytoskeleton to extracellular ligands. They also participate in wound healing, cell migration, killing of target cells, and in phagocytosis. Leukocyte adhesion deficiency syndrome occurs when the β subunit of LFA-1 and Mac-1 is missing. VLA proteins facilitate binding of cells to collagen (VLA-1, VLA-2, and VLA-3), laminin (VLA-1, VLA-2, and VLA-6), and fibronectin (VLA-3, VLA-4, and VLA-5). The cell-to-cell contacts formed by integrins are critical for many aspects of the immune response such as antigen presentation, leukocyte-mediated cytotoxicity, and myeloid cell phagocytosis. Integrins comprise an essential part of an

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adhesion receptor cascade that guides leukocytes from the bloodstream across endothelium and into injured tissue in response to chemotactic signals. Substrate adhesion molecules (SAM)  are extracellular molecules that share a variety of sequence motifs with other adhesion molecules. Most prominent among these are segments similar to the type III repeats of fibronectin and immunoglobulin-like domains. In contrast to other morphoregulatory molecules, SAMs do not have to be made by the cells that bind them. SAMs can link and influence the behavior of one another. Examples include glycoproteins, collagens, and proteoglycans.  uring chemokine-induced lymphocyte polarization, the D cytoskeletal protein moesin is important for the redistribution of adhesion molecules to the cellular uropod. Homing-cell adhesion molecule (H-CAM)  is also known as CD44, gp90 hermes, GP85/Pgp-1, and ECMRIII. It is a lymphocyte transmembrane glycoprotein with a molecular weight of 85 to 95 kDa and is expressed in macrophages, granulocytes, fibroclasts, endothelial cells, and epithelial cells. H-CAM has been found to bind to extracellular matrix molecules such as collagen and hyaluronic acid. H-CAM is also an important signal transduction protein during lymphocyte adhesion as it has been demonstrated that phosphorylation by kinase C and acylation by acyl-transferases enhance H-CAM’s interaction with cytoskeletal proteins. ITIM/ITAM immunoreceptors:  ITAMs (immunoreceptor tyrosine-based activation motif; consensus sequence YxxI/Lx6–12 YxxlI/L) and ITIMs (immunoreceptor tyrosinebased inhibition motif; S/I/V/LxYxxI/V/L) are phosphorylation motifs found in a large number of receptors or adaptor proteins. Phosphorylated ITAMs serve as docking sites for tandem SH2 domains of Syk family kinases, whereas phosphorylated ITIMs recruit tyrosine phosphatases. Signaling through ITAM-bearing receptors usually results in cell activation, while engagement of ITIM-bearing receptors is usually inhibitory. Most of these receptors are involved in tumor development and regulation of the immue system, although also function in tissues such as bone and brain. Immunoreceptor tyrosine-based activation motif (ITAM):  Amino acid sequences in the intracellular portion of signaltransducing cell surface molecules that are sites of tyrosine phosphorylation and of association with tyrosine kinases and phosphotyrosine-binding proteins that participate in signal transduction. Examples include Igα, Igβ, CD3 chains, and several Ig Fc receptors. Following receptor–ligand binding and phosphorylation, docking sites are formed for other molecules that participate in maintaining cell-activating signal transduction mechanisms. ITAMs:  Abbreviation for immunoreceptor tyrosine-based activation motifs.

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gastrointestinal mucosa. Because of its anticoagulant properties, heparin is useful for treatment of thrombosis and phlebitis.

Antigen Presenting Cell ICAM-1

ICAM-3

LFA-3 (CD58)

ICAM-2

LFA-1

LFA-1

LFA-1

CD2

T cell

Figure 2.3  Adhesion receptors.

Immunoreceptor tyrosine-based inhibition motif (ITIM):  Motifs with effects that oppose those of immunoreceptor tyrosine-based activation motifs (ITAMs). These amino acids in the cytoplasmic tail of transmembrane molecules bind phosphate groups added by tyrosine kinases. This six-amino acid (isoleucine-X-tyrosine-X-X-leucine) motif is present in the cytoplasmic tails of immune system inhibitory receptors that include Fc RIIB on B lymphocytes and the killer inhibitory receptor (KIR) on the NK cells. Following receptor–ligand binding and phosphorylation on their tyrosine residue, a docking site is formed for protein tyrosine phosphatases that inhibit other signal transduction pathways, thereby negatively regulating cell activation. Adhesion receptors  (Figure 2.3) are proteins in cell membranes that facilitate the interaction of cells with matrix. They play a significant role in adherence and chemoattraction in cell migration. They are divided into three groups that include the immunoglobulin superfamily which contains the T cell receptor/CD3, CD4, CD8, MHC class I, MHC class II, sCD2/LFA-2, LFA-3/CD58, ICAM-1, ICAM-2, and VCAM-2. The second group of adhesion receptors is made up of the integrin family which contains LFA-1, Mac-1, p150,95, VLA-5, VLA-4/LPAM-1, LPAM-2, and LPAM-3. The third family of adhesion receptors consists of selectin molecules that include Mel-14/LAM-1, ELAM-1, and CD62. Heparin  is a glycosaminoglycan comprised of two types of disaccharide repeating units. One is comprised of d-glucosamine and d-glucuronic acid, whereas the other is comprised of d-glucosamine and l-iduronic acid. Heparin is extensively sulfated and is an anticoagulant. It unites with an antithrombin III which can unite with and block numerous coagulation factors. It is produced by mast cells and endothelial cells. It is found in the lungs, liver, skin, and

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Heparan sulfate  is a glycosaminoglycan that resembles heparin and is comprised of the same disaccharide repeating unit. Yet, it is a smaller molecule and less sulfated than heparin. An extracellular substance, heparan sulfate is present in the lungs, arterial walls, and on numerous cell surfaces. Reactive oxygen intermediates (ROIs)  are free radicals, including nitric oxide, derived from nitrogen, that destroy microorganisms within phagosomes. These are highly reactive compounds that include superoxide anion (O2), singlet oxygen, hydroxyl radicals (OH), and hydrogen peroxide (H2O2) that are produced in cells and tissues. Phagocytes use ROIs to form oxyhalides that injure ingested microorganisms. Release from cells may induce inflammatory responses leading to tissue injury. Also termed “reactive oxygen species” (ROS). 4-1BB  is a TNF receptor family molecule that binds specifically to 4-1BB ligand. 4-1BB ligand (4-1BBL)  is a TNF family molecule that binds to 4-1BB. Integrin family of leukocyte adhesive proteins:  The CD11/ CD18 family of molecules. Integrins, HGF/SF activation of:  Integrins and growth factor receptors can share common signaling pathways. Each type of receptor can impact the signal and ultimate response of the other. An example of a growth factor that has been shown to influence members of the integrin family of cell adhesion receptors is hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a multifunctional cytokine that promotes mitogenesis, migration, invasion, and morphogenesis. HGF/SF-dependent signaling can modulate integrin function by promoting aggregation and cell adhesion.  orphogenic responses to HGF/SF are dependent on adheM sive events. HGF/SF-induced effects occur via signaling of the MET tyrosine kinase receptor, following ligand binding. HGF/SF binding to MET leads to enhanced integrinmediated B cell and lymphoma cell adhesion. Blocking experiments with monoclonal antibodies directed against integrin subunits indicate that α4 β1 and α5 β1 integrins on hematopoietic progenitor cells are activated by HGF/SF to induce adhesion to fibronectin. The HGF/SF-dependent signal transduction pathway can also induce ligand-binding activity in functionally inactive αv β3 integrins. These effects elicited by HGF/SF highlight the importance of growth factor regulation of integrin function in both normal and tumor cells. LFA-1, LFA-2, LFA-3:  See leukocyte functional antigens.

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Atlas of Immunology, Third Edition Adhesion to Artificial Membranes LFA-1

LFA-1

T cell

LFA-1

CD4+ T cell (Helper T)

LFA-1 LFA-1

T cell receptor CD2

CD4

Antigen peptide

ICAM-1 Class II

LFA-3

Antigen-presenting cell

Figure 2.5  Lymphocyte function-associated antigen-3.

ICAM-1

Figure 2.4  Lymphocyte function-associated antigen-1.

Lymphocyte function-associated antigen-1 (LFA-1)  (Figure 2.4) is a leukocyte integrin that facilitates lymphocyte adhesion to endothelial cells and antigen-presenting cells. The glycoprotein is comprised of a 180-kDa α chain and a 95-kDa β chain expressed on lymphocyte and phagocytic cell membranes. LFA-1’s ligand is the intercellular adhesion molecule 1 (ICAM-1). It facilitates natural killer cell and cytotoxic T cell interaction with target cells. Complement receptor 3 and p150,95 share the same specificity of the 769-amino acid residue β chain found in LFA-1. A gene on chromosome 16 encodes the α chain whereas a gene on chromosome 21 encodes the β chain. This leukocyte integrin (β2) adhesion molecule has a critical role in adhesion of leukocytes to each other and to other cells as well as microbial recognition by phagocytes. FLA-1 binds not only ICAM-1 but also ICAM-2 or ICAM-3. LFA-1dependent cell adhesion is dependent on temperature, magnesium, and cytoskeleton. LFA-1 induces costimulatory signals that are believed to be significant in leukocyte function. LFA-1 function is critical to most aspects of the immune response. Also referred to as CD11a/CD18. See CD11a and CD18. Lymphocyte function-associated antigen-2 (LFA-2):  See CD2 (Figures 2.7 to 2.9). LFA-2  is a T cell antigen that is the receptor molecule for sheep red cells and is also referred to as the T11 antigen. The molecule has a 50-kDa mol wt. The antigen also seems to be involved in cell adherence, probably binding LFA-3 as its ligand. LFA-3  is a 60-kDa polypeptide chain expressed on the surfaces of B cells, T cells, monocytes, granulocytes, platelets, fibroblasts, and endothelial cells of vessels. LFA-3 is the

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ligand for CD2 and is encoded by genes on chromosome 1 in man. Lymphocyte function-associated antigen-3 (LFA-3)  (Figures  2.5 and 2.6) is an immunoglobulin superfamily cell adhesion molecule found on antigen-presenting cells, among others, that facilitates their adhesion to T cells. It is a 60-kDa polypeptide chain expressed on the surfaces of B cells, T cells, monocytes, granulocytes, platelets, fibroblasts, and endothelial cells of vessels. LFA-3 or CD58 is expressed either as a transmembrane or a lipid-linked cell surface protein. The transmembrane form consists of a 188amino acid extracellular region, a 23-amino acid transmembrane hydrophobic region, and a 12-amino acid intracellular hydrophilic region ending in the C-terminus. LFA-3 expression by antigen-presenting cells that include dendritic cells, macrophages, and B lymphocytes point to a possible role in regulating the immune response. Intercellular adhesion molecules (ICAMs)  are leukocyte integrin ligands that facilitate the binding of lymphocytes and other leukocytes to various cells, such as antigen-presenting cells and endothelial cells. Intercellular adhesion molecule-1 (ICAM-1)  (Figure 2.10) is a 90-kDa cellular membrane glycoprotein that occurs in multiple cell types including dendritic cells and endothelial cells. It is the lymphocyte function-associated antigen-1 (LFA-1) ligand. The LFA-1 molecules on cytotoxic T lymphocytes (CTL) interact with ICAM-1 molecules found on CTL target cells. Interferon γ, tumor necrosis factor, and IL-1 can elevate ICAM-1 expression. ICAM-1 is a member of the immunoglobulin gene superfamily of cell adhesion molecules. It plays a major role in the inflammatory response and in T cell-mediated host responses serving as a costimulatory molecule on antigen-presenting cells to activate MHC class II restricted T cells and on other types of cells in association with MHC class I to activate cytotoxic T cells. On endothelial cells, it facilitates migration of activated leukocytes to the site of injury. It is the cellular receptor for a subgroup of rhinoviruses.

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ICAM-1

D1

D2

D3

ICAM-2

D4

D5

D1

D2

Figure 2.8  CD2 space fill. Resolution 2.0 Å.

LFA-3

D1

D2

superfamily that is important in cellular interactions. It is a cell surface molecule that serves as a ligand for leukocyte integrins. ICAM-2 facilitates lymphocytes binding to antigen-presenting cells or to endothelial cells. It binds to LFA-1, a T lymphocyte integrin. ICAM-2:  See intercellular adhesion molecule.

LFA-3

CD2

D1

Intercellular adhesion molecule-3 (ICAM-3)  is a leukocyte cell surface molecule that plays a critical role in the interaction of T lymphocytes with antigen presenting cells. The interaction of the T lymphocyte with an antigen presenting cell through union of ICAM-1, ICAM-2, and ICAM-3 with LFA-1 molecules is also facilitated by the interaction of the T-cell surface molecule CD2 with LFA-3 present on antigen-presenting cells.

D2

D12

D2

ICAM-3:  See intercellular adhesion molecule-3.

Key: N-glycosylation site

CD2

Cysteine

Figure 2.6  Immunoglobulin superfamily adhesion receptors.

ICAM-1 (intercellular adhesion molecule-1)  is a γ interferon-induced protein which is needed for the migration of polymorphonuclear neutrophils into areas of inflammation. Intercellular adhesion molecule-2 (ICAM-2)  (Figure 2.11) is a protein that is a member of the immunoglobulin

Figure 2.7  CD2 ribbon diagram. Resolution 2.0 Å.

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Figure 2.9  CD2 ribbon structure.

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Atlas of Immunology, Third Edition Lymphocyte LFA-1

ICAM-1

VCAM ICAM-1

ICAM-2

ICAM-1

LFA-1

Lymphocyte

Endothelial cell

Figure 2.10  ICAM-1.

Figure 2.12  VCAM-1 bound to an endothelial cell.

Very late activation antigens (VLA molecules)  are β-1 integrins that all have the CD19 β chain in common. They were originally described on T lymphocytes grown in longterm culture but were subsequently found on additional types of leukocytes and on cells other than blood cells. VLA proteins facilitate leukocyte adherence to vascular endothelium and extracellular matrix. Resting T lymphocytes express VLA-4, VLA-5, and VLA-6. VLA-4 is expressed on multiple cells that include thymocytes, lymphocytes in blood, B and T cell lines, monocytes, NK cells, and eosinophils. The extracellular matrix ligand for VLA-4 and VLA-5 is fibronectin, and for VLA-6 it is laminin. The binding of these molecules to their ligands gives T lymphocytes costimulator signals. VLA-5 is present on monocytes, memory T lymphocytes, platelets, and fibroblasts. It facilitates B and T cell binding to fibronectin. VLA-6, which is found on platelets, T cells, thymocytes, and monocytes, mediates platelet adhesion to laminin. VLA-3, a laminin receptor, binds collagen and identifies fibronectin. It is present on B cells, the thyroid, and the renal glomerulus. Platelet VLA-2 binds to collagen only, whereas endothelial cell VLA-2 combines with collagen and laminin. Lymphocytes bind through VLA-4 to high endothelial venules and to endothelial cell surface proteins (VCAM-1) in areas of inflammation. VLA-1, which is present on activated T cells, monocytes, melanoma cells, and smooth muscle cells, binds collagen and laminin.

VLA receptors  refer to a family of integrin receptors found on cell surfaces. They consist of α and β transmembrane chain heterodimers. There is a VLA-binding site at the arginine-glycine-aspartamine sequences of vitronectin and fibronectin. VLA receptors occur principally on T lymphocytes. They also bind laminin and collagen. They participate in cell–extracellular matrix interactions. Vascular cell adhesion molecule-1 (VCAM-1)  (Figures 2.12 and 2.13) is a molecule that binds lymphocytes and monocytes. It is found on activated endothelial cells, dendritic cells, tissue macrophages, bone marrow fibroblasts, and myoblasts. VCAM-1 belongs to the immunoglobulin gene superfamily and is a ligand for VLA-4 (integrin α4/β1) and integrin α4/β7. It plays an important role in leukocyte recruitment to inflammatory sites and facilitates lymphocyte, eosinophil, and monocyte adhesion to activated endothelium. It participates in lymphocyte–dendritic cell interaction in the immune response. Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31)  is an antigen that is a single-chain membrane glycoprotein with a 140-kDa mol wt. It is found on granulocytes, monocytes, macrophages, B cells, platelets, and endothelial cells. Although it is termed gpIIa’, it is different from the CD29 antigen. At present the function of CD31 is unknown. It may be an adhesion molecule. PECAM (CD31):  An immunoglobulin-like molecule present on leukocytes and at endothelial cell junctions. These molecules participate in leukocyte–endothelial cell interactions, as during an inflammatory response. See also “platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31).”

Figure 2.11  ICAM-2. Ribbon diagram. Resolution 2.2 Å.

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Endothelial leukocyte adhesion molecule-1 (ELAM-1)  facilitates focal adhesion of leukocytes to blood vessel walls. It is induced by endotoxins and cytokines and belongs to the adhesion molecule family. ELAM-1 is considered to play a

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Molecules, Cells, and Tissues of the Immune Response

hypertension, and uremia. It may have a role in the development of congestive heart failure.

NH2 S S S

C2 CHO-N

S S S S S

S S S CHO-N

CHO-N

NH2

C2

C2

C2

S S

S S S S S

S

N-CHO

N-CHO CHO-N

S S

C2

C2

N-CHO

CHO-N C2

VCAM-1/CD106

C2

S S S S S

S N-CHO C2 N-CHO S

S

S S

CHO-N

CHO-N

NH2

S

S

N-CHO

19aa

S

C2

N-CHO C2

CHO-N CHO-N

S S

N-CHO

C2

C2

C2

N-CHO

C2

N-CHO

C2

N-CHO

S S S

S

S

S S C2

S

S

C2

S

28aa

ICAM-1/CD54

Gatekeeper effect  refers to contraction of endothelium mediated by IgE, permitting components of the blood to gain access to the extravascular space as a consequence of increased vascular permeability. Fibronectin  is an adhesion-promoting dimeric glycoprotein found abundantly in the connective tissue and basement membrane. The tetrapeptide Arg-Gly-Asp-Ser facilitates cell adhesion to fibrin, Clq, collagens, heparin, and types I-, II-, III-, V-, and VI-sulfated proteoglycans. Fibronectin is also present in plasma and on normal cell surfaces. Approximately 20 separate fibronectin chains are known. They are produced from the fibronectin gene by alternative splicing of the RNA transcript. Fibronectin is comprised of two 250-kDa subunits joined near their carboxy-terminal ends by disulfide bonds. The amino acid residues in the subunits vary in number from 2145 to 2445. Fibronectin is important in contact inhibition, cell movement in embryos, cell-substrate adhesion, inflammation, and wound healing. It may also serve as an opsonin.

117aa

PECAM-1/CD31

Figure 2.13  Schematic representation of VCAM-1.

significant role in the pathogenesis of atherosclerosis and infectious and autoimmune diseases. Neutrophil and monocyte adherence to endothelial cells occurs during inflammation in vivo where there is leukocyte margination and migration to areas of inflammation. Endothelial cells activated by IL-1 and TNF synthesize ELAM-1, at least in culture. A 115-kDa chain and a 100-kDa chain comprise the ELAM-1 molecule. ELAM-1 (endothelial leukocyte adhesion molecule-1)  is a glycoprotein of the endothelium that facilitates adhesion of neutrophils. Structurally, it has an epidermal growth factor-like domain, a lectin-like domain, amino acid sequence homology with complement-regulating proteins, and six tandem-repeated motifs. Tumor necrosis factor, interleukin-1, and substance P induce its synthesis. Its immunoregulatory activities include attraction of neutrophils to inflammatory sites and mediating cell adhesion by sialyl-Lewis X, a carbohydrate ligand. It acts as an adhesion molecule or addressin for T lymphocytes that home to the skin. Endothelin  is a peptide comprised of 21 amino acid residues that is derived from aortic endothelial cells and is a powerful vasoconstrictor. A gene on chromosome 6 encodes the molecule. It produces an extended pressor response, stimulates release of aldosterone, inhibits release of renin, and impairs renal excretion. It is elevated in myocardial infarction and cardiogenic shock, major abdominal surgery, pulmonary

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Fibrinogen  is one of the largest plasma proteins and has a mol wt of 330 to 340 kDa, comprising more than 3000 amino acid residues. The concentration in the plasma ranges between 200 and 500 mg/l00 ml. The molecule contains 3% carbohydrate, about 28 to 29 disulfide linkages, and one free sulfhydryl group. Fibrinogen exists as a dimer and can be split into two identical sets comprising three different polypeptide chains. Fibrinogen is susceptible to enzymatic cleavage by a variety of enzymes. The three polypeptide chains of fibrinogen are designated Aα, Bβ, and γ. By electron microscopy the dried fibrinogen molecule shows a linear arrangement of three nodules, 50 to 70 Å in diameter, connected by a strand about 15 Å thick. Fibrinopeptides  are released by the conversion of fibrinogen into fibrin. Thrombin splits fragments from the N-terminal region of Aα and Bβ chains of fibrinogen. The split fragments are called fibrinopeptide A and B, respectively, and are released in the fluid phase. They may be further degraded and may apparently have vasoactive functions. The release rate of fibrinopeptide A exceeds that of fibrinopeptide B and this differential release may play a role in the propensity of nascent fibrin to polymerize. Fibrin  is a protein responsible for the coagulation of blood. It is formed through the degradation of fibrinogen into fibrin monomers. Polymerization of the nascent fibrin molecules (comprising the α, β, and γ chains) occurs by end-to-end as well as lateral interactions. The fibrin polymer is envisaged as having two chains of the triad structure lying side by side in a staggered fashion in such a way that two terminal nodules are associated with the central nodule of a third molecule. The chains may also be twisted around each other. The fibrin

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Heparin binding region

Cell attachment region RGD NH2

Somatomedin Connector 1 B domain domain

2

3

1

2

3

4

Fibril

10K COOH

Hemopexin domains

= Cysteine residues

Triple helix

Figure 2.14  Vitronectin.

polymer thus formed is stabilized under the action of a fibrinstabilizing factor, another component of the coagulation system. Fibrinogen may also be degraded by plasmin. In this process, a number of intermediates, designated as fragments X, Y, D, and E, are formed. These fragments interfere with polymerization of fibrin by binding to nascent intact fibrin molecules, thus causing a defective and unstable polymerization. Fibrin itself is also cleaved by plasmin into similar but shorter fragments collectively designated fibrin degradation products. Of course, any excess of such fragments will impair the normal coagulation process—an event with serious clinical significance. Abzymes, such as thromboplastin activator linked to an antibody specific for antigens in fibrin that are not present in fibrinogen, are used clinically to lyse fibrin clots obstructing coronary arteries in myocardial infarction patients. Vitronectin  (Figure  2.14), a cell adhesion molecule that is a 65-kDa glycoprotein, is found in the serum at a concentration of 20 mg/l. It combines with coagulation and fibrinolytic proteins and with C5b67 complex to block its insertion into lipid membranes. Vitronectin appears in the basement membrane, together with fibronectin in proliferative vitreoretinopathy. It decreases nonselective lysis of autologous cells by insertion of soluble C5b67 complexes from other cell surfaces. Vitronectin is also called epibolin and protein S.

α-Chain

Gly

X

Y

Gly

X

Y

Gly

X

Y

Figure 2.15  Collagen.

Laminin  (Figure 2.17) is a relatively large (820-kDa) basement membrane glycoprotein comprised of three polypeptide subunits. It belongs to the integrin receptor family which includes a 400-kDa α heavy chain and two 200-kDa light chains designated β-1 and β-2. By electron microscopy the molecule is arranged in the form of a cross. The domain structures of the α and β chains resemble one another. There are six primary domains. Domains I and II have repeat sequences forming α helices. Domains III and V are comprised of cysteine-rich repeating sequences. The globular regions are comprised of domains IV and VI. There is an additional short cysteine-rich α domain between domains I and II in the β-1 chain. There is a relatively large globular segment linked to the C-terminal of domain I, designated the “foot” in the α chain. Five “toes” on the foot contain repeat sequences. Laminins have biological functions and characteristics that include facilitation of cellular adhesion and linkage to other basement membrane constituents such as collagen type IV, heparan, and glycosaminoglycans. Laminins also facilitate neurite regeneration, an activity associated with the foot of the molecule. There is more than

I n plasma, 65-kDa and 75-kDa glycoproteins that facilitate adherence of cells as well as the ability of cells to spread and to differentiate are known as serum spreading factors. Collagen  (Figure 2.15) is a 285-kDa extracellular matrix protein that contains proline, hydroxyproline, lysine, hydroxylysine, and glycine 30%. The structure consists of a triple helix of 95-kDa polypeptides forming a tropocollagen molecule that is resistant to proteases. Collagen types other than IV form fibrils with quarter stagger overlap between molecules that provide a fibrillar structure which resists tension. Several types of collagen have been described and most of them can be cross-linked through lysine side chain. Tenascin  (Figure  2.16) is a matrix protein produced by embryonic mesenchymal cells. It facilitates epithelial tissue differentiation and consists of six 210-kDa proteins that are all alike.

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Figure 2.16  Tenascin.

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1

NH3+

α Collagen IB binding site NH3+

β1 Chain (215kD)

–OOC

α Chain (400kD)

NH3+

β2 Chain (205kD)

COO–

Figure 2.18  Mac-1.

COO–

Heparan sulfate proteoglycan binding site

Figure 2.17  Laminin.

one form of laminin, each representing different gene products, even though they possess a high degree of homology. S-laminin describes a form found only in synaptic and nonmuscle basal lamina. This is a single 190-kDa polypeptide (in the reduced form) and is greater than 1000 kDa in the nonreduced form. It is associated with the development or stabilization of synapses. S-laminin is homologous to the β-1 chain of laminin. Laminin facilitates cell attachment and migration. It plays a role in differentiation and metastasis and is produced by macrophages, endothelial cells, epithelial cells, and Schwann cells. The laminin receptor  is a membrane protein comprised of two disulfide bond-linked subunits, one relatively large and one relatively small. Its function appears to be for attachment of cells and for the outgrowth of neurites. It may share structural similarities with fibronectin and vitronectin, both of which are also integrins. MAC-1  (Figure 2.18) is found on mononuclear phagocytes, neutrophils, NK cells, and mast cells. It is an integrin molecule comprised of an alpha chain (CD11b) linked noncovalently to a beta chain (CD18) that is the same as the beta chains of LFA-1 and of p150,95. MAC-1 facilitates phagocytosis of microbes that are coded with iC3b. It also facilitates neutrophil and monocyte adherence to the endothelium.

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β

CD11:  A “family” of three leukocyte-associated single chain molecules that has been identified in recent years (sometimes referred to as the LFA/Mac-1 family). They all consist of two polypeptide chains; the larger of these chains (α) is different for each member of the family; the smaller chain (β) is common to all three molecules (see CD11a, CD11b, CD11c). CD11a  α chain of the LFA-1 molecule with a 180-kDa mol wt. It is present on leukocytes, monocytes, macrophages, and granulocytes but negative on platelets. LFA-1 binds the intercellular adhesion molecules ICAM-1 (CD54), ICAM-2, and ICAM-3. A human T lymphocyte  encircled by a ring of sheep red blood cells is referred to as an E rosette. This was used previously as a method to enumerate T lymphocytes (Figure 2.19). GlyCAM-1  is a vascular addressin molecule resembling mucin that is present on high endothelial venules in lymphoid tissues. L-selectin molecules on lymphocytes in the peripheral blood bind GlyCAM-1 molecules, causing the lymphocytes to exit the blood circulation and circulate into the lymphoid tissues. LAM-1 (leukocyte adhesion molecule-1)  is a homing protein found on membranes, which combines with target cellspecific glycoconjugates. It helps to regulate migration of leukocytes through lymphocyte binding to high endothelial venules and to regulate neutrophil adherence to endothelium at inflammatory sites. CD44  is a transmembrane molecule with 80- to 90-kDa mol wt. It is found on some white and red cells and is weakly

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ELAM-1

Mel-14 (LAM-1)

CD2 T cell

Figure 2.19  E rosette.

Lectin-like domain EGF-like domain Short consensus repeat

expressed on platelets. It functions probably as a homing receptor. CD44 is a receptor on cells for hyaluronic acid; it binds to hyaluronate. It mediates leukocyte adhesion. CD44 is a ubiquitous multistructural and multifunctional cell surface glycoprotein that participates in adhesive cell-to-cell and cell-to-matrix interactions. It also plays a role in cell migration and cell homing. Its main ligand is hyaluronic acid (HA), hyaluronate, hyaluronan. CD44 is expressed by numerous cell types of lymphohematopoietic origin including erythrocytes, T and B lymphocytes, natural killer cells, macrophages, Kupffer cells, dendritic cells, and granulocytes. It is also expressed in other types of cells such as fibroblasts and CNS cells. Besides hyaluronic acid, CD44 also interacts with other ECM ligands such as collagen, fibronectin, and laminin. In addition to function stated above, CD44 facilitates lymph node homing via binding to high endothelial venules, presentation of chemokines or growth factors to migrating cells, and growth signal transmission. CD44 concentration may be observed in areas of intensive cell migration and proliferation as in wound healing, inflammation, and carcinogenesis. Many cancer cells and their metastases express high levels of CD44. It may be used as a diagnostic or prognostic marker for selected human malignant diseases. E-selectin (CD62E)  (Figure  2.20) is a molecule found on activated endothelial cells, which recognizes sialylated Lewis

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Figure 2.20  Selectins.

X and related glycans. Its expression is associated with acute cytokine-mediated inflammation. CD62E  is a 140-kDa antigen present on endothelium. CD62E is endothelium leuckocyte adhesion molecule (ELAM). It mediates neutrophil rolling on the endothelium. It also binds sialyl-Lewis X. P-selectin (CD62P)  (Figure 2.21) is a molecule found in the storage granules of platelets and the Weibel-Palade bodies of endothelial cells. Ligands are sialylated Lewis X and related glycans. P-selectins are involved in the binding of leukocytes to endothelium and platelets to monocytes in areas of inflammation. Weibel-Palade bodies  are P-selectin granules found in endothelial cells. P-selectin is translocated rapidly to the cell surface following activation of an endothelial cell by such mediators as histamine and C5a. CD34  is a vascular addressin present on lymph node high endothelial venules.

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cites. They govern the selective homing of leukocytes to specific locations in the body. The cellular adhesion molecules present on high endothelial venules (HEVs) facilitate extravasation of lymphocytes at particular anatomical sites in the body. HEVs of the various lymphoid organs and tissues express different addressins on their surfaces and signal only lymphocytes with appropriate complementary homing receptors.

Figure 2.21  P-selectin NMR.

CD34  is a molecule (105- to 120-kDa mol wt) that is a single chain transmembrane glycoprotein present on immature hematopoietic cells and endothelial cells as well as bone marrow stromal cells. Three classes of CD34 epitopes have been defined by differential sensitivity to enzymatic cleavage with neuraminidase and with glycoprotease from Pasteurella haemolytica. Its gene is on chromosome 1. CD34 is the ligand for L-selectin (CD62L). CD62P  is a 75- to 80-kDa antigen present on endothelial cells, platelets, and megakaryocytes. CD62P is an adhesion molecule that binds sialyl-Lewis X. It is a mediator of platelet interaction with monocytes and neutrophils. It also mediates neutrophil rolling on the endothelium. It is also referred to as P-selectin, GMP-140, PADGEM, or LECAM-3. L-selectin (CD62L)  is an adhesion molecule of the selectin family found on lymphocytes that is responsible for the homing of lymphocytes to lymph node high endothelial venules where it binds to CD34 and GlyCAM-1. This induces the migration of lymphocytes into tissues. L-selectin is also found on neutrophils where it acts to bind the cells to activated endothelium early in the inflammatory process.

LPAM-1  is a combination of α4 and β7 integrin chains that mediate the binding of lymphocytes to the high endothelial venules of Peyer’s patch in mice. The addressin for LPAM-1 is MadCAM-1. MadCAM-1  (Figure 2.22) facilitates access of lymphocytes to the mucosal lymphoid tissue, as in the gastrointestinal tract. Cadherins  are one of four specific families of cell adhesion molecules that enable cells to interact with their environment. Cadherins help cells to communicate with other cells in immune surveillance, extravasation, trafficking, tumor metastasis, wound healing, and tissue localization. Cadherins are calcium dependent. The five different cadherins include N-cadherin, P-cadherin, T-cadherin, V-cadherin, and E-cadherin. Cytoplasmic domains of cadherins may interact with proteins of the cytoskeleton. They may bind to other receptors based on homophilic specificity, but they still depend on intracellular interactions linked to the cytoskeleton. E-cadherin  and its associated cytoplasmic proteins α-, β-, and γ-catenin play an important role in epithelial cell– cell adhesion and in the maintenance of tissue architecture. Naive T Cell L-selectin

CD62L  a 150-kDa antigen present on B and T cells, monocytes, and NK cells, is a leukocyte adhesion molecule (LAM). It mediates cell rolling on the endothelium. It also binds CD34 and GlyCAM. CD62L is also referred to as L-selectin, LECAM-1, or LAM-1. Addressin  is a molecule such as a peptide or protein that serves as a homing device to direct a molecule to a specific location (an example is ELAM-1). Lymphocytes from Peyer’s patches home to mucosal endothelial cells bearing ligands for the lymphocyte homing receptor. Mucosal addressin cell adhesion molecule-1 (MadCAM) is the Peyer’s patch addressin in the intestinal wall that links to the integrin α4β7 on T lymphocytes that home to the intestine. Thus, endothelial cell addressins in separate anatomical locations bind to lymphocyte homing receptors leading to organ-specific lymphocyte homing. Vascular addressins  are mucin-like molecules on endothelial cells that bind selected leukocytes to particular anatomical

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MadCAM-1 Mucosal endothelium

Figure 2.22  MadCAM-1.

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PMN Leukocytes

Micropore filter

f. Met - Leu - Phe

Chemotactic factor

Chemotaxis  (Figure 2.23) is the process whereby chemical substances direct cell movement and orientation. The orientation and movement of cells in the direction of a chemical’s concentration gradient are positive chemotaxis, whereas movement away from the concentration gradient is termed negative chemotaxis. Substances that induce chemotaxis are referred to as chemotaxins and are often small molecules, such as C5a, formyl peptides, lymphokines, bacterial products, leukotriene B4, etc., that induce positive chemotaxis of polymorphonuclear neutrophils, eosinophils, and monocytes. These cells move into inflammatory agents by chemotaxis. A dual chamber device called a Boyden chamber is used to measure chemotaxis in which phagocytic cells in culture are separated from a chemotactic substance by a membrane. The number of cells on the filter separating the cell chamber from the chemotaxis chamber reflects the chemotactic influence of the chemical substance for the cells.

 irected migration of cells, known as chemotaxis, is mediD ated principally by the complement components C5a and C5a-des Arg. Neutrophil chemoattractants also include bacterial products such as N-formyl methionyl peptides, fibrinolysis products, oxidized lipids such as leukotriene B4, and stimulated leukocyte products. Interleukin 8 is chemotactic for polymorphonuclear neutrophils. Chemokines that are chemotactic for polymorphonuclear neutrophils include epithelial cell-derived neutrophil activating peptide (ENA-78), neutrophil activating peptide 2 (NAP-2), growth-related oncogene (GRO-α, GRO-β, and GRO-γ), and macrophage inflammatory protein-2α and β (MIP-2α and MIP-2β). Polypeptides with chemotactic activity mainly for mononuclear cells (β chemokine) include monocyte chemoattractant protein-1, 2, and 3 (MCP-1, MCP-2, and MCP-3), macrophage inflammatory protein-1 (MIP-1) α and β, and RANTES. These chemotactic factors are derived from both inflammatory and noninflammatory cells including neutrophils, macrophages, smooth muscle cells, fibroblasts, epithelial cells, and endothelial cells. MCP-1 participates in the recruitment of monocytes in various pathologic or physiologic conditions. Neutrophil chemotaxis assays are performed using the microchamber technique. Chemotactic assays are also useful to reveal the presence of chemotaxis inhibitors in serum.

Chemotactic factors  include substances of both endogenous and exogenous origin. Among them are bacterial extracts, products of tissue injury, chemical substances, various proteins, and secretory products of cells. The most important among them are those generated from complement and described as anaphylatoxins. This name is related to their concurrent ability of stimulating the release of mediators from mast cells. Some chemotactic factors act specifically in directing migration of certain cell types. Others have a broader spectrum of activity. Many of them have additional activities besides acting as chemotactic factors. Such effects of aggregation and adhesion of cells, discharge of lysosomal

Chemotactic receptors  are specific cellular receptors for chemotactic factors. In bacteria, such receptors are designated sensors and signalers and are associated with various transport mechanisms. The cellular receptors for chemotactic factors have not been isolated and characterized. In leukocytes, the chemo­tactic receptor appears to activate a serine proesterase enzyme, which sets in motion the sequence of events related to cell locomotion. The receptors appear specific for the chemotactic factors under consideration, and apparently the same receptors mediate all types of cellular responses inducible by a given chemotactic factor. However, these responses can be dissociated from each other,

Figure 2.23  Chemotaxis.

Downregulation or mutation of the E-cadherin/catenin genes can disrupt intercellular adhesion, which may lead to cellular transformation and tumor progression.

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enzymes, and phagocytosis by phagocytic cells may be concurrently stimulated. Participation in various immunologic phenomena such as cell triggering of cell–cell interactions is known for certain chemotactic factors. The structure of chemotactic factors and even the active region in their molecules have been determined in many instances. However, advances in the clarification of their mechanism of action have been facilitated by the use of synthetic oligopeptides with chemotactic activities. The specificity of such compounds depends both on the nature of the amino acid sequence and the position of amino acids in the peptide chain. Methionine at the NH2-terminal is essential for chemotactic activity. Formylation of Met leads to a 3,000- to 30,000-fold increase in activity. The second position from the NH2-terminal is also essential, and Leu, Phe, and Met in this position are essentially equivalent. Positively charged His and negatively charged Glu in this position are significantly less active, substantiating the role of a neutral amino acid in the second position at the N-terminal.

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suggesting that binding to the putative receptor initiates a series of parallel, interdependent, and coordinated biochemical events leading to one or another type of response. Using a synthetic peptide N-formyl-methionyl-leucyl-phenylalanine, about 2000 binding sites have been demonstrated per PMN leukocyte. The binding sites are specific, have a high affinity for the ligand, and are saturable. Competition for the binding sites is shown only by the parent or related compounds; the potency of the latter varies. Positional isomers may inhibit binding. Full occupancy of the receptors is not required for a maximal response, and occupancy of only 10 to 20% of them is sufficient. The presence of spare receptors may enhance the sensitivity in the presence of small concentrations of chemo­ tactic factors and may contribute to the detection of a gradient. There also remains the possibility that some substances with chemotactic activity do not require specific binding sites on cell membranes. Chemokinesis  refers to the determination of the rate of movement or random motion of cells by chemical substances in the environment. The direction of cellular migration is determined by chemotaxis, not chemokinesis.

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EMF-1 (embryo fibroblast protein-1)  is a chemokine of the α family (CXC family). It has been found in chicken fibroblasts and mononuclear cells, yet no human or murine homolog is known. Cultured chick embryo fibroblasts (CEFs) abundantly express the avian gene 9E3/CEF-4. The EMF-1 gene was isolated from RSV-transformed CEF identified by differential screening of a cDNA library. EMF-1 is characterized as a chemokine because its sequence resembles that of CTAP-III and PF4. RSV-infected cells represent the tissue source. Fibroblasts and mononuclear cells are the target cells. Expression of EMF-1 together with high collagen levels and in wounded tissues suggests that it has a role in the wound response and/or repair. EMF-1 is chemotactic for chicken peripheral blood mononuclear cells. ENA-78 (epithelial derived neutrophil attractant-78)  is a chemokine of the α family (CXC family) and is related to NAP-2, GRO-α, and IL-8. Tissue sources include epithelial cells and platelets. Neutrophils are the target cells. ENA-78 is increased in peripheral blood, synovial fluid, and synovial tissue from rheumatoid arthritis patients. ENA-78 mRNA levels are elevated in acutely rejecting human renal allografts compared with renal allografts that are not being rejected.

Leukotaxis  is chemotaxis of leukocytes. A Boyden chamber  (Figure  2.24) is a two-compartment structure used in the laboratory to assay chemotaxis. The two chambers in the apparatus are separated by a micropore filter. The cells to be tested are placed in the upper chamber and a chemotactic agent such as F-met-leu-phe is placed in the lower chamber. As cells in the upper chamber settle to the filter surface, they migrate through the pores if the agent below chemoattracts them. On staining of the filter, cell migration can be evaluated.

A chemotactic peptide  is a peptide that attracts cell migration such as formyl-methionyl-leucyl-phenylalanine. Chemotactic deactivation  represents the reduced chemo­ tactic responsiveness to a chemotactic agent caused by prior incubation of leukocytes with the same agent but in the absence of a concentration gradient. It can be tested by adding first the chemotactic factor to the upper chamber, washing, and then testing the response to the chemotactic factor placed in the lower chamber (no gradient being present). The mechanism of deactivation has been postulated as obstruction of the membrane channels involved in cation fluxes. Deactivation phenomena are used to discriminate between chemokinetic factors which enhance random migration and true chemotactic factors which cause directed migration. Only true chemotactic factors are able to induce deactivation. A chemoattractant  is a substance that attracts leukocytes and which may induce significant physiologic alterations in cells that express receptors for them.

Neutrophil suspension Micropore filter

Chemotactic solution

Figure 2.24  Boyden chamber.

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Formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe)  is a synthetic peptide that is a powerful chemotactic attractant for leukocytes, facilitating their migration. It may also induce neutrophil degranulation. This peptide resembles chemotactic factors released from bacteria. Following interaction with neutrophils, leukocyte migration is enhanced and complement receptor 3 molecules are increased in the cell membrane. f-Met peptides  are bacterial tripeptides such as formyl-MetLeu-Phe that are chemotactic for inflammatory cells, inducing leukocyte migration.

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ACT-2  is a human homolog of murine MIP-1b that chemoattracts monocytes but prefers activated CD4+ cells to CD8+ cells. T cells and monocytes are sources of ACT-2.

employed to transfer the A1AT gene to lung epithelial cells, after which A1AT mRNA and functioning A1AT become demonstrable.

Adhesins  are bacterial products that split proteins. They combine with human epithelial cell glycoprotein or glycolipid receptors, which could account for the increased incidence of pulmonary involvement attributable to Pseudomonas aeruginosa in patients who are intubated.

α helix:  A spiral or coiled structure present in many proteins and polypeptides. It is defined by intrachain hydrogen bonds between -CO and -NH groups that hold the polypeptide chain together in a manner that results in 3.6 amino acid residues per helical turn. There is a 1.5-Å rise for each residue. The helix has a pitch of 5.4 Å. The helical backbone is formed by a peptide group and the α carbon. Hydrogen bonds link each -CO group to the -NH group of the fourth residue forward in the chain. The α helix may be left- or right-handed. Righthanded α helices are the ones found in proteins.

Annexins (lipocortins)  are proteins with a highly conserved core region comprised of four or eight repeats of about 70 amino acid residues and a highly variable N-terminal region. The core region mediates Ca2+ -dependent binding to phospholipid membranes and forms a Ca2+ channel-like structure. Physical and structural features of annexin proteins suggest that they regulate many aspects of cell membrane function, including membrane trafficking, signal transduction, and cell–matrix interactions. Their actions resemble some of those of glucocorticoids, including antiinflammatory, antiedema, and immunosuppressive effects. Apolipoprotein (APO-E)  is a plasma protein involved in many functions including lipid transport, tissue repair, and the regulation of cellular growth and proliferation. There are three major isoforms of APO-E encoded by the epsilon 2, 3, or 4 alleles (APO-E2, APO-E3, APO-E4). APO-E3 is the most common variant. There is much interest in the APO-E4 variant as it may be implicated in Alzheimer’s disease. Other APO-E polymorphisms have been implicated in disorders of lipid metabolism and heart disease. β cells  are insulin secreting cells in the islet of Langerhans of the pancreas. β-pleated sheet  is a protein configuration in which the β  sheet polypeptide chains are extended and have a 35-nm axial distance. Hydrogen bonding between NH and CO groups of separate polypeptide chains stabilize the molecules. Adjacent molecules may be either parallel or antiparallel. The β-pleated sheet configuration is characteristic of amyloidosis and is revealed by Congo red staining followed by polarizing light microscopy, which yields an apple-green birefringence and ultrastructurally consists of nonbranching fibrils. β barrel:  See β sheet. α-1 antitrypsin (A1AT):  A glycoprotein in circulating blood that blocks trypsin, chymotrypsin, and elastase, among other enzymes. The gene on chromosome 14 encodes 25 separate allelic forms which differ according to electrophoretic mobility. The PiMM phenotype is physiologic. The PiZZ phenotype is the most frequent form of the deficiency which is associated with emphysema, cirrhosis, hepatic failure, and cholelithiasis, with an increased incidence of hepatocellular carcinoma. It is treated with prolastin. Adenoviruses may be

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Canonical structure:  A frequently encountered molecular sequence or molecular arrangement. Chaperones  are a group of proteins that includes BiP, a protein that binds the immunoglobulin heavy chain. Chaperones aid the proper folding of oligomeric protein complexes. They prevent incorrect conformations or enhance correct ones. Chaperones are believed to combine with the surfaces of proteins exposed during intermediate folding and to restrict further folding to the correct conformations. They take part in transmembrane targeting of selected proteins. Chaperones hold some proteins that are to be inserted into membranes in intermediate conformation in the cytoplasm until they interact with the target membrane. Besides BiP, they include heat shock proteins 70 and 90 and nucleoplasmins. The coagulation system  is a cascade of interaction among 12 proteins in blood serum that culminates in the generation of fibrin, which prevents bleeding from blood vessels whose integrity has been interrupted. The clotting system  is a mixture of cells, their fragments, zymogens, zymogen activation products and naturally occurring inhibitors, adhesive and structural proteins, phospholipids, lipids, cyclic and noncyclic nucleotides, hormones, and inorganic cations, all of which normally maintain blood flow. With disruption of the monocellular layer of endothelial cells lining a vessel wall, the subendothelial layer is exposed, bleeding occurs, and a cascade of events is initiated that leads to clot formation. These homeostatic reactions lead to formation of the primary platelet plug followed by a clot that mainly contains crosslinked fibrin (secondary hemostasis). After the blood vessel is repaired, the clot is dissolved by fibrinolysis. See also coagulation system. Hageman factor (HF)  is a zymogen in plasma that is activated by contact with a surface or by the kallikrein system at the beginning of the intrinsic pathway of blood coagulation. This is an 80-kDa plasma glycoprotein which, following activation, is split into an α and β chain. When activated, this substance is a serine protease that transforms prekallikrein into kallikrein. HF is coagulation factor XII.

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Endocrine:  An adjective describing regulatory molecules such as hormones that reach target cells from cells that are the site of their synthesis through the bloodstream.

Prekallikrein  is a kallikrein precursor. The generation of kallikrein from prekallikrein can activate the intrinsic mechanism of blood coagulation.

Ectopic:  The location of expression of a structure or protein away from its normal position in the body or tissue.

Protein kinase C (PKC)  is a serine/threonine kinase that Ca2+ activates in the cytoplasm of cells. It participates in T and B cell activation and is a receptor for phorbol ester that acts by signal transduction, leading to hormone secretion, enzyme secretion, neurotransmitter release, and mediation of inflammation. It is also involved in lipogenesis and gluconeogenesis. PKC participates also in differentiation of cells and in tumor promotion.

Hormones:  Messenger chemical molecules synthesized in the body by an organ, by cells of an organ, or by diffusely located cells, which have a precise regulatory action on the function of a certain organ or organs on cell types. Substances secreted by various endocrine glands and transported in the bloodstream to target organs on which their effects are produced. Also applied to various substances not produced by special glands but having a similar action. F-actin:  Actin molecules in a dual-stranded helical polymer. Together with the tropomyosin–tropinin regulatory complex, it constitutes the thin filaments of skeletal muscle. Hemophilia  is an inherited coagulation defect attributable to blood clotting factor VIII, factor IX, or factor XI deficiency. Hemophilia A patients are successfully maintained by the administration of exogenous factor VIII, which is now safe. Before mid-1985, factor products were a source of several cases of AIDS transmission when factor VIII was extracted from the blood of HIV-positive subjects by accident. Hemophilia B patients are treated with factor IX. Hemophilia A and B are cross linked, but hemophilia C is autosomal. Lectins  are glycoproteins that bind to specific sugars and oligosaccharides and link to glycoproteins or glycolipids on the cell surface. They can be extracted from plants or seeds, as well as from other sources. They are able to agglutinate cells such as erythrocytes through recognition of specific oligosaccharides and occasionally will react with a specific monosaccharide. Many lectins also function as mitogens and induce lymphocyte transformation, during which a small resting lymphocyte becomes a large blast cell that may undergo mitosis. Well-known mitogens used in experimental immunology include phytohemagglutinin, pokeweed mitogen, and concanavalin A. C-type lectin  is a type of lectin whose binding to carbohydrate ligands is calcium dependent. Lectin-like receptors  are macrophage and monocyte surface structures that bind sugar residues. The ability of these receptors to anchor polysaccharides and glycoproteins facilitates attachment during phagocytosis of microorganisms. Steroid hormones elevate the number of these cell-surface receptors. Ischemia  is deficient blood supply to a tissue as a consequence of vascular obstruction. Isoforms  are different versions of a protein encoded by alleles of a gene or by different but closely related genes.

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Protein S  is a 69-kDa plasma protein that is vitamin-K dependent and serves as a cofactor for activated protein C. It occurs as an active single chain protein or as a dimeric protein that is disulfide-linked and inactive. Protein S, in the presence of phospholipid, facilitates protein C inactivation of factor Va and combines with C4b-binding proteins. Protein S deficiency, which is transmitted as an autosomal dominant, is characterized clinically by deep vein thrombosis, pulmonary thrombosis, and thrombophlebitis. Laurell rocket electrophoresis is used to assay protein S. Phosphatase  is an enzyme that deletes phosphate groups from protein amino acid residue side chains. Lymphocyte protein phosphatases control signal transduction and transcription factor activity. Protein phosphatases may show specificity for either phosphotyrosine residues or phosphoserine and phosphothreonine residues. Small G proteins  are monomeric G proteins, including Ras, that function as intracellular signaling molecules downstream of many transmembrane signaling events. In their active form they bind GTP and hydrolyze it to GDP to become inactive. Stress proteins  are characterized into major families generally according to molecular weight. Within a family, heat shock proteins show a high degree of sequence homology throughout the phylogenetic spectrum and are among the most highly conserved proteins in nature. Heat shock protein 70 from mycobacteria and humans reveals 50% sequence homology. In spite of this homology, there are subtle differences in the functions, inducibility, and cellular location among related heat shock proteins for a given species. Even though major stress proteins accumulate to very high levels in stressed cells, they are present at low to moderate levels in unstressed cells pointing to the fact that they play a role in normal cells. In addition to increased synthesis, many heat shock proteins change their intracellular distribution in response to stress. An important characteristic of heat shock proteins is their capacity to function as molecular chaperones, which describes their capacity to bind to denatured proteins, preventing their aggregation, and this helps explain the function of heat shock proteins under normal conditions and in stress situations.

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Ubiquitin  is a 7-kDa protein found free in the blood or bound to cytoplasmic, nuclear, or membrane proteins united through isopeptide bonds to numerous lysine residues. Ubiquitin combines with a target protein and marks it for degradation. It is a 76-amino acid residue polypeptide found in all eukaryotes, but not in prokaryotes. Ubiquitin is found in chromosomes covalently linked to histones, although the function is unknown. It is present on the lymphocyte homing receptor gp90Mel-14. Ubiquitination  is the covalent linkage of several copies of ubiquitin, a small polypeptide, to a protein. Protein that has been ubiquitinated is marked for proteolytic degradation by proteasomes, which is involved in class 1 MHC antigen processing and presentation. Zymogen  refers to the inactive state in which an enzyme may be synthesized. Proteolytic cleavage of the zymogen may lead to active enzyme formation. Adaptor proteins  are critical linkers between receptors and downstream signaling pathways that serve as bridges or scaffolds for recruitment of other signaling molecules. They are functionally heterogeneous, yet share an SH domain that permits interaction with phosphotyrosine residues formed by receptor-associated tyrosine kinases. During lymphocyte activation, they may be phosphorylated on tyrosine residues, which enables them to combine with other homology-2 (SH2) domain-containing proteins. LAT, SLP-76, and Grb-2 are examples of adaptor molecules that participate in T cell activation. Neuropilin  is a cell-surface protein that is a receptor for the collapsin/semaphorin family of neuronal guidance proteins. Heat shock proteins (HSPs):  A restricted number of highly conserved cellular proteins that increase during metabolic stress such as exposure to heat, inflammation or tumor transformation. Inside cells, HSPs escort peptides from the proteasome to TAP in the course of endogenous antigen processing. Stressed cells may release HSPs as “danger signals” linking Toll-like receptors. They may facilitate exogenous antigen processing and cross-presentation. Heat shock proteins affect protein assembly into protein complexes, proper protein folding, protein uptake into cellular organelles, and protein sorting. The main group of hsps are 70-kDa proteins. Heat shock (stress) proteins are expressed by many pathogens and are classified into four families based on molecular size, i.e., hsp90, hsp70, hsp60, and small hsp (9 cycles

+ AB

AB

AB

Figure 4.7  Congenic strains.

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K2

K Class I l-E Aβ3

Aβ2

l-E Aβ

21OHB

Aα

Eβ

21OHA

C4

TNF

Eβ2

TNF

D

Eα

Class II

Slp

Bf

C2 Class III

D2

D3

D4

L

Q1

Q2

Q4

Q5

Q6

Q7

Q8/9 Q10

Class I

Figure 4.8  H-2 histocompatibility system is the major histocompatibility complex in the mouse.

progeny are inbred through brother–sister matings to yield a homozygous inbred strain. Mutation and genetic linkage may lead to random differences at a few other loci in the congenic strain. Designations for congenic strains consist of the symbol for the background strain followed by a period and then the symbol for the donor strain.

The H-2I region  is a murine H-2 MHC region where the genes encoding class II molecules are found.

H-2  (Figure 4.8) designates the major histocompatibility complex in the mouse. H-2 genes are located on chromosome 17. They encode somatic cell surface antigens as well as the host Ir genes. Each of these has a length of 600 bp. There are four regions in the H-2 complex designated K, I, S, and D. K region genes encode class I histocompatibility molecules designated K. I region genes encode class II histocompatibility molecules designated I-A and I-E. S region genes encode class III molecules designated C2, C4, factor B, and P-450 cytochrome (21-hydroxylase). D region genes encode class I histocompatibility molecules designated D and L. Antigens that represent the H-2 type of a particular inbred strain of mice are encoded by H-2 alleles. Thus, differences in the antigenic structure between inbred mice of differing H-2 alleles is of critical importance in the acceptance or rejection of tissue grafts exchanged between them. K, D, and L subregions of H-2 correspond to A, B, and C subregions of HLA in humans. The I-A and I-E regions are equivalent to the human HLA-D region.

H-2L  is the murine class I histocompatibility antigen found on spleen cells that serves as a target epitope in graft rejection.

H-2 complex:  The murine major histocompatibility complex. It is comprised of K, P, A, E, S, D, Q, T, and M loci that contain multiple genes. See H2 histocompatibility system. The H-2 locus  is the mouse major histocompatibility region on chromosome 17. H-2 restriction  is MHC restriction involving the murine H-2 MHC.

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H-2D and H-2K  are murine H-2 MHC loci whose products are class I antigens. Both H-2D and H-2K loci have multiple alleles.

HLA  is the abbreviation for human leukocyte antigen. The HLA histocompatibility system in humans represents a complex of MHC class I molecules distributed on essentially all nucleated cells of the body and MHC class II molecules that are distributed on B lymphocytes, macrophages, and a few other cell types. These are encoded by genes at the major histocompatibility complex. The HLA locus in humans is found on the short arm of chromosome 6. This has now been well defined, and in addition to encoding surface isoantigens, genes at the HLA locus also encode Ir genes. The class I region consists of HLA-A, HLA-B, and HLA-C loci and the class II region consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. Class II molecules play an important role in the induction of an immune response since antigen-presenting cells must complex an exogenous antigen with class II molecules to present it to CD4+ T lymphocytesin the presence of interleukin-1. Class I molecules are important in presentation of intracellular antigen to CD8+ T lymphocytes as well as in effector functions of target cells (Figure  4.9). Class III molecules encoded by genes located between those that encode class I and class II molecules include C2, BF, C4a, and C4b. Class I and class II molecules play an important role in the transplantation of organs and tissues. HLA-C molecules act as

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Major Histocompatibility Complex

Extracellular matrix

Cytosol

Golgi

ER lumen Calnexin β 2m

Class I heterodimer

Protein

Class I heterodimer

TAP1

TAP2 Peptides

Class I H chain

Proteasome

Figure 4.9  Class I MHC assembly.

inhibitors of the lytic capacity of natural killer (NK) cells and non-MHC-restricted T cells. The microlymphocytotoxicity assay is used for HLA-A, -B, -C, -DR, and -DQ typing but is gradually being replaced by molecular (DNA) typing. The primed lymphocyte test is used for DP typing. Uppercase letters designate individual HLA loci, as in HLA-B, and alleles are designated by numbers, as in HLA-B*0701.

sequences that distinguish among class II α genes or class II β genes. These allelic variation sites have been suggested to form epitopes which represent individual structural differences in immune recognition.

HLA-A  is a class I histocompatibility antigen in humans. It is expressed on nucleated cells of the body. Tissue typing to identify an individual’s HLA-A antigens employs lymphocytes.

HLA-D region  (Figure  4.11a, Figure  4.11b) refers to the human MHC class II region comprised of three subregions designated DR, DQ, and DP. Multiple genetic loci are present in each of these. DN (previously DZ) and DO subregions are each comprised of one genetic locus. Each class II HLA molecule is comprised of one α and one β chain that constitute a heterodimer. Genes within each subregion encode a particular class II molecule’s α and β chains. Class II genes that encode α chains are designated A, whereas class II genes that encode β chain are designated B. A number is used following A or B if a particular subregion contains two or more A or B genes.

The HLA locus  is the major histocompatibility locus in man. HLA allelic variation:  (Figure 4.10) Genomic analysis has identified specific individual allelic variants to explain HLA associations with rheumatoid arthritis, type I diabetes mellitus, multiple sclerosis, and celiac disease. There is a minimum of six α and eight β genes in distinct clusters, termed HLA-DR, -DQ, and -DP within the HLA class II genes. DO and DN class II genes are related but mapped outside the DR, DQ, and DP regions. There are two types of dimers along the HLA cell surface HLA-DR class II molecules. The dimers are made up of either DR α polypeptide associated with DRβ1 polypeptide or DR with DRβ2 polypeptide. Structural variation in class II gene products is linked to functional features of immune recognition leading to individual variations in histocompatibility, immune recognition, and susceptibility to disease. There are two types of structural variations which include variation among DP, DQ, and DR products in primary amino acid sequence by as much as 35% and individual variation attributable to different allelic forms of class II genes. The class II polypeptide chain possesses domains which are specific structural subunits containing variable

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HLA class III:  See MHC genes and class III MHC molecules.

Homozygous typing cells (HTCs)  are cells obtained from a subject who is homozygous at the HLA-D locus. HTCs facilitate MLR typing of the human D locus. HLA disease association:  Certain HLA alleles occur in a higher frequency in individuals with particular diseases than in the general population. This type of data permits estimation of the relative risk of developing a disease with every known HLA allele. For example, there is a strong association between ankylosing spondylitis, which is an autoimmune disorder involving the vertebral joints, and the class I MHC allele, HLA-B27. There is a strong asso-ciation between products of the polymorphic class II alleles HLA-DR and -DQ and certain autoimmune diseases since class II MHC

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Name HLA-A HLA-B HLA-C HLA-E HLA-F HLA-G HLA-H

Previous equivalents E, "6.2" F, "5.4" G, "6.0" H, AR,"12.4"

HLA-J

cda 12

HLA-K

HLA-70

HLA-L

HLA-92

HLA-DRA HLA-DRB1

DRα DRβI, DR1B

HLA-DRB2 HLA-DRB3

DRβII DRβIII, DR3B

HLA-DRB4 HLA-DRB5 HLA-DRB6

DRβIV, DR4B DRβIII DRBX, DRBσ

HLA-DRB7

DRBψ1

HLA-DRB8

DRBψ2

HLA-DRB9 HLA-DQA1 HLA-DQB1 HLA-DQA2

M4.2 β exon DQα1, DQ1A DQβ1, DQ1B DXα, DQ2A

HLA-DQB2

DXβ, DQ2B

HLA-DQB3

DVβ, DQB3

HLA-DOB HLA-DMA HLA-DMB HLA-DNA HLA-DPA1 HLA-DPB1 HLA-DPA2 HLA-DPB2

DOβ RING6 RING7 DZα, DOα DPα1, DP1A DPβ2, DP2B DPα2, DP2A DPβ2, DP2B

Molecular characteristics Class I α-chain Class I α-chain Class I α-chain associated with class I 6.2-kB Hind III fragment associated with class I 5.4-kB Hind III fragment associated with class 6.0 Hind III fragment Class 1 pseudogene associated with 5,4-kB Hind III fragment Class 1 pseudogene associated with 5.9-kB Hind III fragment Class 1 pseudogene associated with 7.0-kB Hind III fragment Class 1 pseudogene associated with 9.2-kB Hind III fragment DR α chain DR β1 chain determining specificities DR1, DR2, DR3, DR4, DR5, etc. pseudogene with DR β-like sequences DR β3 chain determining DR52 and Dw24, Dw25, Dw26 specificities DR β4 chain determining DR53 DR β5 chain determining DR51 DRB pseudogene found on DR1, DR2, and DR10 haplotypes DRB pseudogene found on DR4, DR7 and DR9 haplotypes DRB pseudogene found on DR4, DR7 and DR9 haplotypes DRB pseudogene, isolated fragment DQ α chain as expressed DQ β chain as expressed DQ α-chain-related sequence, not known to be expressed DQ β-chain-related sequence, not known to be expressed DQ β-chain-related sequence, not known to be expressed DO β chain DM α chain DM β chain DN α chain DP α chain as expressed DP β chain as expressed DP α-chain-related pseudogene DP β-chain-related pseudogene

TAP-1 TAP-2 LMP2 LMP7

RING4, Y3, PSF1 RING11, Y1, PSF2 RING12 RING10

ABC (ATP binding cassette) transporter ABC (ATP binding cassette) transporter Proteasome-related sequence Proteasome-related sequence

Figure 4.10  Names for genes in the HLA region.

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DPA2

DPA1

DPB2 DPB1

DNA

0

LMP

DMB DMA

LMP

TAP

500

DQA2 DQB2 TAP DOB DQB2 DQB3 DQA1

DRB

DRA

1000

Proteasome genes

(a) Leader sequence W

X1

X2

Y

Transmembrane and cytoplasmic

TATA

α5'

3' Regulatory sequences

α1

α2 3' Untranslated sequence

(b) Figure 4.11a and b  MHC class II.

molecules are of great importance in the selection and activation of CD4+ T lymphocytes which regulate the immune responses against protein antigens. For example, 95% of Caucasians with insulin-dependent (type I) diabetes mellitus have HLA-DR3 or HLA-DR4, or both. There is also a strong association of HLA-DR4 with rheumatoid arthritis. Numerous other examples exist and are the targets of current investigations, especially in extended studies employing DNA probes. Calculation of the relative risk (RR) and absolute risk (AR) can be found in this atlas under the definitions of those terms. The HLA-DP subregion  is a site of two sets of genes designated HLA-DPA1 and HLA-DPB1 and the pseudogenes HLA-DPA2 and HLA-DPB2. DPα and DPβ chains encoded by the corresponding genes DPA1 and DPB1 unite to produce the DPαβ molecule. DP antigen or type is determined principally by the very polymorphic DPβ chain in contrast to the much less polymorphic DPα chain. DP molecules carry DPw1 to DPw6 antigens. The HLA-DQ subregion  contains two sets of genes, designated DQA1 and DQB1, and DQA2 and DQB2. DQA2 and DQB2 are pseudogenes. DQα and DQβ chains, encoded by DQA1 and DQB1 genes, unite to produce the DQαβ molecule. Although both DQα and DQβ chains are polymorphic, the DQβ chain is the principal factor in determining the DQ antigen or type. DQαβ molecules carry DQw1 to DQw9 specificities. The HLA-DR subregion  is the site of one HLA-DRA gene. Although DRB gene number varies with DR type, there are usually three DRB genes, termed DRB1, DRB2, and DRB3 (or DRB4). The DRB2 pseudogene is not expressed. The DRα chain, encoded by the DRA gene, can unite with products of DRB1 and DRB3 (or DRB4) genes, which are the DRβ-1 and DRβ-3 (or DRβ-4) chains. This yields two separate DR

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molecules, DRαβ-1 and DRαβ-3 (or DRαβ-4). The DRβ chain determines the DR antigen (DR type) since it is very polymorphic, whereas the DRα chain is not. DRαβ-1 molecules carry DR specificities DR1 to DRw18. Yet, DRαβ-3 molecules carry the DRw52, and the DRαβ-4 molecules carry the DRw53 specificity. HLA-DR antigenic specificities  reflect the epitopes on DR gene products. Selected specificities have been mapped to define loci. HLA serologic typing requires the identification of a prescribed antigenic determinant on a particular HLA molecular product. One typing specificity can be present on many different molecules. Different alleles at the same locus may encode these various HLA molecules. Monoclonal antibodies are now used to recognize certain antigenic determinants shared by various molecules bearing the same HLA typing specificity. Monoclonal antibodies have been employed to recognize specific class II alleles with disease associations. HLA-DM  (Figure 4.12 to Figure 4.14) is an invariant MHC class II molecule of humans that facilitates the loading of antigenic peptides onto MHC class II molecules. As a result of proteolysis of the invariant chain a small fragment called the class II-associated invariant chain peptide, or CLIP, remains bound to the MHC class II molecule. CLIP peptide is replaced by antigenic peptides, but in the absence of HLA-DM this does not occur. The HLA-DM molecule must therefore play some part in removal of the CLIP peptide and in the loading of antigenic peptides. HLA-DO  is a negative modulator of HLA-DM-mediated MHC class II peptide loading. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. Therefore, HLA-DO affects the peptide repertoire presented to the immune system by MHC class II molecules.

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MHC class II HLA-DR NN S

α1

S

β1

α2

Clip

β2 S S

S S

Papain cleavage site

Papain cleavage site

HLA-DM

Figure 4.14  HLA-DM. C

C

Figure 4.12  Class II MHC molecules are glycoprotein histocompatibility antigens that play a critical role in immune system cellular interactions. Each class II MHC molecule is comprised of a 32- to 34-kDa α chain and a 29- to 32-kDa β chain, each of which possesses N-linked oligosaccharide groups, amino termini that are extracellular, and carboxyl termini that are intracellular. Approximately 70% of both α and β chains are extracellular.

Peptide-binding cleft

β1

α1

N

β2

C

Figure 4.13  MHC class II molecular structure.

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α2

HLA nonclassical class I genes  include genes located within the MHC class I region that encode products that can be associated with β2 microglobulin. However, their function and tissue distribution are different from those of HLA-A, -B, and -C molecules. Examples include HLA-E, -F, and -G. Of these, only HLA-G is expressed on the cell surface. It is uncertain whether or not these HLA molecules are involved in peptide binding and presentation like classical class I molecules. HLA-G  is either is nonpolymorphic or has very limited polymorphism. It is a nonclassical (Class Ib) major histocompatibility complex (MHC) molecule expressed in immuneprivileged tissues. It is a Class I HLA antigen with extensive variability in the α-2 domain. It is found on trophoblasts, i.e., placenta cells and trophoblastic neoplasms. HLA-G is expressed only on cells such as placental extravillous cytotrophoblasts and choriocarcinoma that fail to express HLA-A, -B, and -C antigens. HLA-G expression is most pronounced during the first trimester of pregnancy. Fetal trophoblasts do not express the Class Ia MHC molecule’s HLA-A and HLA-B, allowing the fetus to escape the maternal T cell response. Evidence suggests that HLA-G protects the fetus from attack by natural killer (NK) cells, macrophages, and monocytes by interacting with the inhibitory receptors on leukocyte immunoglobulin-like receptor-1 (LIR-1 or ILT-2), leukocyte immunoglobulin-like receptor 2 (LIR02 or ILT-4), and killer immunoglobulin-like receptor 2DL4 (KIR2DLA). Trophoblast cells expressing HLA-G at the maternal–fetal junction may protect the semiallogeneic fetus from “rejection.” Prominent HLA-G expression suggests maternal immune tolerance. HLA-G exerts tolerogenic functions involved in transplant acceptance as well as in tumoral and viral immune escape.

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HLA-E  is a human leukocyte antigen (HLA) class I nonclassical molecule. It is a relatively invariant human MHC class I molecule that serves as a ligand for NK cell receptors. HLA-F  is a human leukocyte antigen (HLA) class I nonclassical molecule. It is believed to be a peptide-binding molecule that may reach the cell surface where it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors, thereby altering the activation threshold of immune effector cells. It is a predominantly empty, intracellular, TAPassociated MHC class Ib protein with a restricted expression pattern. The C2 and B genes  are situated within the MHC locus on the short arm of chromosome 6. They are termed MHC class III genes. TNF-α and TNF-β genes are situated between the C2 and HLA-B genes. Another gene, designated FD, lies between the Bf and C4a genes. C2 and B complete primary structures have been deduced from cDNA and protein sequences. C2 is comprised of 732 residues and is an 81-kDa molecule, whereas B contains 739 residues and is an 83-kDa molecule. Both proteins have a three-domain globular structure. During C3 convertase formation, the amino terminal domains C2b or Ba are split off. They contain consensus repeats that are present in CR1, CR2, H, DAF, and C4bp, which all combine with C3 and/or C4 fragments and regulate C3 convertases. The amino acid sequences of the C2 and B consensus repeats are known. C2b contains sites significant for C2 binding to C4b. Ba, resembling C2b, manifests binding sites significant in C3 convertase assembly. Available evidence indicates that C2b possesses a C4b-binding site and that Ba contains a corresponding C3b-binding site. In considering assembly and decay of C3 convertases, initial binding of the three-domain structures C2 or B to activatorbound C4b or C3b, respectively, requires one affinity site on the C2b/Ba domain and another on one of the remaining two domains. A transient change in C2a and Bb conformation results from C2 or B cleavage by C1s or D. This leads to

I.

greater binding affinity and Mg+2 sequestration, and acquisition of proteolytic activity for C3, C2a, or Bb dissociation leads to C3 convertase decay. Numerous serum-soluble and membrane-associated regulatory proteins control the rate of formation and association of C3 convertases. Genomic analysis has identified specific individual allelic variants  to explain HLA associations with rheumatoid arthritis, type I diabetes, multiple sclerosis, and celiac disease. There are a minimum of six α and eight β genes in distinct clusters, termed HLA-DR, -DQ, and -DP within the HLA class II genes. DO and DN class II genes are related but mapped outside the DR, DQ, and DP regions. There are two types of dimers along the HLA cell-surface HLA-DR class II molecules. The dimers are made up of either DRαpolypeptide associated with DRβ1-polypeptide or DR with DRβ2-polypeptide. Structural variation in class II gene products is linked to functional features of immune recognition leading to individual variations in histocompatibility, immune recognition, and susceptibility to disease. There are two types of structural variations which include variations among DP, DQ, and DR products in primary amino acid sequence by as much as 35% and individual variation attributable to different allelic forms of class II genes. The class II polypeptide chain possesses domains which are specific structural subunits containing variable sequences that distinguish among class IIα genes or class IIβ genes. These allelic variation sites have been suggested to form epitopes that represent individual structural differences in immune recognition. MHC restriction  (Figure 4.15) is the recognition of antigen in the context of either class I or class II molecules by the T cell receptor for antigen. In the afferent limb of the immune response, when antigen is being presented at the surface of a macrophage, dendritic cell, or other antigen-presenting cell to CD4+ T lymphocytes, this presentation must be in the context of MHC class II molecules for the CD4+ lymphocyte to recognize the antigen and proliferate in response to it. By contrast,

II. HLA-A1

HLA-A1

CD8 HLA-A1

TcR

III.

HLA-A1

HLA-A2

HLA-A2

HLA-A2

Virus

Lysis

HLA-A2

TcR

No lysis

Lysis

Figure 4.15  MHC restriction.

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α S V S

β

S S V

S V S

S C2 S

V

C2

S S

SS SS

COOH COOH

COOH

Figure 4.16  Structure of CD4.

Figure 4.17  Structure of CD8.

cytotoxic (CD8+) T lymphocytes recognize foreign antigen such as viral antigens on infected target cells only in the context of class I MHC molecules. Once this recognition system is in place, the cytotoxic T cell can fatally injure the target cell through release of perforin and granzyme molecules that penetrate the target cell surface. T cells recognize a firm peptide antigen only in the context of a specific allelic form of an MHC molecule to which it is bound.

interactions. It is physically associated with the intracellular tyrosine protein kinase, known as p561ck, which phosphorylates nearby proteins. This antigen is thereby relaying a signal to the cells. Cross-linking of CD4 may induce activation of this enzyme and phosphorylation of CD3.

Major histocompatibility complex restriction:  See MHC restriction. CD4  (Figure  4.16) is a single-chain glycoprotein, also referred to as the T4 antigen, that has a 56-kDa mol wt and is present on approximately two-thirds of circulating human T cells, including most T cells of helper/inducer type. The antigen is also found on human monocytes and macrophages. The molecule is a receptor for gp120 of HIV-1 and HIV-2 (AIDS viruses). This antigen binds to class II MHC molecules on APC and may stabilize antigen-presenting cell and T-cell

The CD4 molecule  exists as a monomer and contains four immunoglobulin-like domains. The first domains of CD4 form a rigid rod-like structure that is linked to the two carboxyl-terminal domains by a flexible link. The binding site for MHC class II molecules is thought to involve the D1 and D2 domains of CD4. CD8  (Figures  4.17 and 4.18) is an antigen, also referred to as the T8 antigen, that has a 32- to 34-kDa mol wt. The CD8 antigen consists of two polypeptide chains α and β which may exist in the combination α/α homodimer or α/β heterodimers. Most antibodies are against the α-chain. This antigen binds to class I MHC molecules on APC and may stabilize APC/class I cell interactions.

Figure 4.18  The outline structure of the CD4 and CD8 coreceptor molecules.

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Major Histocompatibility Complex

D1 D2

CD4

α

CD8

β

× A

B

C

D

D3 D4

Figure 4.19  This structure consists of the N-terminal 114 residues of CD8. These residues make up a single immunoglobulin axis which coincides with a crystallographic twofold axis.

AC (25%)

AD (25%)

BC (25%)

BD (25%)

Figure 4.20  Each set of alleles is referred to as a haplotype.

The CD8 molecule  is a heterodimer of an α- and β-chain that are covalently associated by a disulfide bond. The two chains of the dimer have similar structures, each having a single domain resembling an immunoglobulin variable domain and a stretch of peptide believed to be in a relatively extended conformation (Figure 4.19). Each set of alleles is referred to as a haplotype  (Figure 4.20). These are the phenotypic characteristics encoded by closely linked genes on one chromosome inherited from one parent. An individual inherits one haplotype from the mother and one haplotype from the father. It frequently describes several major histocompatibility complex (MHC) alleles on a single chromosome. Selected haplotypes are in strong linkage disequilibrium between alleles of different loci. According to Mendelian genetics, 25% of siblings will share both haplotypes. In an outbred population, the offspring are generally heterozygous at many loci and will express both maternal and paternal MHC alleles. The alleles are therefore codominantly expressed, that is, both maternal and paternal gene products are expressed in the same cells. In inbred mice,

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however, each H-2 locus is homozygous because the maternal and paternal haplotypes are identical and all offspring express identical haplotypes. An ancestral haplotype  is an MHC haplotype that numerous families share, indicating that they probably have common ancestors. It is also called “HLA supratype” or “common extended haplotype.” Balancing selection:  A form of evolutionary selection that maintains various phenotypes in a population, such as MHC isoforms. Peptide-binding motif:  The assemblage of anchor residues of an MHC isoform that are common to the peptide aminoacid sequences that interact with the isoform. Directional selection:  A form of natural selection in which older alleles are replaced with newer variants, as in the MHC, leading to change.

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5

Antigen Processing and Presentation

T lymphocytes recognize antigens only in the context of selfMHC molecules on the surface of accessory cells. During processing, intact protein antigens are degraded into peptide fragments. Most epitopes that T cells recognize are peptide chain fragments. B cells and T cells often recognize different epitopes of an antigen leading to both antibody and cell-mediated immune responses. Before antigen can bind to MHC molecules, it must be processed into peptides in the intracellular organelles. CD4+ helper T lymphocytes recognize antigens in the context of class II MHC molecules, a process known as class II MHC restriction. By contrast, CD8+ cytotoxic T lymphocytes recognize antigens in the context of class I molecules, which is known as class I MHC restriction. Following the generation of peptides by proteolytic degradation in antigen-presenting cells, peptide–MHC complexes are presented on the surface of antigen-presenting cells where they may be recognized by T lymphocytes. Antigens derived from either intracellular or extracellular proteins may be processed to produce peptides from either self or foreign proteins that are presented by surface MHC molecules to T cells. In the class II MHC processing pathway, professional antigen-presenting cells such as macrophages, dendritic cells, or B lymphocytes incorporate extracellular proteins into endosomes where they are processed (Figures  5.1 and 5.2). Enzymes within the vesicles of the endosomal pathway cleave proteins in the acidic environment. Class II MHC heterodimeric molecules, united with invariant chain, are shifted to endosomal vesicles from the endoplasmic reticulum. Following cleavage of the invariant chain, DM molecules remove a tiny piece of invariant chain from the MHC molecules’ peptide-binding groove. Following complexing of extracellular-derived peptide with the class II MHC molecule, the MHC–peptide complex is transported to the cell surface where presentation to CD4+ T cells occurs. Proteins in the cytosol, such as those derived from viruses, may be processed through the class I MHC route of antigen presentation. The multiprotein complex in the cytoplasm, known as the proteasome effects, involve proteolytic degradation of proteins in the cytoplasm to yield many of the peptides that are presented by class I MHC molecules. TAP molecules transport peptides from the cytoplasm to the endoplasmic reticulum where they interact and bind to class I MHC dimeric molecules. Once the class I MHC molecules have become stabilized through peptide binding, the complex leaves the endoplasmic reticulum entering the Golgi apparatus en route to the surface of the cell. Thus, mechanisms are provided through MHC-

restricted antigen presentation to guarantee that peptides derived from extracellular microbial proteins can be presented by class II MHC molecules to CD4+ helper T cells and that peptides derived from intracellular microbes can be presented by class I MHC molecules to CD8+ cytotoxic T lymphocytes. The generation of microbial peptides produced through antigen processing to combine with self MHC molecules is critical to the development of an appropriate immune response. Antigen presentation  (Figures 5.3 and 5.4) is the display of peptide antigens on the cell surface together with either MHC class I or class II molecules, which permits T cells to recognize antigen on a target cell or antigen-presenting cell surface. T lymphocytes recognize antigens only in the context of self-MHC molecules on the surface of antigenpresenting cells. During processing, intact protein antigens are degraded into peptide fragments. Most epitopes that T cells recognize are peptide chain fragments. B cells and T cells often recognize different epitopes of an antigen leading to both antibody and cell-mediated immune responses. Before antigen can bind to MHC molecules, it must be processed into peptides in the intracellular organelles. CD4+ helper T lymphocytes recognize antigens in the context of class II MHC molecules, a process known as class II MHC restriction. By contrast, CD8+ cytotoxic T lymphocytes recognize antigens in the context of class I molecules, which is known as class I MHC restriction. Following the generation of peptides by proteolytic degradation in antigen-presenting cells, peptide-MHC complexes are presented on the surface of antigen-presenting cells where they may be recognized by T lymphocytes. Antigens derived from either intracellular or extracellular proteins may be processed to produce peptides from either self or foreign proteins that are presented by surface MHC molecules to T cells. In the class II MHC processing pathway, professional antigen-presenting cells, such as macrophages, dendritic cells, or B lymphocytes, incorporate extracellular proteins into endosomes where they are processed. Enzymes within the vesicles of the endosomal pathway cleave proteins in the acidic environment. Present:  Action taken by antigen-presenting cells. Refer to antigen presentation. Antigen processing  is the degradation of proteins into peptides capable of binding to MHC molecules for presentation in the peptide binding groove of either MHC class I or MHC 201

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is requisite for processing and presentation of exogenous antigen.

TCR THcell Endosome MHCII Lysosome Immunogen

CD4

Antigen presenting cell

Figure 5.1  Capture, processing, and presentation of antigen by an antigen-presenting cell.

class II molecules for presentation to T lymphocytes. For presentation by MHC molecules, antigens must be processed into peptides. Exogenous antigen processing and presentation  refers to epitopes that originate outside the animal body, are taken up by antigen-presenting cells, degraded via the endocytic pathway, and bound to MHC class II molecules in an endolysosomal vesicle. This is followed by exhibition of the peptide–MHC class II complex at the cell surface. This pathway is confined almost exclusively to antigen-presenting cells.

Macropinocytosis:  Antigen uptake through engulfment by a cell of extracellular fluid droplets containing soluble macromolecules and creating macropinosomes that join the endocytic pathway. Referred to also as “cell drinking.” Macropinosome:  A structure produced by invagination of a plasma cell membrane to produce a vesicle containing an extracellular fluid droplet. Refer to macropinocytosis. The DRiP pathway  is the defective ribosomal products pathway. It is a form of antigen processing whereby misfolded polypeptides are ubiquinated rapidly and digested by standard proteasomes, rendering the resulting DRiP peptides capable of association with MHC class I molecules. An antigenic peptide  is a peptide that is able to induce an immune response and one that complexes with major histocompatibility complex (MHC), thereby permitting its recognition by a T cell receptor.

Endogenous antigen processing and presentation  is a mechanism whereby cytosolic endogenous antigens are degraded into peptides by proteasomes and bound to MHC class I molecules in the rough endoplasmic reticulum. This is followed by exhibition of the peptide–MHC class I complex at the cell surface. This “cytosolic antigen processing pathway” occurs in nearly all nucleated cell types.

The peptide-binding cleft  is that part of a major histocompatibility molecule that binds peptides for display to T lymphocytes. Paired α-helices on a floor of an eight-stranded β-pleated sheet comprise the cleft. Situated in and around this cleft are polymorphid residues that are the amino acids which differ among various MHC alleles.

The endocytic pathway  uses membrane-bound vesicles, such as endosomes and endolysosomes, within the cell that harbor hydrolytic enzymes and additional molecules requisite for digestion of internalized substances. This pathway

Determinant selection model:  Concept that immune response variability to a given antigen among different individuals is attributable to each person’s MHC alleles’ ability to successfully bind and present that antigen’s determinants.

Class I

αβli

TGN Golgi

Endoplasmic reticulum

Class II

αβ

Early endosome

Late endosome

Lysosome

Figure 5.2  Processing pathways for class II-restricted antigen presentation.

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Target cell

N

CD8+

CD8+ T cell

Antigen

β2 microglobulin

C

T cell receptor

MHC class I

Antigen presenting macrophage CD4+ T cell

β α Antigen MHC class II

T cell receptor

Figure 5.4  Antigen presentation.

N C

Figure 5.3  Presentation of MHC histocompatibility antigen HLA-B 270S complexed with nonapeptide ARG-ARG-ILELYS0ALA-ILE-THR-LEU-LYS. The C-terminal amino acid of the antigen-binding domain is protected by an N-methyl group. Three water molecules bridge the binding of the peptide to the histocompatibility protein.

Pulsing:  Antigen presenting cell peptide groove loading with antigen by electroporation. Anchor residues  are amino acid side chains of the peptide whose side chains fit into pockets in the peptide-binding cleft of the MHC molecule. The side chains anchor the peptide in the cleft of the MHC molecule by binding two complementary amino acids in the MHC molecule. CD1  is an antigen that is a cortical thymocyte marker, which disappears at later stages of T cell maturation. The antigen is also found on interdigitating cells, fetal B cells, and Langerhans cells. These chains are associated with β2-microglobulin and the antigen is thus analogous to classical histocompatibility antigens, but coded for by a different chromosome. More recent studies have shown that the molecule is coded for by at least five genes on chromosome 1, of which three produce recognized polypeptide products. CD1 may participate in antigen presentation.

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Direct antigen presentation  refers to cell surface allogeneic MHC molecule presentation by graft cells to T lymphocytes of the graft recipient, leading to T lymphocyte activation. This process does not require processing. Direct recognition of foreign MHC molecules is a cross-reaction between a normal TCR that recognizes self-MHC molecules plus foreign antigen and allogeneic MHC molecule–peptide complex. The powerful T cell response to allografts is due in part to direct presentation. A proteasome  (Figure  5.6) is a 650-kDa organelle in the cytoplasm termed the low molecular mass polypeptide complex. The proteasome is believed to generate peptides by degradation of proteins in the cytosol. It is a cylindrical structure comprised of as many as 24 protein subunits. The proteasome participates in degradation of proteins in the cytosol that are covalently linked, ubiquinated, prior to presentation to MHC class I-restricted T lymphocytes. Proteasomes that include MHC gene encoded subunits are especially adept at forming peptides that bind MHC class I molecules. A strategic part of the endogenous antigen processing and presentation pathway. In resting cells the standard proteasome digests mostly undesired self proteins. In dendritic cells and other cells accompanying inflammation, the immunoproteasome functions in dendritic cells to digest foreign proteins. Immunoproteasome:  See proteasome. Indirect antigen presentation:  In organ or tissue transplantation, the mechanism whereby donor allogeneic MHC molecules are present in microbial proteins. The recipient

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Peptide

HLA heavy chain

113Å

13Å

α

27Å β 148Å Beta–2 Microglobulin

β

α Peptide

Catalytic sites

HLA heavy chain

Beta–2 Microglobulin

Figure 5.5  A schematic backbone structure of human class I histocompatibility antigen (HLA-A 0201) complexed with a decameric peptide from hepatitis B nucleocapsid protein (residues 18 to 27). Determined by x-ray crystallography.

professional antigen-presenting cells process allogeneic MHC proteins. The resulting allogeneic MHC peptides are presented in association with recipient self-MHC molecules to host T lymphocytes. By contrast, recipient T cells recognize unprocessed allogeneic MHC molecules on the surface of the graft cells in direct antigen presentation. The immunological synapse  is the nanometer scale gap between a T cell and an antigen-presenting cell, which is the site of interaction between T cell antigen receptors and major histocompatibility complex molecule–peptide complexes that initiate adaptive immune responses. Refer to SMAC (supramolecular activation complex).

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Figure 5.6  Longitudinal and transverse section through the 20S proteasome. The 20S proteasome is composed of two outer and two inner rings. The two outer rings each comprise seven copies of the 25.9-kDa α subunit.

SMAC  is the supramolecular activation complex. The point of contact between a T cell and an antigen-presenting cell where there is accumulation of stabilized lipid rafts and activated T cell receptors. It is also termed “the immunological synapse.” The SMAC possesses a concentric triple ring construct comprised of the cSMAC, inner pSMAC and outer pSMAC (the “c” refers to central and the “p” refers to peripheral). pSMAC  is the peripheral supramolecular activation complex. The interior pSMAC encircles the cSMAC and accommodates CD2/LFA-3 pairs. The exterior pSMAC encircles the inner pSMAC, accommodates LFA-1/ICAM-1 pairs, and is united to the actin cytoskeleton. cSMAC  is the central supramolecular activation complex, constituting the center ring of the SMAC. It is comprised of activated T cell receptors, pMHC, coreceptors and related kinases, as well as lipid rafts. Erp57  is a chaperone molecule that participates in the loading of peptide onto MHC class I molecules in the endoplasmic reticulum. Transporter associated with antigen processing (TAP)  refers to a TAP-binding heterodimeric protein in the rough endoplasmic reticulum membrane that transports peptides from the cytosol to the endoplasmic reticulum lumen. It is comprised of TAP 1 and TAP 2 subunits that bind peptides to class I MHC molecules.

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TAP  is an abbreviation for “transporter of antigen processing.” TAP is a heterodimeric protein situated in the rough endoplasmic reticulum membrane that conveys HSPchaperoned peptides from the cytosol into the rough endoplasmic reticulum lumen where they are loaded onto MHC class I molecules. It is a critical constituent of endogenous antigen processing and presentation. A presentosome  is a molecular assemblage on both sides of the endoplasmic reticulum membrane that aid processing of endogenous antigen. Proteasomal degradation yields peptides that are complexed with HSP70, HSP90, or HSP110 in the cytosol for conveyance to TAP. Peptides are united to gp96 or PDI in the endoplasmic reticulum on the other side of TAP. Agrin  is an aggregating protein crucial for formation of the neuromuscular junction. It is also expressed in lymphocytes and is important in reorganization of membrane lipid microdomains in setting the threshold for T cell signaling. T cell activation depends on a primary signal delivered through the T cell receptor and a secondary costimulatory signal mediated by coreceptors. Costimulation is believed to act through the specific redistribution and clustering of membrane and intracellular kinase-ridge lipid raft microdomains at the contact site between T cells and antigen-presenting cells. This site is known as the immunological synapse. Endogenous mediators of raft clustering in lymphocytes are essential for T cell activation. Agrin induces the aggregation of signaling proteins and the creation of signaling domains in both immune and nervous systems through a common lipid raft pathway. A lipid raft  is a plasma membrane subdomain rich in cholesterol and glycosphingolipid-rich where cellular activation molecules are concentrated. Proteins in an activated T cell requisite for T cell receptor signaling interact with the rafts and produce a large stable immunosome that attracts and activates more molecular signals. The rafts and signaling molecules act with the T cell receptors’ cytoplasmic region to transduce T cell receptor signaling to the nucleus. Proteasome genes  are two genes in the MHC class II region that encode two proteasome subunits. The proteasome is a protease complex in the cytosol that may participate in the generation of peptides from proteins in the cytosol. LMP genes  are two genes located in the MHC class II region in humans and mice that code for proteasome subunits. They are closely associated with the two TAP genes. LMP-2 and LMP-7  are catalytic subunits of the organelle (proteasome) that degrades cytosolic proteins into peptides in the class I MHC pathway of antigen presentation. MHC genes encode these two subunits which are upregulated by IFN-γ and are especially significant in the generation of class I MHC-binding peptides.

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Tapasin:  TAP-associated protein is a chaperone molecule that participates in the assembly of peptide-MHC class I molecule complexes in the endoplasmic reticulum. A rough endoplasmic reticulum protein that unites TAP and the MHC class I α chain at two separate binding sites. Believed to stabilize empty MHC class I heterodimers in a configuration appropriate for peptide loading. Cells deficient in this protein have unstable MHC class I cell surface molecules. Tapasin (TAP-associated protein)  is a molecule that is critical in MHC class I molecule assembly. Without this protein, MHC class I molecules are unstable on the cell surface. The transporter in antigen processing (TAP) 1 and 2 genes  (Figure 5.7) are in the MHC class II region that must be expressed for MHC class I molecules to be assembled efficiently. TAP 1 and TAP 2 are postulated to encode components of a heterodimeric protein pump that conveys cytosolic peptides to the endoplasmic reticulum. Here they associate with MHC class I heavy chains. TAP 1 and TAP 2 genes:  See transporter in antigen processing 1 and 2 genes. An antigen-presenting cell (APC)  is a cell that can process a protein antigen, break it into peptides, and present it in conjunction with major histocompatibility complex (MHC antigens) on the cell surface where it may interact with appropriate T  cell receptors. Professional antigenpresenting cells include dendritic cells, macrophages, and B cells that are capable of initiating T lymphocyte responsiveness to antigen. These cells display antigenic peptide fragments in association with the proper class of MHC molecules and also bear costimulatory surface molecules. Dendritic cells are the most important professional antigen-presenting cells for initiating primary T lymphocyte responses. This is facilitated in part by their continuous high level expression of costimulatory B7 molecules. Dendritic reticulum cells, macrophages, Langerhans cells and B cells process and present antigen to immunoreactive lymphocytes such as CD4 + T helper/inducer cells (Figure  5.8). An MHC transporter-gene-encoded peptide supply factor may mediate peptide antigen presentation. In addition to the three types of professional APCs Tap 1 1

2

6 7 3 4 5

A NH2 B

C

F Human tap 1

D

8

9

E

10

10

Tap 2

9 8

E

7 6

D

5 4

C

F Human tap 2

32

1

B

A NH2

COOH COOH

Figure 5.7  Topology of Tap 1 and Tap 2 proteins.

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T cell

T cell receptor

CD4

Processed antigen Histopope

MHC class II molecule

Figure 5.8  Antigen-presenting cell.

mentioned above, follicular dendritic cells are the main antigen-presenting cells for B cells. Nonprofessional antigen-presenting cells include keratinocytes and selected epithelial, endothelial, and mesenchymal cells and can act as antigen-presenting cells when activated during inflammation. Antigen-presenting cells include those that present exogenous antigen processed in their endosomal compartment and are presented together with MHC class II molecules. Other antigen-presenting cells present antigen that has been endogenously produced by the body’s own cells with processing in an intracellular compartment and presentation together with MHC class I molecules. A third group of APCs present exogenous antigen that is taken into the cell and processed followed by presentation together with MHC class I molecules. In addition to processing and presenting antigenic peptides in association with MHC class II molecules, an antigen-presenting cell must also deliver a costimulatory signal that is necessary for T cell activation. Nonprofessional APCs that function in antigen presentation for only brief periods include thymic epithelial cells and vascular endothelial cells. Professional antigen-presenting cells  are dendritic cells, macrophages, and B cells that are capable of initiating responsiveness of naïve T lymphocyte responsiveness to antigen. These cells display antigenic peptide fragments in association with the proper class of MHC molecules and also bear costimulatory surface molecules. Dendritic cells are the most important professional APCs for initiating primary T lymphocyte responses. Among the three major antigen-presenting cells, dendritic cells are the only ones that continuously express high levels of costimulatory B7 and can present antigen via both class I MHC molecules and class II MHC molecules. Thus, they can activate both CD8 and CD4 T cells directly. APC  is the abbreviation for antigen-presenting cell. Professional APC:  Refers to professional antigen-presenting cell.

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APC licensing  is the postulate that total activation of a cytotoxic T cell stimulated by antigen is enabled through costimulation by a dendritic cell whose costimulatory molecules have been upregulated following previous CD40CD40L-mediated interaction with an antigen-activated TH lymphocyte. Cathepsins  are thiol and aspartyl proteases that have broad substrate specificities. Cathepsins represent the most abundant proteases of endosomes in antigen-presenting cells. They are believed to serve an important function in the generation of peptide fragments from exogenous protein antigens that bind to class II MHC molecules. CD2  is a T cell adhesion molecule that binds to the LFA-3 adhesion molecule of antigen-presenting cells. Also called LFA-2. Mature dendritic cells  are antigen-presenting cells of secondary lymphoid tissues that bear costimulatory molecules among other cell surface molecules that render them capable of presenting antigen to naïve T cells that become activated. A circulating dendritic cell  is one that has taken up antigen and is migrating to a secondary lymphoid tissue such as a lymph node. CD8  is a cell surface glycoprotein on T cells that recognizes antigens presented by MHC class I molecules. It binds to MHC class I molecules on antigen-presenting cells and serves as a coreceptor to facilitate the T cell’s response to antigen. The CD8  molecule is a heterodimer of an α and β chain that are covalently associated by disulfide bond. The two chains of a dimer have similar structures, each having a single domain resembling an immunoglobulin variable domain and a stretch of peptide believed to be in a relatively extended conformation. CD8 T cells  comprise the T cell subset that expresses CD8 coreceptor and recognizes peptide antigens presented by MHC class I molecules. CD20  is a B cell marker with a molecular weight of 33, 35, and 37 kDa that appears relatively late in the B cell maturation (after the pro-B cell stage) and then persists for some time before the plasma cell stage. Its molecular structure resembles that of a transmembrane ion channel. The gene is on chromosome 11 at band q12-q13. It may be involved in regulating B cell activation. CD21  is a 145-kDa glycoprotein. Component of the B cell receptor. CD21 is a membrane molecule that participates in transmitting growth-promoting signals to the interior of the B cell. It is the receptor for the C3d fragment of the third component of complement, CR2. The CD21 antigen is a

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Cell type Dendritic cells (Langerhans cells, lymphoid dendritic cells) wMacrophages

Class II Constitutive

Costimulators Constitutive

Inducible by IFN

Inducible by LPS

B-lymphocytes

Constitutive

Constitutive

Vascular endothelial cells

Inducible by IFN

Constitutive

Principal functions Inflammation of CD4 T cell response; allograft rejection Development of CD4 effector T cells Stimulation by CD4 helper T cells in humoral immune responses Recruitment of antigenspecific T cells to site of antigen-exposure or inflammation

Figure 5.9  Properties and functions of antigen-presenting cells.

restricted B cell antigen expressed on mature B cells. It is present at high density on follicular dendritic cells (FDC), the accessory cells of the B zones. Also called complement receptor 2 (CR2). CD22  is a molecule with an α130- and β140-kDa mol wt that is expressed in the cytoplasm of B cells of the pro-B and pre-B cell stage and on the cell surface on mature B cells with surface Ig. The antigen is lost shortly before the terminal plasma cell phase. The molecule has five extracellular immunoglobulin domains and shows homology with myelin adhesion glycoprotein and with N-CAM (CD56). It participates in B cell adhesion to monocytes and T cells. Also called BL-CAM. CD28  is a T-cell low-affinity receptor that interacts with B7 costimulatory molecules to facilitate T lymphocyte activation. More specifically, B7-1 and B7-2 ligands are expressed on the surface of activated antigen-presenting cells (APCs). Signals from CD28 to the T cell elevate expression of high affinity IL-2 receptor and increase the synthesis of numerous cytokines, including IL-2. CD28 regulates the responsiveness of T cells to antigen when they are in contact with APCs. It serves as a costimulatory receptor because its signals are synergistic with those provided by the T cell antigen receptor (TCR-CD3) in promoting T cell activation and proliferation. Without the signal from TCR-CD3, CD28’s signal is only able to stimulate minimal T cell proliferation and may even lead to T cell unresponsiveness. Tp44 (CD28)  is a T lymphocyte receptor that regulates cytokine synthesis, thereby controlling responsiveness to antigen. Its significance in regulating activation of T lymphocytes is demonstrated by the ability of monoclonal antibody against CD28 receptor to block T cell stimulation by specific antigen. During antigen-specific activation of T lymphocytes, stimulation of the CD28 receptor occurs when it combines with the B7/BB1 coreceptor during the interaction between

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T and B lymphocytes. CD28 is a T lymphocyte differentiation antigen that four-fifths of CD3/Ti positive lymphocytes express. It is a member of the immunoglobulin superfamily. CD28 is found only on T lymphocytes and on plasma cells. There are 134 extracellular amino acids with a transmembrane domain and a brief cytoplasmic tail in each CD28 monomer. A costimulator  is an antigen-presenting cell surface molecule that supplies a stimulus, serving as a second signal required for activation of naïve T lymphocytes, in addition to antigen (the “first signal”). An example of a costimulator is the B7 molecule on professional antigen-presenting cells that binds to the CD28 molecule on T lymphocytes. Another example is the interaction of CD40 with CD40L on B cells. Costimulatory molecules  are membrane bound or secreted products of accessory cells that activate signal transduction events in addition to those induced by MHC/TCR interactions. They are required for full activation of T cells, and it is thought that adjuvants may work by enhancing the expression of costimulator molecules by accessory cells. The interaction of CD28/CTLA-4 with B7 to induce full transcription of IL-2 mRNA is an example of costimulator mechanisms. A costimulatory signal  is an extra signal requisite to induce proliferation of antigen-primed T lymphocytes. It is generated by the interaction of CD28 on T cells with B7 on antigen-presenting cells or altered self cells. In B cell activation an analogous second signal is illustrated by the interaction of CD40 on B cells with CD40L on activated TH cells. A costimulatory blockade  is the deliberate interruption of costimulatory signal transmission that leads to anergy and antigen-specific T lymphocytes. Cross-priming  is the activation or priming of a naïve CD4+ cytotoxic T lymphocyte specific for antigens of a third cell such as a virus-infected cell or tumor cell by a professional

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antigen-presenting cell. Cross-priming takes place when a professional antigen-presenting cell ingests an infected cell and the microbial antigens are processed and presented in association with class I MHC molecules. The professional antigen-presenting cell also costimulates the T cells. Also referred to as cross-presentation. Cross-presentation  refers to the exogenous peptide antigen presentation on a dendritic cell(s) or macrophage(s) MHC class I molecules. There may be fusion of a phagosome containing exogenous antigen with an endoplasmic reticulumderived vesicle bearing constituents of the endogenous antigen processing apparatus. Peptide regurgitation or peptide interception may also represent a mechanism of cross-presentation. Peptide interception  is a possible cross-presentation mechanism. An MHC class II compartment holding exogenous peptides combines with a vesicle harboring MHC class I recycling from the cell surface. A reduced pH in the MHC class II compartment causes release of the endogenous peptide from the recycling MHC class I molecule’s groove and permits loading of an exogenous MHC class II compartment peptide, independent of TAP, onto MHC class I.

associated ligands and facilitating in signaling for activation. CD4 and CD8 are T cell coreceptors that bind nonpolymorphic parts of a MHC molecule concurrently with the TCR binding to polymorphic residues and the bound peptide. It is a structure on the surface of a lymphocyte that binds to a part of an antigen simultaneously with membrane immunoglobulin (Ig) or T cell receptor (TCR) binding of antigen and which transmits signals required for optimal lymphocyte activation. CD4 and CD8 represent T cell coreceptors that bind nonpolymorphic regions of a major histocompatibility complex (MHC) molecule simultaneously with the binding of the T cell receptor to polymorphic residues and the exhibited peptide. Contact between the pMHC and the T cell receptor is stabilized by the binding, which also seeks protein tyrosine kinases that engage in intracellular signaling. CD19CD21-CD81 complex acts as a B cell coreceptor.

Peptide regurgitation  is a possible mechanism of cross‑ presentation. Following internalization of extracellular proteins and processing into peptides within dendritic cell endosomes, the peptides are set free or “regurgitated” back into the extracellular environment. The extracellular exogenous peptide may negotiate an exchange of peptides at the cell surface without intracellular processing. The exogenous peptide displaces MHC-associated peptides displayed on the surface.

MHC  restriction is the recognition of an antigen in the context of either class I or class II molecules by the T cell receptor for antigen. In the afferent limb of the immune response, when antigen is being presented at the surface of a macrophage or other antigen-presenting cell to CD4+ T lymphocytes, this presentation must be in the context of MHC class II molecules for the CD4+ lymphocyte to recognize the antigen and proliferate in response to it. By contrast, cytotoxic (CD8+) T lymphocytes recognize foreign antigen, such as viral antigens on infected target cells, only in the context of class I MHC molecules. Once this recognition system is in place, the cytotoxic T cell can fatally injure the target cell through release of perforin molecules that penetrate the target cell surface. T cells recognize a firm peptide antigen only in the context of a specific allelic form of an MHC molecule to which it is bound.

A coreceptor  is a cell surface protein that increases the sensitivity of an antigen receptor to antigen by binding to

CTLA-4  is a molecule that is homologous to CD28 and expressed on activated T cells (Figure  5.10). The genes for

CTLA-4

CD28

CTLA-4

Activated T Cell CD28

CTLA-4 T-cell receptor

Antigen

B7

B7

B7

B7

B7

CD4 MHC class II

Figure 5.10  Participation of CTLA-4 molecules during antigen presentation.

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Antigen Processing and Presentation Naive T Cell

CD28

T-Cell receptor Antigen CD4

B7

Antigen presenting cell

MHC class II

Activated T cell

CTLA-4

B7

Antigen presenting cell

Figure 5.11  B7.

CD28 and CTLA-4 are closely linked on chromosome 2. The binding of CTLA-4 to its ligand B7 is an important costimulatory mechanism (see CD28 and costimulatory molecules). CTLA-4 is a high affinity receptor for B7 costimulatory molecules on T lymphocytes (Figure  5.12). CTLA4-Ig is a soluble protein composed of the CD28 homolog CTLA and the constant region of an IgG1 molecule. It is used experimentally to inhibit the immune response by blocking CD28-B7 interaction. CTLA4-Ig  is a soluble protein composed of the CD28 homolog CTLA and the constant region of an IgG1 molecule. It is used experimentally to inhibit the immune response by blocking CD28–B7 interaction.

Immune costimulatory molecules:  B7-1 and B7-2, together with their receptors CD28 and CTLA-4, constitute one of the dominant costimulatory pathways that regulate T- and B-cell responses. Although both CTLA-4 and CD28 can bind to the same ligands, CTLA-4 binds to B7-1 and B7-2 with a 20- to 100-fold higher affinity than CD28 and is involved in the downregulation of the immune response. B7-1 is expressed on activated B and activated T cells and macrophages. B7-2 is constitutively expressed on interdigitating dendritic cells, Langerhans cells, peripheral blood dendritic cells, memory B cells, and germinal center B cells. It has been observed that both human and mouse B7-1 and B7-2 can bind to either human or mouse CD28 and CTLA-4, suggesting that there are conserved amino acids which form the B7-1/B7-2/CD28/CTLA-4 critical binding sites. B7, B7-2:  B7 is a homodimeric immunoglobulin superfamily protein whose expression is restricted to the surface of cells that stimulate growth of T lymphocytes and is the ligand for CD28. B7 is expressed on the surface of professional antigen-presenting cells and is important in costimulatory mechanisms (Figure 5.11). Some APCs may upregulate expression of B7 following activation by various stimuli including IFN-α, endotoxin, and MHC class II binding. B7 is also termed CD80. B7-2 is a costimulatory molecule whose sequence resembles that of B7. Dendritic cells, monocytes, activated T cells, and activated B lymphocytes may express B7-2. B7 is also referred to as BB1, B7.1, or CD80. An invariant (Ii) chain  is a nonpolymorphic, 31-kDa glycoprotein that associates with class II histocompatibility molecules in the endoplasmic reticulum (Figure  5.13). It inhibits the linking of endogenous peptides with the class II molecule, conveying it to appropriate intracellular compartments. Truncation of the invariant chain stimulates a second signal that may function in the trans-Golgi network, prior to the conveyance of MHC class II molecules to the cell surface.

Figure 5.12  CTLA-4/B7-2 complex.

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Figure 5.13  Invariant chain promotes assembly of class II heterodimers from free chains.

Ii:  See invariant chain. A class II vesicle (CIIV)  is a murine B cell membranebound organelle that is critical in the class II MHC pathway of antigen presentation. It contains all constituents requisite for the formation of peptide antigen and class II MHC molecular complexes, including the enzymes that degrade protein antigens, class II molecules, invariant chain, and HLA-DM. CIIV  is an abbreviation for “class II vesicle.” Refer also to MIICs. MIICs  are MHC class compartments or class II vesicles. These specialized late endosomal spaces comprise a segment of the exogenous (endocytic) antigen processing pathway. Endolysosomal MHC-CLIP complexes enter MIICs where CLIP exchange and peptide loading occur. Desetope  is a term derived from “determinant selection.” It describes that region of class II histocompatibility molecules that reacts with the antigen during antigen presentation (Figure 5.14 and Figure 5.15). Allelic variation permits these contact residues to vary, which is one of the factors in

T cell

T-cell receptor Processed antigen MHC class II

Paratope Restitope Epitope Histotope Desetope Agretope

Antigen-presenting cell

Figure 5.14  The schematic representation of the interaction of the class II MHC, processed peptide antigen, and T cell receptor molecules during antigen presentation.

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op

e

Resitope

ot

α chain

e op et p e gr A seto e D

β chain

Processed antigen

CD4

ist

2

H

HSP

T ce ll Pa receptor r Ep ato ito pe pe

T cell

Intact Ii

MHC class II molecule

Antigen 1 Co-stimulatory presenting cell signal Binding of the MHC complex

Figure 5.15  Costimulator for the activation of T cells.

histocompatibility molecule selection of a particular epitope that is being presented. An agretope  refers to the region of a protein antigen that combines with an MHC class II molecule during antigen presentation. This is then recognized by the T cell receptor MHC class II complex. Amino acid sequences differ in their reactivity with MHC class II molecules. A histotope is the portion of an MHC class II histocompatibility molecule that reacts with a T lymphocyte receptor. A restitope is that segment of a T cell receptor that makes contact and interacts with a class II histocompatibility antigen molecule during antigen presentation. CLIP  is the processed fragment of invariant chain. In the MHC class II transport pathway, the peptide binding groove must be kept free of endogenous peptides. The cell uses one protein, called invariant chain (and its processed fragment CLIP), to block the binding site until needed. HLA-DM facilitates release of CLIP peptides and their exchange for antigenic peptides as they become available. As long as CLIP remains in the binding groove, antigenic peptides cannot bind. A superantigen  is an antigen such as a bacterial toxin that is capable of stimulating multiple T lymphocytes, especially CD4 + T cells, leading to the release of relatively large quantities of cytokines. A protein that unites simultaneously with an invariant site on the MHC protein outside the peptide-binding groove and to the CDR2 of selected TCRβ chains. Selected bacterial toxins may stimulate all T lymphocytes in the body that contain a certain family of V β T cell receptor (TCR) genes. Superantigens may induce proliferation of 10% of CD4 + T cells by combining with the T cell receptor V β and to the MHC HLA-DR α-1 domain. Superantigens are thymus-dependent (TD) antigens that do not require phagocytic processing. Instead of fitting into

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T cell

T cell receptor (TCR)

Superantigen

MHC class II molecule

Antigen presenting cell

Figure 5.16  Superantigen.

the TCR internal groove where a typical processed peptide antigen fits, superantigens bind to the external region of the αβ TCR and simultaneously link to DP, DQ, or DR molecules on antigen-presenting cells (Figure  5.16). Superantigens react with multiple TCR molecules whose peripheral structure is similar. Thus, they stimulate multiple T cells that augment a protective T and B cell antibody response. This enhanced responsiveness to antigens such as toxins produced by staphylococci and streptococci is an important protective mechanism in the infected individual (Figure  5.17). Several staphylococcal enterotoxins are superantigens and may activate many T cells resulting in the release of large quantities of cytokines and producing a clinical syndrome resembling septic shock. MHC-I antigen presentation:  Proteins in the cytosol, such as those derived from viruses, may be processed through the class I MHC route of antigen presentation. The multiprotein complex in the cytoplasm, known as the proteasome effects, involves proteolytic degradation of proteins in the cytoplasm to yield many of the peptides that are presented by class I MHC molecules. TAP molecules

CD4 or CD8 LFA-1

MHC

LFA-3 Antigen presenting cell

CD2 TCR

T cell

Antigen peptide

Superantigen

Figure 5.17  Cellular and molecular interactions in antigen presentation.

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A granuloma  is a tissue reaction characterized by altered macrophages (epithelioid cells), lymphocytes, and fibroblasts caused by hyperactivated macrophages that fuse together to isolate a persistent pathogenic microorganism or a nondegradable foreign body (Figure 5.18). It also contains CD4 + and CD8 + T lymphocytes. Its production is dependent on TNF synthesized by activated T H1 effector lymphocytes. The cells form microscopic masses of mononuclear cells. Giant cells form from some of these fused cells. Granulomas may be of the foreign body type, such as those surrounding silica or carbon particles, or of the immune type that encircle particulate antigens derived from microorganisms. Activated macrophages trap antigen which may cause T cells to release lymphokines causing more macrophages to accumulate. This process isolates the microorganism. Granulomas appear in cases of tuberculosis and develop under the influence of helper T cells that react against Mycobacterium tuberculosis. Some macrophages and epithelioid cells fuse to form multinucleated giant cells in immune granulomas. There may also be occasional neutrophils and eosinophils. Necrosis may develop. It is a delayed type of hypersensitivity reaction that persists as a consequence of the continuous presence of foreign body or infection. HAM-1 and HAM-2 (histocompatibility antigen modifier):  These are two murine genes that determine formation of permeases that are antigen transporters (oligopeptides) from the cytoplasm to a membrane-bound compartment where antigen complexes with MHC class I and class II molecules. In man, the equivalents of HAM-1 and HAM-2 are termed ATP-binding cassette transporters.

α helices ICAM-1

transport peptides from the cytoplasm to the endoplasmic reticulum, where they interact and bind to class I MHC dimeric molecules. Once the class I MHC molecules have become stabilized through peptide binding, the complex leaves the endoplasmic reticulum, entering the Golgi apparatus en route to the surface of the cell. Thus, mechanisms are provided through MHC-restricted antigen presentation to guarantee that peptides derived from extracellular microbial proteins can be presented by class II MHC molecules to CD4 + helper T cells and that peptides derived from intracellular microbes can be presented by class I MHC molecules to CD8 + cytotoxic T lymphocytes. The generation of microbial peptides produced through antigen processing to combine with self MHC molecules is critical to the development of an appropriate immune response.

T lymphocyte–B lymphocyte cooperation  refers to the association of B cell and helper T cell through a number of receptor–ligand interactions at the surfaces of both cell types which leads to B cell proliferation and differentiation into plasma cells that synthesize and secrete specific antibody specific for thymus-dependent antigen (Figure  5.19). B cell immunoglobulin receptors react with protein antigens. This is followed by endocytosis, antigen processing,

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IL-12 a

APC

Antigen

T helper (CD4 + TH1 cell)

T

Interleukin 2 TNF Granuloma forms

INFγ

Lympocyte Macrophage Monocyte Epithelioid cell Fibroblast

Giant cell

Figure 5.18  Granuloma.

and presentation to helper T lymphocytes. Their antigenspecific T cell receptors recognize processed antigens only in the context of MHC class II molecules on the B cell surface during antigen presentation. CD4 + helper T cells secrete lympho­k ines, including IL-2, which promote B cell growth and differentiation into plasma cells that secrete specific antibodies. T cells are required for B cells to be able to switch from forming IgM to synthesizing IgG or IgA. B and T lymphocytes recognize different antigens. B cells may recognize peptides, native proteins, or denatured proteins. T cells are more complex in their recognition system in that a peptide antigen can be presented to them only in the context of MHC class II or class I histocompatibility molecules. Hapten–carrier complexes have been successfully used in delineating the different responses of B and T cells to each part of this complex. Immunization of a rabbit or other animal with a particular hapten–carrier complex will induce a primary immune response, and a second injection of the same hapten–carrier conjugate will induce a secondary immune response. However, linkage of the same hapten to a different carrier elicits a much weaker secondary response in an animal primed with the original hapten–carrier complex. This is termed the carrier effect. B lymphocytes recognize the hapten, and T lymphocytes the carrier.

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By light microscopy, resting lymphocytes  appear as a distinct and homogeneous population of round cells, each with a large, spherical or slightly kidney-shaped nucleus which occupies most of the cell and is surrounded by a narrow rim of basophilic cytoplasm with occasional vacuoles. The nucleus usually has a poorly visible single indentation and contains densely packed chromatin. Occasionally, nucleoli can be distinguished. The small lymphocyte variant, which is the predominant morphologic form, is slightly larger than an erythrocyte. Larger lymphocytes, ranging between 10 and 20 μm in diameter, are difficult to differentiate from monocytes. They have more cytoplasm and may show azurophilic granules. Intermediate-size forms between the two are described. By phase contrast microscopy, living lymphocytes show a feeble motility with ameboid movements that give the cells a hand-mirror shape. The mirror handle is called a uropod. In large lymphocytes, mitochondria and lysosomes are better visualized, and some cells show a spherical, birefringent, 0.5-μm diameter inclusion, called a gall body. Lymphocytes do not spread on surfaces. The different classes of lymphocytes cannot be distinguished by light microscopy. By scanning electron microscopy, B lymphocytes sometimes show a hairy (rough) surface, but this is apparently an

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CD72

CD5

B7

CD28

CD54

CD11a/CD18

MHC Class II

Ag

artifact. Electron microscopy does not provide additional information except for visualization of the cellular organelles which are not abundant. This suggests that the small, resting lymphocytes are end-stage cells. However, under appropriate stimulation, they are capable of considerable morphologic changes. Cooperation  refers cooperation.

TCR CD4 T cell CD54

CD11a/CD18

T

lymphocyte–B

lymphocyte

Cooperativity  refers to the effect observed when two binding sites are linked to their ligand to yield an effect of binding to both that is greater than the sum of each binding site acting independently.

CD3

B cell

to

Cognate interaction  refers to the interaction of processed antigen on a B cell surface interacting with a T cell receptor for antigen resulting in B cell differentiation into an antibodyproducing cell. Cognate recognition  refers to cognate interaction.

CD19 Ig Igα

Ag

Igβ

Figure 5.19  T–B cell interactions.

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B Lymphocyte Development and Immunoglobulin Genes

The bursa of Fabricius  (Figure 6.1) is located near the terminal portion of the cloaca and, like the thymus, is a lympho-epithelial organ. The bursa begins to develop after the 5th day of incubation and becomes functional around the 10th to 12th day. It has an asymmetric sac-like shape and a star-like lumen, which is continuous with the cloacal cavity. The epithelium of the intestine covers the bursal lumen but lacks mucous cells. The bursa contains abundant lymphoid tissue, forming nodules beneath the epithelium. The nodules show a central medullary region containing epithelial cells and project into the epithelial coating. The center of the medullary region is less structured and also contains macrophages, large lymphocytes, plasma cells, and granulocytes. A basement membrane separates the medulla from the cortex; the latter comprises mostly small lymphocytes and plasma cells. The bursa is well developed at birth but begins to involute around the 4th month; it is vestigial at the end of the first year. There is a direct relationship between the hormonal status of the bird and involution of the bursa. Injections of testosterone may lead to premature regression or even lack of development, depending on the time of hormone administration. The lymphocytes in the bursa originate from the yolk sac and migrate there via the bloodstream. They comprise B cells which undergo maturation to immunocompetent cells capable of antibody synthesis. Bursectomy at the 17th day of incubation induces agammaglobulinemia, with the absence of germinal centers and plasma cells in peripheral lymphoid organs. A bursacyte  is a lymphocyte that undergoes maturation and differentiation under the influence of the bursa of Fabricius in avian species. This cell synthesizes the antibody which provides humoral immunity in this species. A bursacyte is a B lymphocyte. The anatomical site in mammals and other nonavian species that resembles the bursa of Fabricius in controlling B cell ontogeny is termed a bursa equivalent. Mammals do not have a specialized lymphoid organ for maturation of B lymphocytes. Although lymphoid nodules are present along the gut, forming distinct structures called Peyer’s patches, their role in B-cell maturation is no different from that of lymphoid structures in other organs. After commitment to B-cell lineage, the B cells of mammals leave the bone marrow in a relatively immature stage. Likewise, after education in the thymus, T  cells migrate from the thymus also in a relatively immature stage. Both populations continue their maturation process away from the site of origin and are subject to influences originating in the environment in which they reside.

Bursectomy  refers to the surgical removal or ablation of the bursa of Fabricius, an outpouching of the hindgut near the cloaca in birds. Surgical removal of the bursa prior to hatching or shortly thereafter, followed by treatment with testosterone in vivo, leads to failure of the B cell limb of the immune response responsible for antibody production. B lymphocytes  are lymphocytes of the B cell lineage that mature under the influence of the bursa of Fabricius in birds and in the bursa equivalent (bone marrow) in mammals. B cells occupy follicular areas in lymphoid tissues and account for 5 to 25% of all human blood lymphocytes that number 1000 to 2000 cells per mm3. They comprise most of the bone marrow lymphocytes, one-third to one-half of the lymph node and spleen lymphocytes sites, but less than 1% of those in the thymus. Nonactivated B cells circulate through the lymph nodes and the spleen. They are concentrated in follicles and marginal zones around the follicles. Circulating B cells may interact and be activated by T cells at extrafollicular sites where the T cells are present in association with antigen-presenting dendritic cells. Activated B cells enter the follicles, proliferate, and displace resting cells. They form germinal centers and differentiate into both plasma cells that form antibody and long-lived memory B cells. Those B cells synthesizing antibodies provide defense against microorganisms including bacteria and viruses. Surface and cytoplasmic markers reveal the stage of development and function of lymphocytes in the B cell lineage. Pre-B cells contain cytoplasmic immunoglobulins, whereas mature B cells express surface immunoglobulin and complement receptors. B lymphocyte markers include CD9, CD19, CD20, CD24, Fc receptors, B1, BA-1, B4, and Ia. Refer also to B cell. Hematogones  are early precursor B cells that express immature cell surface antigens such as HLA-DR, CD10, CD19, and CD20. Morphologically they appear as small compact lymphocytes. Large pre-B cells  are immature B cells with a surface pre-B cell receptor which is deleted on transition to small pre-B cells, which undergo light-chain gene rearrangement. Small pre-B cells  are pre-B cells undergoing light-chain gene rearrangements. Pre-B cells  are in a stage of B cell development characterized by rearrangement of heavy-chain genes but not lightchain genes. They develop (Figure 6.2 and Figure 6.3) from 215

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Bursa fabricius

pre-B

Virgin B

Mature B

Activated B

Cytoplasmic IgM

Cytoplasmic Ig surface IgM

Cytoplasmic Ig surface IgM

Cytoplasmic Ig surface IgM

Figure 6.3  Pre-B cell development.

transmit signals that induce further pre-B cell maturation into immature B cells.

Figure 6.1  The bursa of Fabricius is an outpouching of the hindgut located near the cloaca in avian species that governs B cell ontogeny. This specific lymphoid organ is the site of migration and maturation of B lymphocytes.

lymphoid stem cells in the bone marrow. These are large, immature lymphoid cells that express cytoplasmic T chains but no light chains or surface immunoglobulin, and are found in fetal liver and adult bone marrow. They are the earliest cells of the B cell lineage. Antigen is not required for early differentiation of the B cell series. Pre-B cells differentiate into immature B cells, followed by mature B cells that express surface immunoglobulin. Pre-B cell immunoglobulin genes contain heavy chain V, D, and J gene segments that are contiguous. No rearrangement of light-chain gene segments has yet occurred. In addition to their cytoplasmic IgM, pre-B cells are positive for CD10, CD19, and HLA-DR markers. Pre-B cells have rearranged heavy- but not light-chain genes. They express cytoplasmic Ig μ heavy chains and surrogate light chains but not Ig light chains. The pre-B cell receptor consists of μ chains and surrogate light chains. These receptors Immature B cell Mature B cell – IgM+ IgD

IgM+

IgD+

Bone marrow stromal cell (Fibroblast) IL-7

Pre-B cell

+ B220+CD43– +

CD24 FLT3 lgM–

FL SDF-1

B220+ CD43– CD24+ IgM–

Late Pro-B cell

B220+ CD43+ CD24–FLT3+ Pre- or early IgM– Pro- B cell

B220+ – + CD43 CD24 + FLT3

FL IL-7 IL-11 SDF-1

Bone marrow stromal cell (Fibroblast)

Primitive B cell progenitor

Figure 6.2  FLT-3 and bone marrow B cell lymphopoiesis.

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A pre-B cell receptor  is a maturing B cell lymphocyte receptor comprised of a μ heavy chain, an invariant surrogate light chain, and the Igα/Igβ heterodimer. It is expressed at the pre-B cell stage, and its appearance marks the end of heavychain gene rearrangement. The two proteins comprising the surrogate light chain include the λ5 protein, which is homologous to the λ light-chain C domain, and the Vpre-B protein which is homologous to a Vdomain. Association of the pre-B cell receptor with the Igαand the Igβ signal transduction proteins forms the pre-B cell receptor complex. Stimulation of proliferation and continued maturation of developing B cells require pre-B cell receptors. 𝛌5:  See pre-B cell receptor. Lymphocyte maturation  is the development of pluripotent bone marrow precursor cells into T or B lymphocytes that express antigen receptors that are present in peripheral lymphoid tissue. B cell maturation takes place in the bone marrow and T cell maturation is governed by the thymus. 𝛌5 B cell development:  See Vpre-B protein. Terminal deoxynucleotidyl transferase (TdT)  is an enzyme catalyzing the attachment of mononucleotides to the 3′ terminus of DNA. It thus acts as a DNA polymerase. Tdt is an enzyme present in immature B and T lymphocytes, but not demonstrable in mature lymphocytes. TdT is present both in the nuclear and soluble fractions of thymus and bone marrow. The nuclear enzyme is also able to incorporate ribonucleotides into DNA. In mice, two forms of TdT can be separated from a preparation of thymocytes. They are designated peak I and peak II. They have similar enzymatic activities and appear to be serologically related but display significant differences in their biologic properties. Peak I appears constant in various strains of mice and at various ages. Peak II varies greatly. In some strains, peak II remains constant up to 6 to 8 months of age; in others, it declines immediately after birth. A total of 80% of bone marrow TdT is associated with a particular fraction of bone marrow cells separated on a discontinuous BSA gradient. This fraction represents 1 to 5% of the total marrow cells, but is O antigen negative. These cells become O positive after treatment with a thymic hormone, thymopoietin, suggesting that they are precursors of thymocytes. Thymectomy is associated with rapid loss of peak II and a

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slower loss of peak I in this bone marrow cell fraction. TdT is detectable in T cell leukemia, 90% of common acute lymphoblastic leukemia cases, and half of acute undifferentiated leukemia cells. Approximately one-third of chronic myeloid leukemia cells in blast crisis and a few cases of pre-B cell acute lymphoblastic leukemia cases show cells that are positive for TdT. This marker is very infrequently seen in cases of chronic lymphocytic leukemia. In blast crisis, some cells may simultaneously express lymphoid and myeloid markers. Indirect immunofluorescence procedures can demonstrate TdT in immature B and T lymphocytes. It inserts nontemplated nucleotides (N-nucleotides) into the junctions between gene segments during T cell receptor and immunoglobulin heavy-chain gene rearrangement.

antigen. They constitute the final step in B cell maturation of the bone marrow and reside in peripheral lymphoid organs.

Vpre-B:  See pre-B cell receptor.

CD5  was initially described as an alloantigen termed Ly-1 on murine T cells. Subsequently a pan T cell marker of similar molecular mass was found on human lymphocytes using monoclonal antibodies. This was named CD5. Thus, CD5 is homologous at the DNA level with Ly-1. CD5 is a 67-kDa type I transmembrane glycoprotein comprised of a single polypeptide of approximately 470 amino acids. The signal peptide is formed by the first 25 amino acids. CD5 is expressed on the surface of all αβ T cells but is absent or of low density on γδ T cells. It has been discovered on many murine B cell lymphomas as well as on endothelial cells of blood vessels in the pregnant sheep uterus. CD72 on B cells is one of its three ligands. CD5 is present on thymocytes and most peripheral T cells. It is believed to be significant for the activation of T cells and possibly B1 cells. CD5 is also present on a subpopulation of B cells termed B1 cells that synthesize polyreactive and autoreactive antibodies as well as the “natural antibodies” present in normal serum. Human chronic lymphocytic leukemia cells express CD5, which points to their derivation from this particular B cell subpopulation.

Vpre-B and 𝛌5  are proteins produced at the early pro-B cell stage of B cell development that are required for regulation of μ chain expression on the cell surface. Vpre-B and λ5 take the place of immunoglobulin light chains in pre-B cells. These proteins have an important role in B cell differentiation. B cells  (Figure 6.4) are lymphocytes that are derived from the fetal liver in the early embryonal stages of development and from the bone marrow thereafter. Plasma cells that synthesize antibody develop from precursor B cells. An immature B lymphocyte  is a B cell that has rearranged a heavy- and a light-chain variable-region gene and expresses surface IGM, but not surface IgD. It arises from bone marrow precursors that fail to proliferate or differentiate following exposure to antigen. By contrast, it undergoes apoptosis and is functionally unresponsive. This mechanism plays a role in the negative selection of B cells specific for self antigens in the bone marrow. A naïve B cell  is a mature lymphocyte that has exited the bone marrow but has not yet come into contact with the antigen for which it is specific. A mature B cell  is a B lymphocyte that expresses IgM and IgD and has become functionally capable of responding to

9–12 µm

Figure 6.4  B cell.

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Memory B cells  are long-lived antigen-specific B cells generated during exposure of naïve B lymphocytes to antigen in a primary immune response. Subsequent exposure to the antigen for which they are specific leads to reactivation and differentiation into plasma cells as a secondary or subsequent immune response. B-1 cells  are B lymphocytes that express the CD5 glycoprotein and synthesize antibodies of broad specificities. They comprise a minor population of B cells. Also called CD5 B cells.

CD5 B cells  constitute an atypical, self-renewing class of B lymphocytes that reside mainly in the peritoneal and pleural cavities in adults and which have a far less diverse receptor repertoire than do conventional B cells. Preprogenitor cells  comprise a pool of cells that represents a second step in the maturation of B cells and is induced by nonspecific environmental stimuli. They are the immature cells that by themselves are unable to mount an immune response but are the pool from which the specific responsive clones will be selected by the specific antigen. They are present both in the bone marrow and peripheral lymphoid organs such as the spleen, but in the latter they form a minor population. They are characterized by the presence of some surface markers, frequently doublet or triplet surface immunoglobulins, and are capable of being stimulated by selected activators. They are sometimes termed B1 cells. B lymphocytes  are lymphocytes of the B cell lineage that mature under the influence of the bursa of Fabricius in birds and in the bursa equivalent (bone marrow) in mammals.

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B cells occupy follicular areas in lymphoid tissues and account for 5 to 25% of all human blood lymphocytes that number 1000 to 2000 cells/mm3. They comprise most of the bone marrow lymphocytes, and one-third to one-half of the lymph node and spleen lymphocytes, but less than 1% of those in the thymus. Nonactivated B cells circulate through lymph nodes and spleen. They are concentrated in follicles and marginal zones around the follicles. Circulating B cells may interact and be activated by T cells at extrafollicular sites where the T cells are present in association with antigen-presenting dendritic cells. Activated B cells enter the follicles, proliferate, and displace resting cells. They form germinal centers and differentiate into both plasma cells that form antibody and long-lived memory B cells. Those B cells synthesizing antibodies provide defense against microorganisms including bacteria and viruses. Surface and cytoplasmic markers reveal the stage of development and function of lymphocytes in the B cell lineage. Pre-B cells contain cytoplasmic immunoglobulins, whereas mature B cells express surface immunoglobulin and complement receptors. B lymphocyte markers include CD9, CD19, CD20, CD24, Fc receptors, B1, BA-1, B4, and Ia. Refer also to B cell. Secondary lymphoid follicle:  Areas of secondary lymphoid tissues populated by proliferating B cells responding to antigen. B1a B cells (CD5)  are a small population of B cells that express CD5 but to a lesser degree than CD5 expression on T cells. CD5 is a negative regulator of T cell receptor signaling. CD5 participates in B cell receptor-induced apoptosis of B1a cells. B-2 cells  are B lymphocytes that fail to express the CD5 glycoprotein and synthesize antibodies of narrow specificities. They comprise most of the B cell population. Unprimed  refers to animals or cells that have not come into contact previously with a particular antigen. Tec kinase  is a family of src-like tyrosine kinases that have a role in activation of lymphocyte antigen receptors through activation of PLC-γ. Btk in B lymphocytes, which is mutated in X-linked agammaglobulinemia (XLA), human immunodeficiency disease, and ltk in T lymphocytes, are examples of other Tec kinases. Syk PTK  is a 72-kDa phosphotyrosine kinase found on B cells and myeloid cells that is homologous to the ZAP-70 PTK found on T cells and NK cells. Both Syk and ZAP-70 play roles in the functions of distinct antigen receptors. B-lymphocyte tolerance  refers to the immunologic nonreactivity of B lymphocytes induced by relatively large doses of antigen. It is of relatively short duration. By contrast, T cell tolerance requires less antigen and is of longer duration.

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Exclusive B cell tolerance leaves T cells immunoreactive and unaffected. B cell tolerance  is manifested as a decreased number  of antibody-secreting cells following antigenic stimulation, com­pared with a normal response. Hapten-specific tolerance can be induced by inoculation of deaggregated haptenated-gammaglobulins (Ig). Induction of tolerance requires membrane Ig crosslinking. Tolerance may have a duration of 2 months in B cells of the bone marrow and 6 to 8 months in T cells. Whereas prostaglandin E enhances tolerance induction, IL-1, LPS, or 8-bromoguanosine block tolerance instead of an immunogenic signal. Tolerant mice carry a normal complement of hapten-specific B cells. Tolerance is not attributable to a diminished number or isotype of antigen receptor. It has also been shown that the six normal activation events related to membrane Ig turnover and expression do not occur in tolerant B cells. Whereas tolerant B cells possess a limited capacity to proliferate, they fail to do so in response to antigen. Antigenic challenge of tolerant B cells induces them to enlarge and increase expression, yet they are apparently deficient in a physiologic signal required for progression into a proliferative stage. A B lymphocyte hybridoma  is a clone formed by the fusion of a B lymphocyte with a myeloma cell. Activated splenic B  lymphocytes from a specifically immune mouse are fused with myeloma cells by polyethylene glycol. Thereafter, the cells are plated in multi-well tissue culture plates containing HAT medium. The only surviving cells are the hybrids, since the myeloma cells employed are deficient in hypoxanthine-guanine phosphoribosyl transferse and fail to grow in HAT medium. Wells with hybridomas are screened for antibody synthesis. This is followed by cloning, which is carried out by limiting dilution or in soft agar. The hybridomas are maintained either in tissue culture or through inoculation into the peritoneal cavity of a mouse that corresponds genetically to the cell strain. The antibody-producing B lymphocyte confers specificity and the myeloma cell confers immortality upon the hybridoma. B lymphocyte hybridomas produce monoclonal antibodies. The B lymphocyte receptor  (Figure 6.5) is an immunoglobulin anchored to the B lymphocyte surface. Its combination with antigen leads to B lymphocyte division and differentiation into memory cells, lymphoblasts, and plasma cells. The

B lymphocyte receptor

Figure 6.5  B-lymphocyte receptor.

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B Lymphocyte Development and Immunoglobulin Genes

tyrosine kinases. Anti-Ig binding leads to their phosphorylation. Igαand Igβ are required for expression of IgM and IgD on the B cell surface. Disulfide bonds link Igαand Igβ pairs that are associated noncovalently with the membrane Ig cytoplasmic tail to form the B cell receptor complex (BRC). The Igαand Igβ cytoplasmic domains bear immunoreceptor tyrosine based activation motifs (ITAMs) that participate in early signaling when antigens activate B cells.

mIg

Ig–α Ig–β

Cytoplasm

Figure 6.6  Schematic representation of Ig on a cell membrane.

original antigen specificity of the immunoglobulin is maintained in the antibody molecules subsequently produced. B lymphocyte receptor immunoglobulins (Figure 6.6) are to be distinguished from those in the surrounding medium that adhere to the B cell surface through Fc receptors. See membrane immunoglobulin. A B cell antigen receptor  (Figure 6.7) is an antibody expressed on antigen reactive B cells that is similar to secreted antibody but is membrane-bound due to an extra domain at the Fc portion of the molecule. Upon antigen recognition by the membranebound immunoglobulin, noncovalently associated accessory molecules mediate transmembrane signaling to the B cell nucleus. The immunoglobulin and accessory molecule complex is similar in structure to the antigen receptor–CD3 complex of T lymphocytes. The cell surface membrane-bound immunoglobulin molecule serves as a receptor for antigen, together with two associated signal-transducing Igα/Igβ molecules. Igα and Igβ  are proteins on the B cell surface that are noncovalently associated with cell surface IgM and IgD. They link the B cell antigen-receptor complex to intracellular Cell membrane Ig-β L IgM-α H IgM µ chain

Igα/Igβ (CD79a/CD79b):  The Igα/Igβ heterodimer interacts with immunoglobulin heavy chains for signal transduction. In the pro-B cell stage, rearrangement of the immunoglobulin heavy-chain gene leads to expression of surface membrane immunoglobulin (mIgμ). mIgμ associates with Igα/ Igβ and surrogate light chain in pre-B cells or ordinary light chains in B cells to form the precursor B cell receptor and B cell receptor, respectively. Igαand Igβ are expressed before immunoglobulin heavy-chain gene rearrangement. They are products of mb-1 and B29 genes, respectively. Allelic exclusion is mediated through signal transduction via Igαand Igβ and depends on intact tyrosine residues. Igα/Igβ complex:  A B cell receptor complex accessory heterodimer needed to transduce intracellular signaling activated by mIg interaction with antigen. Igαand Igβ cytoplasmic tails contain ITAMs. Specificity  refers to the recognition by an antibody or a lymphocyte receptor of a specific epitope in the presence of other epitopes for which the antigen-binding site of the antibody or of the lymphoid cell receptor is specific. The B cell receptor (BCR) complex  is the antigen receptor of B cells, each of which makes a single type of immunoglobulin. The form of this immunoglobulin on the cell surface is the B cell receptor for the antigen of interest in addition to the membrane-bound immunoglobulin monomer of the intracellular signaling molecules that comprise the accessory Igα/ Igβ complex. Membrane-bound immunoglobulin (mIg)  is an immunoglobulin molecule on the surface that possesses a transmembrane region, extended C-terminal region and lacks a tail piece. It is the antigen-binding structure of the B cell receptor. mIg:  Abbreviation for membrane-bound immunoglobulin.

H IgM-α L Ig-β

Figure 6.7  B cell antigen receptor.

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A B cell coreceptor  (Figure 6.8) is a three-protein complex that consists of CR2, TAPA-1 (Figure  6.9), and CD19 that associates with the B cell receptor and facilitates its response to specific antigen. CR2 unites not only with an activated component of complement, but also with CD23. TAPA-1 is a serpentine membrane protein. The cytoplasmic tail of CD 19 is the mechanism through which the complex interacts with lyn, a tyrosine kinase. Activation of the coreceptor by ligand binding leads to union of phosphatidyl inositol-3′ kinase with

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CR2 S S

CD19

S lyn

S

TAPA-1 PI3 kinase

Figure 6.8  B cell coreceptor.

CD19 resulting in activation. This produces intracellular signals that facilitate B cell receptor signal transduction. TAPA-1  is a serpentine membrane protein that crosses the cell membrane four times. It is one of three proteins comprising the B cell coreceptor. It is also call CD81. CD19  (Figure  6.10) is an antigen with a 90-kDa mol wt that has been shown to be a transmembrane polypeptide with at least two immunoglobulin-like domains. The CD19 antigen is the most broadly expressed surface marker for B cells, appearing at the earliest stages of B cell differentiation. The CD19 antigen is expressed at all stages of B cell maturation, from the pro-B cell stage until just before the terminal differentiation to plasma cells. CD19 complexes with CD21 (CR2) and CD81 (TAPA-1). It is a coreceptor for B lymphocytes. CD20  (Figure 6.11) is a B cell marker with a 33-, 35-, and 37-kDa mol wt that appears relatively late in the B cell maturation (after the pro-B cell stage) and then persists for some time before the plasma cell stage. Its molecular structure resembles that of a transmembrane ion channel. The gene is on chromosome 11 at band q12-q13. It may be involved in regulating B cell activation.

CR1

CR2

CD19

Figure 6.10  CD19.

CD21  (Figure 6.12) is an antigen with a 145-kDa mol wt, that is expressed on B cells and, even more strongly, on follicular dendritic cells. It appears when surface Ig is expressed after the pre-B cell stage and is lost during early stages of terminal B cell differentiation to the final plasma cell stage. CD21 is coded for by a gene found on chromosome 1 at band q32. The antigen functions as a receptor for the C3d complement component and also for Epstein–Barr virus. CD21, together with CD19 and CD81, constitutes the coreceptor for B cells. It is also termed CR2. CD21  is a 145-kDa glycoprotein component of the B cell receptor. CD21 is a membrane molecule that participates in transmitting growth-promoting signals to the interior of the B cell. It is the receptor for the C3d fragment of the third component of complement, CR2. The CD21 antigen is a restricted B cell antigen expressed on mature B cells. It is present at high density on follicular dendritic cells (FDC), the accessory cells of the B zones. Also called complement receptor 2 (CR2). CD22  is a molecule with an α130- and β140-kDa mol wt that is expressed in the cytoplasm of B cells of the pro-B and pre-B cell stage and on the cell surface on mature B cells with surface Ig. The antigen is lost shortly before the terminal plasma cell phase. The molecule has five extracellular

iC3b

C3b

COOH

CR2

TAPA-1

Figure 6.9  Complement receptor complexes on the surface of B cells include CR1, C3b, CR2, CD19, iC3b, CR2 (CD21), and TAPA-1. B cell markers that are used routinely for immunophenotyping by flow cytometry include CD19, CD20, and CD21.

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NH2

COOH

Figure 6.11  CD20.

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C C C C C C C C C C C C C C C

Figure 6.12  CD21.

immunoglobulin domains and shows homology with myelin adhesion glycoprotein and with N-CAM (CD56). It participates in B cell adhesion to monocytes and T cells. Also called BL-CAM. The CD21 antigen  is a restricted B cell antigen expressed on mature B cells. The antigen is present at high denisty on follicular dendritic cells (FDC), the accessory cells of the B zones. It shows moderate labeling of B cells and a strong labeling of FDC in cryostat sections, whereas the staining of B cells is reduced or abolished in paraffin sections. However, the labeling of FDC in paraffin sections is as strong as on cryostat sections. A plasmablast  is an immature cell of the plasma cell lineage that reveals distinctive, clumped nuclear chromatin developing endoplasmic reticulum and a Golgi apparatus. It

Figure 6.14  Plasma cell in peripheral blood smear.

is a B lymphocyte in a lymph node that is beginning to reveal plasma cell features. It manifests increased rough endoplasmic reticulum, Golgi apparatus, and ribosomes. A plasmacyte  is a plasma cell. Plasma cells  (Figure 6.13 to Figure 6.15) are terminally differentiated antibody-producing B cells that fail to express MHC class II or mIg and are incapable of receiving further T cell help. Immunoglobulins are present in their cytoplasm and secretion of immunoglobulin by plasma cells has been directly demonstrated in vitro. Increased levels of immunoglobulins in some pathologic conditions are associated with increased numbers of plasma cells, and conversely, their number at antibodyproducing sites increases following immunization. Plasma cells develop from B cells and are large spherical or ellipsoidal cells, 10 to 20 μm in size. Mature plasma cells have abundant cytoplasm, staining deep blue with Wright’s stain, and have an eccentrically located round or oval nucleus, usually surrounded by a well-defined perinuclear clear zone. The nucleus contains coarse and clumped masses of chromatin, often arranged in a cartwheel fashion. The nuclei of normal, mature plasma cells have no nucleoli, but those of neoplastic plasma cells, such as those seen in multiple myeloma, have conspicuous nucleoli. The cytoplasm of normal plasma cells has conspicuous Golgi complex and rough endoplasmic reticulum and frequently contains

Plasma cell

Figure 6.13  Plasma cell diagram.

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Figure 6.15  Plasma cell cluster in peripheral blood smear.

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vacuoles. The nuclear to cytoplasmic ratio is 1:2. By electron microscopy, plasma cells show very abundant endoplasmic reticulum, indicating extensive and active protein synthesis. Plasma cells do not express surface immunoglobulin or complement receptors, which distinguishes them from B lymphocytes. Plasma cells that are short-lived differentiate quickly without manifesting isotype switching or somatic hypermutation.

Antigen

Activation B´ cell

Antibody-secreting cells  are differentiated B lymphocytes that synthesize the secretory form of immunoglobulin. Antibody-secreting cells result from antigen stimulation. They may be found in the lymph nodes, spleen, and bone marrow. A signal peptide  is the leader sequence, a small sequence of amino acids, that shepherds the heavy or light chain through the endoplasmic reticulum and is cleaved from the nascent chains prior to assembly of a completed immunoglobulin molecule. B cell activation  (Figure  6.16) follows antigen binding to membrane immunoglobulin molecules on B-lymphocyte surfaces. This interaction of antigen and membrane immunoglobulin may lead to two types of response. Either biochemical

IL-5 Proliferation

An example of plasma cell antigen  is a murine plasmacyte membrane alloantigen. It may be designated PC-1, PC-2. Pyroninophilic cells  are cells whose cytoplasm stains red with methyl green pyronin stain. This signifies large quantities of RNA in the cytoplasm, indicating active protein synthesis. For example, plasma cells or other protein-producing cells are pyroninophilic.

IL-4

B cell

B˝ cell

B˝ cell

IL-6

Differentiation Plasma cell

Plasma cell

Figure 6.16  B cell activation.

signals are conveyed to the cells via the B-lymphocyte antigen receptor leading to lymphocyte activation, or antigen is taken into endosomal vesicles where protein antigens are processed and resulting peptides presented at the B-lymphocyte surface to helper T  cells. With respect to B-lymphocyte antigen receptor signaling, the relatively short cytoplasmic tails of membrane IgM and IgD are unable to transduce signals caused by Ig clustering. Therefore, Igαand Igβ that are expressed on mature B cells is a noncovalent association with membrane Ig actually transduce signals (Figure 6.17). B cell activation:  B cell responses to antigen, whether T-independent or T-dependent, result in the conversion of small resting B cells to large lymphoblasts and then either into plasma cells that form specific antibody or into longlasting memory B cells. Thymic independent antigens such T Cell

CD4+

Naive T Cell

CD40L

CTLA-4

CD28

IL-2

(proliferation)

CTLA-4

IL-2Rα

IFN-γ IL-4 CD40L

(Cytokine activation) Naive B Cell

CD40

TCR IL-2

IFN-γ

LFA-1 ICAM-1

NK Cell B7-2

B7-2

Dendritic Cell

Activated B Cell

Antibody Producing B Cell

CD40L

CTLA-4* MHC41

sig ?

CD40

( = Antigen )

ICAM-1

(If antigen is very IFN-γ IL-4 abundant, very little IL-4 is IL-4 produced) IFN-γ

MCH41

*Delayed appearance of CTLA-4 followed by B7-ligation downregulates T cell activity

IgG

IgE

IgA

TGF-β

TH3 Cell

Figure 6.17  Hypothetical B7/CD40 pathway for B cell activation.

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as bacterial polysaccharides can activate B cells independently of T cells by crosslinking of the B cell receptor. By contrast, protein antigens usually require the intimate interaction of B cells with helper T cells. Antigen stimulation of the B cell receptor leads to endocytosis and degradation of the antigen captured by the B cell receptor. Peptides that result from degraded antigen are bound to MHC class II molecules and transported to the cell surface for presentation to T lymphocytes. T cells bearing a specific T cell receptor that recognizes the peptide-MHC complex presented on the B cell surface are activated. Activated T cells help B cells by either soluble mediators such as cytokines, i.e., IL-4, IL-5, and IL-6, or as membrane-bound stimulatory molecules such as CD40 ligand. In germinal centers, B cells are converted to large replicating centroblasts and then to nonreplicating centroblasts. In germinal centers, frequent Ig region mutations and the switch from IgM to IgG, IgA, or IgE production occur. Mutation increases the diversity of antigen-binding sites. Mutations that lead to loss of antigen binding cause the cells to die by apoptosis. The few cells to which mutation gives an immunoglobulin product that has high affinity for antigen are selected for survival. These antigen selected cells differentiate into plasma cells that produce antibody or into small long-lived memory B cells that enter the blood and lymphoid tissues. Virgin B cells  that have never interacted with antigen must have two separate types of signals to proliferate and differentiate. The antigen provides the first signal through interaction with surface membrane Ig molecules on specific B lymphocytes. Helper T cells and their lymphokines provide the second type of signal needed. Whereas polysaccharides and lipids, as nonprotein antigens, induce IgM antibody responses without antigen-specific T cell help, protein antigens, which are helper T cell dependent, lead to the production of immunoglobulin of more than one isotype and of high affinity in addition to immunologic memory. Igαand Igβ together with membrane Ig molecules constitute the B lymphocyte antigen receptor (BCR) complex. The BCR complex is a B lymphocyte surface multiprotein complex that identifies antigen and transduces activating signals into the cell. The BCR comprises membrane immunoglobulin, which binds antigen, and Igαand Igβ proteins that initiate signals. Lymphocyte antigen receptor complex:  Igα and Igβ function in B lymphocytes as CD3 and proteins do in T  cells. Required for signal transduction are immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of Igαand Igβ. Crosslinking of the B cell receptor complex by antigen leads to increased cell size and cytoplasmic ribonucleic acid with increased biosynthetic organelles including ribosomes as resting cells enter G1 stage in the cell cycle. Class II molecules and B7-2 and B7-1 costimulators show increased expression. B cells stimulated by antigen are then able to activate helper T cells. Expression of receptors for T cell cytokines increases, thereby facilitating the ability

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of antigen-specific B cells to receive T cell help. The effect of B cell receptor complex signaling on proliferation and differentiation depends in part on the type of antigen. Following activation as a result of combination with antigen, cell proliferation and differentiation are facilitated by interaction with helper T lymphocytes. Helper T cells must recognize antigen and there must be interaction between protein antigen-specific B cells and T lymphocytes for antibody to be formed. When B cells, acting as antigen-presenting cells, interact with helper T lymphocytes that are specific for the peptide being presented, there are numerous ligand–receptor interactions that facilitate transmission of signals to B cells that are required to generate a humoral immune response. Among these are B7 molecules: CD28 and CD40:CD40 ligand interactions. Cytokines play important roles in antibody production by switching from one heavy-chain isotype to another and by providing amplification mechanisms through augmentation of B lymphocyte proliferation and differentiation. Germinal centers are the sites of synthesis of antibodies of high affinity and of memory B cells. Clonotypic  is an adjective that defines the features of a specific B cell population’s receptors for antigens that are products of a single B-lymphocyte clone. Following release from the B cells, these antibodies should be very specific for antigen, have a restricted spectrotype, and should possess at least one unique private idiotypic determinant. Clonotypic may also be used to describe the features of a particular clone of T lymphocytes’ specific receptor for antigen with respect to idiotypic determinants, specificity for antigen, and receptor similarity from one daughter cell of the clone to another. Anti-B cell receptor idiotype antibodies  interact with antigenic determinants (idiotopes) at the variable N-terminus of the heavy and light chains comprising the paratope region of an antibody molecule where the antigen-binding site is located. The idiotope antigenic determinants may be situated either within the cleft of the antigen-binding region or located on the periphery or outer edge of the variable region of heavy- and light-chain components. Antiidiotypic antibodies also block T cell receptors for antigens for which they are specific. Patching  (Figure 6.18) describes the accumulation of membrane receptor proteins cross-linked by antibodies or lectins on a lymphocyte surface prior to capping. The antigen–antibody Treatment with anti-Ig

Diffuse

Patch

Figure 6.18  Patching.

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Colocalization  is a mechanism of differential redistribution of membrane components into patches and caps, which has been employed to investigate possible interactions between various plasma membrane components or between the cell membrane and cytoplasmic structures.

Figure 6.19  Capping.

complexes are internalized following capping, which permits antigen processing and presentation in the context of MHC molecules. Membrane protein redistribution into patches is passive, not requiring energy. The process depends on the lateral diffusion of membrane constituents in the plane of the membrane. Capping  (Figure  6.19) refers to the migration of antigens on the cell surface to a cell pole following cross-linking of antigens by a specific antibody. These antigen–antibody complexes coalesce or aggregate into a “cap” produced by interaction of antigen with cell surface IgM and IgD molecules at sites distant from each other, as revealed by immunofluorescence. Capping is followed by interiorization of the antigen. Following internalization, the cell surface is left bereft of immunoglobulin receptors until they are reexpressed. The capping phenomenon  is the migration of surface membrane proteins toward one pole of a cell following crosslinking by a specific antibody, antigen, or mitogen. Bivalent or polyvalent ligands cause the surface molecules to aggregate into patches. This passive process is referred to as patching. The ligand–surface molecule aggregates, in patches, move to a pole of the cell where they form a cap. If a cell with patches becomes motile, the patches move to the rear, forming a cluster of surface molecule–ligand aggregates that constitutes a cap. The process of capping requires energy and may involve interaction with microfilaments of the cytoskeleton. In addition to capping in lymphocytes, the process occurs in numerous other cells. Cocapping:  If two molecules are associated in a membrane, capping of one induced by its ligand may lead also to capping of the associated molecule. Antibodies to membrane molecule x may induce capping of membrane molecule y as well as of x if x and y are associated in the membrane. In this example, the capping of the associated y molecule is termed cocapping.

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Clustering:  Monomeric antigens, monovalent lectins, and monovalent antibody to mIg or any other membrane component neither cap on the cell surface nor do they produce large clusters. Multivalent ligand binding is necessary for clustering as well as for capping. Clustering, unlike capping, is a passive redistribution process. Spotting or patching requires the cell to be neither living nor metabolically active. Clustering is affected not only by the factors that control phenomena occurring in a three-dimensional fluid aqueous phase but also by physicochemical properties of the plasma membrane. The outcome of cluster formation is influenced by physiological interactions between the membrane proteins themselves. Receptor-mediated endocytosis  is internalization into endosomes of cell surface receptor bound molecules such as B cell receptors to which antigens are bound and internalized. The interaction of a soluble macromolecule to its corresponding cell surface receptor leads to internalization through clathrin polymerization. When the clathrin-coated pit is invaginated, the receptor and bound macromolecule are enclosed in a clathrin-coated vesicle, which is subjected to the endocytic processing pathway. Immune response (Ir) genes  regulate immune responsiveness to synthetic polypeptide and protein antigens as demonstrated in guinea pigs and mice. This property is transmitted as an autosomal dominant trait that maps to the major histocompatibility complex (MHC) region. Ir genes control helper T-lymphocyte activation, which is required for the generation of antibodies against protein antigens. T lymphocytes with specific receptors for antigen fail to recognize free or soluble antigens, but they do recognize protein antigens noncovalently linked to products of MHC genes termed class I and class  II MHC molecules. The failure of certain animal strains to respond may be due to ineffective antigen presentation in which processed antigen fails to bind properly to class II MHC molecules or due to an ineffective interaction between the T cell receptor and the MHC class II-antigen complex. Immunoglobulin genes  (Figure  6.20) encode heavy and light polypeptide chains of antibody molecules and are found on different chromosomes, i.e., chromosome 14 for heavy-chain, chromosome 2 for κ light chain, and chromosome 22 for λ light chain. The DNA of the majority of cells does not contain one gene that encodes a complete immunoglobulin heavy or light polypeptide chain. Separate gene segments that are widely distributed in somatic cells and germ cells come together to form these genes. In B cells, gene rearrangement leads to the creation of an antibody

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Gamma globulin (Immunoglobulins)

Beta globulin

Alpha-2-globulin

–

Alpha-1-globulin

Cathode

+ Albumin

Anode

Figure 6.20  Electrophoresis of serum protein showing the gammaglobulin region that contains the immunoglobulins.

gene that codes for a specific protein. Somatic gene rearrangement also occurs with the genes that encode T cell antigen receptors. Gene rearrangement of this type permits the great versatility of the immune system in recognizing a vast array of epitopes. Three forms of gene segments join to form an immunoglobulin light-chain gene. The three types include light-chain variable region (VL), joining (JL), and constant region (C L) gene segments. VH, JH, and CH as well as D (diversity) gene segments assemble to encode the heavy chain. Heavy and light chain genes have a closely similar organizational structure. There are 100 to 300 Vκ genes, five Jκ genes, and one Cκ gene on the κ locus of chromosome 2. There are 100 VH genes, 30 D genes, 6 JH genes, and 11 CH genes on the heavy-chain locus of chromosome 14. Several Vλ, six Jλ, and six Cλ genes are present on the λ locus of chromosome 22 in humans. VH and VL genes are classified as V gene families, depending on the sequence homology of their nucleotides or amino acids. Immunoglobulin gene superfamily:  See immunoglobulin superfamily. J exon  is a DNA sequence that encodes part of the third hypervariable region of a light or heavy chain located near the 5′ end of the κ, λ, and γ constant region genes. An intron separates the J exon from them. The J exon should not be confused with the J  chain. The H constant region gene is associated with several J exons. The V region gene is translocated to a site just 5′ to one of the J exons during stem cell differentiation to a lymphocyte. A J gene segment  is a DNA sequence that codes for the carboxy terminal 12 to 21 residues of T lymphocyte receptor or immunoglobulin polypeptide chain variable regions. Through gene rearrangement, a J gene segment unites either a V or a D gene segment to intron 5′ of the C gene segment. The J region  is the variable part of a polypeptide chain, comprising a T lymphocyte receptor or immunoglobulin that a J gene segment encodes. The J region of an immunoglobulin light chain is comprised of the third hypervariable region carboxy terminal (1 or 2 residues) and the fourth framework region (12 to 13 residues). The J region of an immunoglobulin

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heavy chain is comprised of the third hypervariable region carboxy terminal portion and the fourth framework region (15 to 20 residues). The heavy chain’s J region is slightly longer than that of the light chain. The variable region carboxy terminal portion represents the J region of the T cell receptor. Junctional diversity:  When gene segments join imprecisely, the amino acid sequence may vary and affect variable region expression. This can alter codons at gene segment junctions. These include the V-J junction of the genes encoding immunoglobulin κ and λ light chains and the V-D, D-J, and D-D junctions of genes encoding immunoglobulin heavy chains, or the genes encoding T cell receptor β and δ chains. P-nucleotides:  Palindromic or p-nucleotides are short inverted repeat nucleotide sequences in VDJ junctions of rearranged immunoglobulin and T cell receptor genes. They are generated from a hairpin intermediate during recombination and contribute to junctional diversity of antigen receptors. Nicking of hairpin loop joining gene segments undergoing V(D J recombination in the intervening DNA instead of at the precise ends of the coding sequences, a recessed strand end and an overhang are formed. The nucleotides that are added to fill in the spaces on both strands are referred to as P nucleotides. The twelve/twenty-three (12/23) rule:  Immunoglobulin or T cell receptor gene segments can be joined only if one has a recognition signal sequence with a 12-bp spacer and the other has a 23-bp spacer. V(D)J recombination takes place only between gene segments whose apposition unites a 12-recognition signal sequence with a 23-recognition signal sequence. RAG recombinases only recognize gene segments with pairing of opposing types of these sequences. V(D)J recombination class switching  is a mechanism to generate multiple-binding specificities by developing lymphocytes through exon recombination from a conservative number of gene segments known as variable (V), joining (J), and diversity (D) gene segments at seven different loci that include μ, κ, and λ for B cell immunoglobulin genes, and α, β, γ, and δ genes for T cell receptors. RAG-1 and RAG-2:  Refer to recombination activating genes 1 and 2. V(D)J recombination  is the formation of unique variable (V) exons by site-specific recombination at the DNA level of pre-existing Ig and TCR loci of V, D, and J gene segments. Accomplished by the recognition, cutting and rejoining of the heptamer-nonamer recombination signal sequences (RSSs) that flank these gene segments. When three segments are involved, the D and J gene segments are linked first, followed by the joining of V to the DJ unit. Inversional joining  is an event that takes place during V(D) J recombination when the two gene segments to be united are in the opposite transcriptional orientation.

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Deletional joining  is an event during V(D)J recombination characterized by both gene segments to be brought into apposition have the same transcriptional orientation. V(J) recombination:  See class switching. RAG recombinases  are recombination activation gene recombinases. RAG-1 and RAG-2 are the main enzymes that facilitate V(D)J gene segment recombination in immunoglobulin and T cell receptor loci. Only developing B and T cells express the RAG genes. Recombination activating genes 1 and 2 (RAG-1 and RAG-2)  are genes that activate Ig gene recombination. Pre-B cells and immature T cells contain them. It remains to be determined whether RAG-1 and RAG-2 encode the recombinases or the regulatory proteins that control recombinase function. These genes encode two proteins, RAG-1 and RAG-2 that are requisite for rearrangements of both Ig and TCR genes. In the absence of these genes neither Ig nor T cell receptor proteins are produced, which blocks the production of mature T and B cells. Recombination recognition sequences  are DNA sequences situated adjacent to the V, D, and J segments in antigen receptor loci that are recognized by the RAG-1/RAG-2 component of V(D)J recombinase. The recognition sequences are comprised of a highly conserved seven nucleotide heptamer situated adjacent to the V, D, or J coding sequence, followed by a 12 or 23 nonconserved nucleotide spacer and a highly conserved nonnucleotide segment termed the nonamer. Productivity testing  is an assay on developing B or T cells of a specific V(D)J combination’s functionality by transcribing the newly constructed gene and translating its mRNA. Together with accessory proteins of the pre-B cell receptor or pre-T cell receptor, the chain of interest is expressed on the cell surface and sends a signal to the cell confirming successful recombination. A signal joint  is a structure produced by the precise joining of recognition signal sequences during somatic recombination that produces T cell receptor (TCR) and immunoglobulin (Ig) genes. The DNA sequence produced by uniting of blunt RSS ends after V(D)J recombination of gene segments. Somatic recombination  refers to DNA recombination whereby functional genes encoding variable regions of antigen receptors are produced during lymphocyte development. A limited number of inherited or germ-line DNA sequences that are first separated from each other are assembled together by enzymatic deletion of intervening sequences and religation. This takes place only in developing B or T cells. Also called somatic rearrangement. Germ-line configuration  refers to the arrangement of immunoglobulin and T cell receptor genes in the DNA of

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germ cells and in almost all somatic cells in which somatic recombination has not taken place. Unproductive rearrangements  are DNA rearrangements of T cell receptor and immunoglobulin genes that produce a gene incapable of encoding a functional polypeptide chain. Switch recombination  is an immunoglobulin isotype switching mechanism. Based on pairing of switch regions located 5′ of each CH exon (other than Cs). The CH switch region, an original component of the V(D)J-C gene, couples with the downstream CH exon’s switch region leading to excision of the intervening CH exons. This is followed by union of the V(D)J exon with the new CH exon. Selected cyto­kines, including TGFβ, INFγ, or IL-4 and germline transcripts govern CH exon selection. A V gene  encodes the variable region of immunoglobulin light or heavy chains. Although it is not in proximity to the C gene in germ-line DNA, in lymphocyte and plasma cell DNA the V gene lies near the 5′ end of the C gene from which it is separated by a single intron. A V gene segment  is a DNA segment encoding the first 95 to 100 amino acid residues of immunoglobulin and T cell polypeptide chain variable regions. There are two coding regions in the V gene segment which are separated by a 100to 400-bp intron. The first 5′ coding region is an exon that codes for a brief untranslated mRNA region and for the first 15 to 18 signal peptide residues. The second 3′ coding region is part of an exon that codes for the terminal 4 signal peptide residues and 95 to 100 variable region residues. A J gene segment encodes the rest of the variable region. A D gene segment is involved in the encoding of immunoglobulin heavy chain and T cell receptor β and δ chains. V(D)J recombinase  is an enzyme required to recombine VDJ segments. The enzyme is able to identify and splice the V (variable), J (joining), and in some cases D (diverse) gene segments that confer antibody diversity. This collection of enzymes makes possible the somatic recombination events that produce functional antigen receptor genes in developing T and B lymphocytes. Some that include RAG-1 and RAG-2 are present only in developing lymphocytes, whereas others are ubiquitous DNA repair enzymes. A vector  is a DNA segment employed for cloning a foreign DNA fragment. A vector should be able to reproduce autonomously in the host cell, possess one or more selectable markers, and have sites for restriction endonucleases in nonessential regions that permit DNA insertion into the vector or replacement of a segment of the vector. Plasmids and bacteriophages may serve as cloning vectors. In allelic exclusion  only one of two genes for which the animal is heterozygous is expressed, whereas the remaining gene is not. Immunoglobulin genes manifest this

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phenomenon. Allelic exclusion accounts for the ability of a B cell to express only one immunoglobulin or the capacity of a T cell to express a T cell receptor of a single specificity. Investigations of allotypes in rabbits established that individual immunoglobulin molecules have identical heavy chains and light chains. Immunoglobulin-synthesizing cells produce only a single class of H chain and one type of light chain at a time. Thus, by allelic exclusion, a cell that is synthesizing antibody expresses just one of two alleles encoding an immunoglobulin chain at a particular locus. The synthesis of a functional μ heavy chain from the IgH locus on one chromosome blocks more V(D)J recombination and μ chain synthesis from the other IgH allele. Allelic exclusion (TCR locus):  The synthesis of a functional TCR β chain from one chromosome’s TCR β locus prevents more V(D)J recombination and synthesis of TCR β chains from the other TCR β allele and V(D)J recombination in the TCR γ locus on both chromosomes. An enhancer  is a segment of DNA containing a group of DNA-binding motifs that can elevate the amount of RNA a cell produces. This DNA sequence activates the beginning of RNA polymerase II transcription from a promoter. Although initially described in the DNA tumor virus SV40, enhancers have now been demonstrated in immunoglobulin μ and κ genes’ J-C intron. Immunoglobulin enhancers function well in B cells, presumably due to precise regulatory proteins that communicate with the enhancer region. Gene rearrangement  refers to genetic shuffling that results in elimination of introns and the joining of exons to produce mRNA. Gene rearrangement within a lymphocyte signifies its dedication to the formation of a single cell type, which may be immunoglobulin synthesis by B lymphocytes or production of a β-chain receptor by T lymphocytes. Neoplastic transformation of lymphocytes may be followed by the expansion of a single clone of cells, which is detectable by Southern blotting. Gene segments  are multiple short DNA sequences in immunoglobulin and T cell receptor genes, which can undergo rearrangements in many different combinations to yield a vast diversity of immunoglobulin or T cell receptor polypeptide chains. One-turn recombination signal sequences  are immunoglobulin gene-recombination signal sequences separated by intervening sequence of 12 bp. The Pax-5 gene  is DNA that encodes B cell specific activator protein (BSAP), which is required as a transcription factor for B lymphocyte development. The one gene, one enzyme theory (historical)  was an earlier hypothesis which proposed that one gene encodes one enzyme or other protein. Although basically true, it is now

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known that one gene encodes a single polypeptide chain, and it is necessary to splice out mRNA introns comprised of junk DNA before mRNA can be translated into a protein. Receptor editing  is the replacement of a light chain or a heavy chain of a self-reactive antigen receptor on an immature B cell with a light chain or heavy chain that does not confer autoreactivity. It is a mechanism whereby rearranged genes in B lineage cells may undergo secondary rearrangement forming different antigenic specificities. This process involves RAG gene reactivation, additional light chain VJ recombinations, and new Ig light chain synthesis, which permits the cell to express a different immunoglobulin receptor that does not react with self. A B cell whose receptors react with self antigen during development in the bone marrow are provided a narrow window of opportunity to rearrange its light-chain gene to prevent apoptosis through alteration of its specificity for antigen as the new light chain replaces the selfreactive one. Phage antibody library:  This is a library of cloned antibody variable region gene sequences that may be expressed as Fab or svFv fusion proteins with bacteriophage coat proteins. These can be exhibited on the phage surface. The phage particle contains the gene encoding a monoclonal recombinant antibody and can be selected from the library by binding of the phage to specific antigen. P-addition  is the appending of nucleotides from cleaved hairpin loops produced by the junction of V-D or D-J gene segments during rearrangements of immunoglobulin or T cell receptor genes. D gene region  is the diversity region of the genome that encodes heavy-chain sequences in the immunoglobulin H chain hypervariable region. D gene  is a small segment of immunoglubulin heavy-chain and T cell receptor DNA that encodes the third hypervariable region of most receptors. The D region  is a segment of an immunoglobulin heavychain variable region or the β or δ chain of the T lymphocyte receptor coded for by a D gene segment. A few residues constitute the D region in the third hypervariable region in most heavy chains of immunoglobulins. The D or diversity region governs antibody specificity and probably T cell receptor specificity as well. A D gene segment  is the DNA region that codes for the D or diversity portion of an immunoglobulin heavy chain or a T lymphocyte receptor β or δ chain. It is the segment that encodes the third hypervariable region situated between the chain regions which the V gene segment and J gene segment encode. This part of the heavy-chain variable region is frequently significant in determining antibody specificity.

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Diversity (D) gene segments  are abbreviated coding sequences between the (V) and constant gene segments in the immunoglubulin heavy chain and T cell receptor β and δ loci together with J segments are recombined somatically with V segments during lymphocyte development. The recombined VDJ DNA encodes the carboxy terminal ends of the antigen receptor V regions, including the third hypervariable (CDR) regions. D segments used randomly contribute to antigen receptor repertoire diversity. D exon  is a DNA sequence that encodes a portion of the immunoglobulin heavy chain’s third hypervariable region. It is situated on the 5′ side of J exons. An intron lies between them. During lymphocyte differentiation, V-D-J sequences are produced that encode the complete variable region of the heavy chain. E2A  is a transcription factor, critical for B lymphocyte development, that is necessary for recombinase-activating gene (RAG) expression and for lambda 5 pre-B cell component expression in B cell development. Also called CD62E. Combinatorial joining  is a mechanism for one exon to unite alternatively with several other gene regions, increasing the diversity of products encoded by the gene. Combinatorial diversity  refers to the numerous different combinations of variable, diversity, and joining segments that are possible as a consequence of somatic recombination of DNA in the immunoglubulin and TCR loci during B cell or T cell development. It serves as a mechanism for generating large numbers of different antigen receptor genes from a limited number of DNA gene segments. A coding joint  is a structure formed when a V gene segment joins imprecisely to a (D)J gene segment in immunoglobulin or T cell receptor genes. A mutation  is a structural change in a gene that leads to a sudden and stable alteration in the genotype of a cell, virus, or organism. It is a heritable change in the genome of a cell, a virus, or an organism apart from that induced through the incorporation of “foreign” DNA. It represents an alteration in DNA’s base sequence. Germ cell mutations may be inherited by future generations, whereas somatic cell mutations are inherited only by the progeny of that cell produced through mitotic division. A point mutation is an alteration in a single base pair. Mutations in chromosomes may be expressed as translocation, deletion, inversion, or duplication. Mutant  is an adjective that describes a mutation that may have occurred in a gene, protein, or cell. The N region  is a brief segment of an immunoglobulin molecule’s or T cell receptor chain’s variable region that

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is not encoded by germ-line genes but instead by brief nucleotide (N) insertions at recombinational junctions. These N-nucleotides may be present both 3′ and 5′ to the rearranged immunoglobulin heavy chain’s D gene segment as well as at the V-J, V-D-J, and D-D junctions of the variable region genes of the T lymphocyte receptor. N-region diversification:  In junction diversity, this is the addition at random of nucleotides that are not present in the genomic sequence at VD, DJ, and VJ junctions. TdT catalyzes N-region diversification, which takes place in TCR αand β genes and in Ig heavy chain genes but not in Ig light chain genes. N-addition:  To append nucleotides by TdT during D-J joining or V to DJ joining. N-nucleotides  are nontemplated nucleotides that TdT adds to the 3′ cut ends of V, D, and J coding segments during rearrangement. They are added to junctions between V, D, and J gene segments in immunoglobulin or T cell receptor genes during lymphocyte development. When as many as 20 of these nucleotides are added, the diversity of the antibody and T cell receptor repertoires is expanded. Immunoglobulin  is the product of a mature B cell product synthesized in response to stimulation by an antigen. Antibody molecules are immunoglobulins of defined specificity produced by plasma cells. The immunoglobulin molecule consists of heavy (H) and light (L) chains fastened together by disulfide bonds. The molecules are subdivided into classes and subclasses based on the antigenic specificity of the heavy chains. Heavy chains are designated by lowercase Greek letters (μ, γ, α, δ, and ε), and the immunoglobulins are designated IgM, IgG, IgA, IgD, and IgE, respectively. The three major classes are IgG, IgM, and IgA, and the two minor classes are IgD and IgE, which together comprise less than 1% of the total immunoglobulins. The two types of light chains (termed κ and λ) are present in all five immunoglobulin classes, although only one type is present in an individual molecule. I gG, IgD, and IgE have two H and two L polypeptide chains, whereas IgM and IgA consist of multimers of this basic chain structure. Disulfide bridges and noncovalent forces stabilize immunoglobulin structure. The basic monomeric unit is Y shaped, with a hinge region rich in proline and susceptible to cleavage by proteolytic enzymes. Both H and L chains have a constant region at the carboxy terminus and a variable region at the amino terminus. The two heavy chains are alike, as are the two light chains in any individual immunoglobulin molecule. Approximately 60% of human immunoglobulin molecules have κ light chains and 40% have λ light chains. The five immunoglobulin classes are termed isotypes based on the heavy-chain specificity of each immunoglobulin class. Two immunoglobulin classes, IgA and IgG, have been further subdivided into subclasses based on H-chain differences. There are four IgG subclasses, designated IgG1

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B Lymphocyte Development and Immunoglobulin Genes

through IgG4, and two IgA subclasses, designated IgA1 and IgA2.  igestion of IgG molecules with papain yields two D Fab and one Fc fragments. Each Fab fragment has one antigen-binding site. By contrast, the Fc fragment has no antigen-binding site but is responsible for fixation of complement and attachment of the molecule to a cell surface. Pepsin cleaves the molecule toward the carboxy terminal end of the central disulfide bond, yielding an F(ab’)2 fragment and a pFc’ fragment. F(ab’)2 fragments have two antigen-binding sites. L chains have a single variable and constant domain, whereas H chains possess one variable and three to four constant domains.  ecretory IgA is found in body secretions such as saliva, S milk, and intestinal and bronchial secretions. IgD and IgM are present as membrane-bound immunoglobulins on B cells, where they interact with antigen to activate B cells. IgE is associated with anaphylaxis, and IgG, which is the only immunoglobulin capable of crossing the placenta, is the major human immunoglobulin. C gene:  DNA encodes the constant region of immunoglobulin heavy and light polypeptide chains. The heavy-chain C gene is comprised of exons that encode the heavy chain’s different homology regions. C gene segment:  DNA encodes for a T cell receptor or an immunoglobulin polypeptide chain constant region. One or more exons may be involved. Constant region gene segments comprise immunoglobulin and T cell receptor gene loci DNA sequences that encode TCR αand β chains and nonvariable regions of immunoglobulin heavy and light polypeptide chains. C segment  is an exon that encodes an immunoglobulin molecule’s constant region domain. Constant exon:  An exon that encodes for the C-terminal part of either an immunoglobulin or a T cell receptor protein. The splicing of a C exon at the mRNA level to a rearranged variable (V) exon yields a transcript of a complete immunoglobulin or T cell receptor gene. C exons of the immunoglobulin heavy chain gene locus are comprised of subexons. V gene:  Gene encoding the variable region of immunoglobulin light or heavy chains. Although it is not in proximity to the C gene in germ-line DNA, in lymphocyte and plasma-cell DNA the V gene lies near the 5′ end of the C gene from which it is separated by a single intron. V gene segment:  DNA segment encoding the first 95 to 100 amino acid residues of immunoglobulin and T cell polypeptide chain variable regions. The two coding regions in the V gene segment are separated by a 100- to 400-bp intron. The first 5′ coding region is an exon that codes for a brief

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untranslated mRNA region and for the first 15 to 18 signal peptide residues. The second 3′ coding region is part of an exon that codes for the terminal 4 signal peptide residues and 95 to 100 variable region residues. A J gene segment encodes the rest of the variable region. A D gene segment is involved in the encoding of immunoglobulin heavy chain and T cell receptor β and δ chains. Membrane immunoglobulin  is cell surface immunoglobulin that serves as an antigen receptor. Virgin B  cells contain surface membrane IgM and IgD molecules. Following activation by antigen, the B cell differentiates into a plasma cell that secretes IgM molecules. Whereas membrane-bound IgM is a four-polypeptide chain monomer, the secreted IgM is a pentameric molecule containing five four-chain unit monomers and one J chain. Other immunoglobulin classes have membrane and secreted types. IgG and IgA membrane immunoglobulins probably serve as memory B cell antigen receptors. That segment of the immunoglobulin introduced into the cell membrane is a hydrophobic heavy-chain region in the vicinity of the carboxy terminus. Within a particular isotype, the heavy chain is of greater length in the membrane form than in the secreted molecule. This greater length is at the carboxy terminal end of the membrane form. Separate mRNA molecules from one gene encode the membrane and secreted forms of heavy chain. Surface immunoglobulin:  All immunoglobulin isotypes may be expressed on the surface of individual B  cells, but only one isotype is expressed at any one time with the exception of unstimulated, mature B lymphocytes which coexpress surface IgM (sIgM) and surface IgD (sIgD). See B lymphocyte receptor. Surrogate light chains  are invariant light chains that are structurally homologous to kappa and lambda light chains and associate with pre-B cell μ heavy chains. They are the same in all B cells. V regions are absent in surrogate light chains. Low levels of cell surface μ-chain and surrogate light-chain complexes are believed to participate in stimulation of kappa or lambda light-chain synthesis and maturation of B cells. This complex of two nonrearranging polypeptide chains (V pre-B and λ5) synthesized by pro-B cells associate with Igμ heavy chain to form the pre-B cell receptor. B cell mitogens  are substances that induce B cell division and proliferation. Protein A  is a Staphylococcus aureus bacterial cell wall protein comprised of a solitary polypeptide chain whose binding sites manifest affinity for the Fc region of IgG. It combines with IgG1, IgG2, and IgG4, but not with the IgG3 subclasses in humans, the IgG subclasses of mice, or the IgG of rabbits and certain other species. It has been used extensively for the isolation of IgG during protein purification and for the protection of IgG in immune (antigen–antibody) complexes. Protein A is antiphagocytic, a property that may be linked to

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its ability to bind an opsonizing antibody’s Fc region. Protein A has been postulated to facilitate escape from the immune response by masking its epitopes with immunoglobulins. It is used in immunology for mitogenic stimulation of human B lymphocytes, investigation of lymphocyte Fc receptors, and agglutination tests, as well as in detection and purification of immunoglobulins by the ELISA technique. B cell-specific activator protein (BSAP)  is a transcription factor that has an essential role in early and later stages of B cell development. It is encoded by the gene Pax-5.

Atlas of Immunology, Third Edition

and induces Bcl-2 expression. Autoimmunity develops in mice when BAFF is overexpressed, and elevated serum level of BAFF have been described in human autoimmune diseases. It is produced mainly by monocytes, macrophages, and dendritic cells but not by B cells. Pro-inflammatory stimuli stimulate its expression by macrophages. It occurs as both a homotrimeric transmembrane molecule and in a soluble homotrimeric form. It binds to three receptors, BAFF-R, TACI and BCMA. BCDF  is B cell differentiation factors.

B lymphocyte stimulatory factors  include interleukins 4, 5, and 6.

BCGF (B cell growth factors)  include interleukins 4, 5, and 6.

B cell differentiation and growth factors  are T lymphocytederived substances that promote differentiation of B lymphocytes into antibody producing cells. They can facilitate the growth and differentiation of B cells in vitro. Interleukins 4, 5, and 6 belong in this category of factors.

AtxBm  is the abbreviation for a so-called B cell mouse, which refers to a thymectomized irradiated adult mouse that has received a bone marrow transplant.

B cell tyrosine kinase (Btk)  is an src-family tyrosine kinase that plays a critical role in the maturation of B lymphocytes. Btk gene mutations lead to cross-linked agammaglobulinemia in which B cell maturation is halted at the pre-B stage. Early B cell factor (EBF)  is a transcription factor required for early B cell development and for RAG expression. EBF (early B cell factor)  is a transcription factor required for early B cell development and for RAG expression. Inositol 1, 4, 5-triphosphate (IP3)  is a signaling molecule in the cytoplasm of lymphocytes activated by antigen that is formed by phospholipase C (PLC γ)-mediated hydrolysis of the plasma membrane. Phospholipid PIP2. IP3’s principle function is to induce the release of intracellular Ca2+ from membrane-bound compartments such as the endoplasmic reticulum (ER). LPS  is the abbreviation for lipopolysaccharide, which may serve as an endotoxin. It is a constituent of Gram-negative bacterial cell walls associated with endotoxin and may lead to endotoxin shock. LPS is used as a polyclonal activator of murine B cells, causing them to divide and differentiate into antibody-forming plasma cells. LPS-binding protein (LBP)  is a substance that can bind a bacterial lypopolysaccharide (LPS) molecule which enables it to interact with CD14 and LPS:LBP-binding protein on macrophages and selected other cells. BAFF  is a B lymphocyte activating factor that is a member of the TNF family. A survival factor needed for the maturation of splenic transitional T1 B cells into transitional T2 B cells. BAFF is necessary for the appearance of mature B cells in the periphery. BAFF signaling is believed to activate NF-qAκB

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Ly antigen  is a murine lymphocyte alloantigen that is expressed to different degrees on mouse T and B  lymphocytes and thymocytes. Also referred to as Lyt antigen. Lyb  is a murine B lymphocyte surface alloantigen. Ly1 B cell  is a murine B lymphocyte that expresses CD5 (Ly1) epitope on its surface. This cell population is increased in inbred strains of mice susceptible to autoimmune diseases, such as the New Zealand mouse strain. Lyb-3 antigen:  Mature murine B cells express a surface marker designated Lyb-3. It is a single membrane-bound 68-kDa polypeptide. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), it appears distinct from the SIg chains δ and μ. It does not contain disulfide bridges. The gene coding for Lyb-3 appears cross-linked and recessive, and mutant mice lacking Lyb-3 antigens are known. Lyb-3 is involved in the cooperation between T and B  cells in response to thymus-dependent antigens and seems to be manifested particularly when the amount of antigen used for immunization is suboptimal. The number of cells carrying Lyb-3 increases with the age of the animal. Ly6  are GPI linked murine cell surface alloantigens found most often on T and B cells but found also on nonlymphoid tissues such as brain, kidney, and heart. Monoclonal antibodies to these antigens indicate TCR dependence. Abelson murine leukemia virus (A-MuLV)  is a B cell murine leukemia-inducing retrovirus that bears the v-abl oncogene. The virus has been used to immortalize immature B lymphocytes to produce pre-B  cell or less differentiated B cell lines in culture. These have been useful in unraveling the nature of immunoglobulin differentiation such as H and L chain immunoglobulin gene assembly, as well as class switching of immunoglobulin.

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B Lymphocyte Development and Immunoglobulin Genes

The Bcl-2  family of proteins consists of proteins which share homology to Bcl-2 in one or more of the Bcl-2 homology regions designated BH1, BH2, BH3, and BH4. Many of the family members have a carboxyl-terminal mitochondrial membrane targeting sequence. All have two central membrane-spanning helices, which are surrounded by additional amphipathic helices. X-ray crystallographic studies have shown that Bcl-X L has structural similarity to diphtheria toxin and colicins. Diphtheria toxin is endocytosed by cells. Acidification of the endosome induces a conformational change in diphtheria toxin that triggers membrane insertion of the membrane spanning domains. Pore formation by dimerization is thought to occur and the toxic subunit diphtheria toxin is translocated from the endosomal lumen into the cytosol. It is clear from reconstitution assays that many members of the Bcl-2 family can form a pore which allows passage of ions or elicit the release of cytochrome c from isolated mitochondria. Passage of ions is more dramatic at low pH. The carboxylmitochondria targeting sequence is not required for in vitro pore formation. Bcl-2  is a 25-kDa human oncoprotein that is believed to play a regulatory role in tissue development and maintenance in higher organisms by preventing the apoptosis of specific cell types. Bcl-2’s inhibitory effect is influenced by the expression of other gene products such as bax, bcl-xs bak, and bad that promote apoptosis. Bcl-2 is situated at the outer membrane of mitochondria, the endoplasmic reticulum, and the nuclear membrane, and may prevent apoptosis either by acting at these locations or as an antioxidant that neutralizes the effects of reactive oxygen species that promote apoptosis or by obstructing mitochondrial channel openings, thereby preventing the release of factors that promote apoptosis. Bcl-2 is involved in the development of the adult immune system as demonstrated by studies with Bcl-2 knockout mice. Failure to induce normal levels of apoptosis due to overexpression of Bcl-2 may contribute to the development of lymphoproliferative disorders and acceleration of autoimmunity under the appropriate genetic background. The role of Bcl-2 in human SLE and PSS has not yet been fully defined.

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Bcl-2 proteins  regulate the rate at which apoptotic signaling events initiate or amplify caspase activity. Bcl-2 alters the apoptotic threshold of a cell rather than inhibiting a specific step in programmed cell death. Cells that overexpress Bcl-2 still carry out programmed cell death in response to a wide spectrum of apoptotic initiators. The dose of the initiator is greater than in the absence of Bcl-2. Several Bcl-2-related proteins have been identified. Whereas some Bcl-2 family members promote cell survival, others enhance the sensitivity of a cell to programmed cell death. Five homologs of Bcl-2 with antiapoptotic properties include Bcl-XL, Bcl-w, Mcl-l, NR-13, and A-1. By contrast, two proapoptotic members of the Bcl-2 family include Bax and Bak. Bcl-2 related proteins are present in the intracellular membranes including the endoplasmic reticulum, outer mitochondrial membrane, and outer nuclear membrane. Bcl-2 proteins are hypothesized to regulate membrane permeability. Bcl-XL  is a Bcl-2 related protein. It is upregulated through the action of the costimulatory molecules CD28 and CD40. Bcl-XL expression is claimed to prevent cell death in response to growth factor limitation or Fas signal transduction. Signal transduction through CD28 or CD40 induces Bcl-XL expression only in antigen-activated cells. Without antigen receptor engagement, CD40 engagement promotes cell death. Bcl-XL induction by either CD28 or CD40 is transient, persisting only 3 to 4 d. Protection of cells from death lasts only as long as Bcl-XL is expressed. E32  is a protein formed early in development of B lymphocytes that has a role in immunoglobulin heavy-chain transcription. Oct-2  is a protein formed early in the development of B lymphocytes that has a role in immunoglobulin heavy-chain transcription. Staphylococcal protein A  is a substance derived from the cell wall of Staphylococcus aureus that interacts with IgG1, IgG2, and IgG4 subclasses. It stimulates human B cell activation.

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7

Immunoglobulin Synthesis, Properties, Structure, and Function

Following enunciation of the clonal selection theory of antibody formation by Burnet in 1957, experimental evidence confirms the validity of this selective theory as opposed to the instructive theory of antibody formation that prevailed during the first half of the 20th century. As immunogeneticists attempted to explain the great diversity of antibodies encoded by finite quantities of DNA, Tonegawa offered a plausible explanation for the generation of antibody diversity in his studies of immunoglobulin gene C, V, J, and D regions and their rearrangement. It is necessary for those segments that encode genes and determine immunoglobulin H and L chains to undergo rearrangement prior to gene transcription and translation. Newly synthesized immunoglobulin molecules have different properties based on their immunoglobulin class or isotype. Nevertheless, antigen-binding specificities reside in the Fab regions of antibody molecules, which governs their interactions with antigens in vitro and in vivo. By contrast, complement binding and activation capabilities, binding to cell surface, and transport through cells reside in the Fc region of the molecule. The fate of immunoglobulin molecules also differs according to the immunoglobulin class, each with its own characteristic half-life. Only IgG is protected from catabolism by binding to a specific receptor. Some antibodies are protective, others cross the placenta from mother to fetus, whereas others participate in hypersensitivity reactions that lead to adverse effects in target tissues. Antibodies are a diverse and unique category of proteins whose antigen-binding diversity is expressed in the 1020 antibody molecules synthesized from the 1012 B lymphocytes found in the human body. Antibodies  are glycoprotein substances produced by B lymphoid lineage cells, termed plasma cells, in response to stimulation with an immunogen. They possess the ability to react in vitro and in vivo specifically and selectively with the antigenic determinants or epitopes eliciting their production or with an antigenic determinant closely related to the homologous antigen. Antibody molecules are immunoglobulins found in the blood and body fluids. Thus, all antibodies are immunoglobulins formed in response to immunogens. Antibodies may be produced by hybridoma technology in which antibody-secreting cells are fused by polyethylene glycol (PEG) treatment with a mutant myeloma cell line. Monoclonal antibodies are widely used in research and diagnostic medicine and have potential in therapy. Antibodies in the blood serum of any given animal species may be grouped according to their physicochemical properties and antigenic characteristics. Immunoglobulins are not restricted to the plasma but may be found in other body

fluids or tissues, such as urine, spinal fluid, lymph nodes, and spleen. Immunoglobulins do not include the components of the complement system. Immunoglobulins (antibodies) constitute approximately 1 to 2% of the total serum proteins in health. γ Globulins are serum proteins that show the lowest mobility toward the anode during electrophoresis when the pH is neutral. γ Globulins comprise 11.2 to 20.1% of the total serum content in man. Antibodies are in the γ globulin fraction of serum. Electrophoretically they are the slowest migrating fraction. Heteroclitic antibody  is an antibody with greater affinity for a heterologous epitope than for the homologous one that stimulated its synthesis. Heterocytotropic antibody  is an antibody that has a greater affinity when fixed to mast cells of a species other than the one in which the antibody is produced. Frequently assayed by skin-fixing ability, as revealed through the passive cutaneous anaphylaxis test. Interaction with the antigen for which these “fixed” antibodies are specific may lead to local heterocytotrophic anaphylaxis. Heterogenetic antibody:  See heterophile antibody. Heterophile antibody  is an antibody found in an animal of one species that can react with erythrocytes of a different and phylogenetically unrelated species. These are often IgM agglutinins. Heterophile antibodies are detected in infectious mononucleosis patients who demonstrate antibodies reactive with sheep erythrocytes. To differentiate this condition from serum sickness, which also is associated with a high titer of heterophile antibodies, the serum sample is absorbed with beef erythrocytes which contain Forssmann antigen. This treatment removes the heterophile antibody reactivity from the serum of infectious mononucleosis patients. High-titer, low-avidity antibodies (HTLA)  are antibodies that induce erythrocyte agglutination at high dilutions in the Coombs’ antiglobulin test. These antibodies cause only weak agglutination and are almost never linked to hemolysis of clinical importance. Examples of HTLA antibodies are antiBga, -Cs, -Ch, -Kna, -JMH, -Rg, and -Yk, among others. Homocytotrophic antibody  is an antibody that attaches better to animal cells of the same species in which it is produced than it does to animal cells of a different species. The term usually refers to an antibody that becomes fixed to mast cells 233

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of an animal of the same species, which results in anaphylaxis with the release of pharmacological mediators of immediate hypersensitivity. These include histamines and other vasoactive amines when the mast cells degranulate. Anti-allotypic antibodies are antibodies  specific for allotopes of an immunoglobulin molecule derived from a member of the same species.  nti-isotypic antibodies are antibodies generated in one speA cies specific for antigenic determinants found only on one immunoglobulin isotype of a different species, i.e., goat antihuman IgM antibodies.  polyreactive antibody is an immunoglobulin molecule that A identifies and unites with several different antigens of significantly different configuration with varying affinity.  olyclonal antibodies are multiple immunoglobulins respondP ing to different epitopes on an antigen molecule. This multiple stimulation leads to the expansion of several antibody-forming clones whose products represent a mixture of immunoglobulins in contrast to proliferation of a single clone which would yield a homogeneous monoclonal antibody product. Thus, polyclonal antibodies represent the natural consequence of an immune response in contrast to monoclonal antibodies, which occur in vivo in pathologic conditions such as multiple myeloma or are produced artificially by hybridoma technology against one of a variety of antigens. The immunoglobulin superfamily  (Figure  7.1) is comprised of several molecules that participate in the immune response and show similarities in structure, causing them to be named the immunoglobulin supergene family. Included are CD2, CD3, CD4, CD7, CD8, CD28, T cell receptor (TCR), MHC class I and MHC class II molecules, leukocyte function associated antigen 3 (LFA-3), the IgG receptor, and a dozen other proteins. These molecules share in common with each other an immunoglobulin-like domain with a length of approximately 100-amino acid residues and a central disulfide bond that anchors and stabilizes antiparallel β strands into a folded structure resembling immunoglobulin. Immunoglobulin superfamily members may share homology with constant or variable immunoglobulin domain regions. Various molecules of the cell surface with polypeptide chains whose folded structures are involved in cell-to-cell interactions belong in this category. Single gene and multigene members are included. Protein molecules sharing 15% amino acid homology with immunoglobulin proteins and possessing one or more immunoglobulin domains belong to this large molecular family. Globulins  are serum proteins comprised of α, β, and γ globulins and classified on the basis of their electrophoretic mobility. All three globulin fractions demonstrate anodic mobility that is less than that of albumin. α globulins have the greatest negative charge, whereas γ globulins have the

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least negative charge. Originally, globulins were characterized based on their insolubility in water, i.e., the euglobulins, or sparing solubility in water, i.e., the pseudoglobulins. Globulins are precipitated in half-saturated ammonium sulfate solution. γ globulin  is an obsolete designation for immunoglobulin. These serum proteins show the lowest mobility toward thev anode during electrophoresis when the pH is neutral. The γ globulin fraction contains immunoglobulins. It is the most cationic of the serum globulins. γ globulin fraction  is the electrophonic fraction of serum in which most of the immunoglobulin classes are found. Cohn fraction II  is, principally, gammaglobulin isolated by ethanol fractionation of serum by the method of Cohn. Antibody-binding site  is the antigen-binding site of an antibody molecule, known as a paratope, which is comprised of heavy chain and light chain variable regions. The paratope represents the site of attachment of an epitope to the antibody molecule. The complementarity-determining hypervariable regions play a significant role in dictating the combining site structure together with the participation of framework region residues. The T cell receptor also has an antigen-binding site in the variable regions of its α and β (or γ and δ) chains. Pyroglobulins  are monoclonal immunoglobulins that undergo irreversible precipitation upon heating to 56°C. These monoclonal immunoglobulins are usually detected during routine inactivation of complement in serum by heating to 56°C in a water bath. Whereas most immunoglobulins are unharmed at this temperature, pyroglobulins precipitate. This may be attributable to formation of hydrophobic bonds linking immunoglobulin molecules as a consequence of diminished heavy chain polarity. Half of the pyroglobulin positive subjects have multiple myeloma, and the remaining half have a lympho­proliferative disorder such as macroglobulinemia, carcinoma, or systemic lupus erythematosus. Their relevance to disease is unknown. Euglobulin  is a type of globulin that is insoluble in water but dissolves in salt solutions. In the past, it was used to designate that part of the serum proteins that could be precipitated by 33% saturated ammonium sulfate at 4°C or by 14.2% sodium sulfate at room temperature. Euglobulin is precipitated from the serum proteins at low ionic strength. Antibody specificity  is a property of antibodies determined by their relative binding affinities, the intrinsic capacity of each antibody combining site, expressed as equilibrium dissociation (Kd) or association (Ka) for their interactions with different antigens. Antiserum  is a preparation of serum containing antibodies specific for a particular antigen, i.e., immunogen. A therapeutic

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Immunoglobulin Synthesis, Properties, Structure, and Function

N N

V

N N

V C

TCR NN

C

β (γ) V

CC C

Class I MHC α

α V (δ)

C C

β2 m

C

N

C C

C C

Class II MHC N N α

C

β C

C

C

C C

CDR N CDR N

CD8 N N

CD3γ(δ, ε) N

? ?

α V

V β

Thy-1

H

C

C

C

C

p-IgR N

NCAM N

V V

H H

V

C

?

H

H

C

PDGFR N

H

H

V

C

H

H

V

FcRII N

C

N

?

C

C

Figure 7.1  Immunoglobulin superfamily.

antiserum may contain antitoxin, antilymphocyte antibodies, etc. An antiserum contains a heterogenous collection of antibodies that bind the antigen used for immunization. Each antibody has a specific structure, antigenic specificity, and cross-reactivity contributing to the heterogeneity that renders an antiserum unique. Polyclonal antiserum is serum  that possesses antibodies synthesized by numerous different B cell clones following stimulation by an antigen. Different epitopes on the antigen molecule stimulate this multiplicity of antibodies. Antiagglutinin  is a specific antibody that interferes with the action of an agglutinin.

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Antitoxin  is an antibody specific for exotoxins produced by certain microorganisms such as the causative agents of diphtheria and tetanus. Prior to the antibiotic era, antitoxins were the treatment of choice for diseases produced by the soluble toxic products of microorganisms, such as those from Corynebacterium diphtheriae and Clostridium tetani. Serum antitoxins are antibodies  specific for exotoxins produced by selected microorganisms, such as the causative agents of diphtheria and tetanus. Prior to the use of antibiotics, antitoxins were the treatment of choice for diseases produced by the exotoxin products of microorganisms,

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Idiotypic determinants (idiotopes)

separate from cellular immunity is useful in understanding and explaining biological mechanisms. Antibody detection:  Techniques employed to detect antibodies include immunoprecipitation, agglutination, complement-dependent assays, labeled antiimmunoglobulin reagents, blotting techniques, and immunohistochemistry. Enzyme-based immunoassays, blotting methods, and immunohistochemistry are routine procedures to detect antibodies and to characterize their specificity.

ss ss

Figure 7.2  Schematic representation of idiotypes present on an immunoglobulin molecule.

such as those from Corynebacterium diphtheriae and Clostridium tetani. Antibody repertoire  refers to all of the antibody specificities that an individual can synthesize.

Anti-DEX antibodies  are murine α1-3 dextran-specific antibodies. Antibody titer  is the amount or level of circulating antibody in a patient with an infectious disease. For example, the reciprocal of the highest dilution of serum (containing antibodies) that reacts with antigen, e.g., agglutination, is the titer. Two separate titer determinations are required to reflect an individual’s exposure to an infectious agent.

Amboceptor (historical):  Paul Ehrlich (circa 1900) considered antisheep red blood cell antibodies, known as amboceptors, to have one receptor for sheep erythrocytes and another receptor for complement. The term gained worldwide acceptance with the popularity of complement fixation tests for syphilis, such as the Wasserman reaction. The term is still used by some when discussing complement fixation.

Antitoxin assay (historical):  Antitoxins are assayed biologically by their capacity to neutralize homologous toxins as demonstrated by production of no toxic manifestations following inoculation of the mixture into experimental animals, e.g., guinea pigs. They may be tested serologically by their ability to flocculate (precipitate) toxin in vitro.

MAC-1  is a monoclonal antibody specific for macrophages.

Univalent antibody  is an antibody molecule with one antigen- binding site. Although incapable of leading to precipitation or agglutination, univalent antibodies or Fab fragments resulting from papain digestion of an IgG molecule might block precipitation of antigen by a typical bivalent antibody.

Humoral immunity  is immunity attributable to specific immunoglobulin antibody and present in the blood plasma, lymph, other body fluids, or tissues. The antibody may also adhere to cells in the form of cytophilic antibody. Antibody or immunoglobulin- mediated immunity acts in conjunction with complement proteins to produce either beneficial (protective) or pathogenic (hypersensitivity tissue-injuring) reactions. Antibodies that are the messengers of humoral immunity are derived from B cells. For purposes of discussion, it is separated from so-called cellular or T cell-mediated immunity, though the two cannot be clearly distinguished since antibodies and T cells often participate in immune reactions together. However, the classification of humoral Antigen binding sites Heavy chain Light chain

B-cell antigen receptor (membrane bound)

Plasma membrane

Antibody units:  See titer. Antitoxin unit:  A unit of antitoxin is that amount of antitoxin present in 1/6000 g of a certain dried unconcentrated horse serum antitoxin which has been maintained since 1905 at the National Institutes of Health in Bethesda, Maryland. The standard antitoxin unit contained sufficient antitoxin to neutralize 100 MLD of the special toxin prepared by Ehrlich and used by him in titration of standard antitoxin. Both the American and international units of antitoxin are the same. Combining site:  See antigen-binding site.

Antibody (secreted)

Figure 7.3  Schematic representation of an antigen receptor on the plasma membrane of a B cell.

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Univalent  is a single binding site.

An antigen-binding site  is the location on an antibody molecule where an antigenic determinant or epitope combines with it. The antigen-binding site is located in a cleft bordered by the N-terminal variable regions of heavy and light chain parts of the Fab region. Also called paratope. Also refers to that part of a T cell receptor that binds antigen specifically.

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An antivenom  is antitoxin prepared specifically for the treatment of bite or sting victims of poisonous snakes or arthropods. Antibodies in this immune serum preparation neutralize the snake or arthropod venom. Also called antivenin or antivenene.

accomplished by gene therapy. Intrabodies can be expressed within the cell in precise locations within the mammalian cells by modifying intrabody genes (in scFv or Fab format) with sequence-encoding classical intracellular trafficking signals.

Titer  is an approximation of the antibody activity in each unit volume of a serum sample. The term is used in serological reactions and is determined by preparing serial dilutions of antibody to which a constant amount of antigen is added. The end point is the highest dilution of antiserum in which a visible reaction with antigen, e.g., agglutination, can be detected. The titer is expressed as the reciprocal of the serum dilution which defines the end point. If agglutination occurs in the tube containing a 1:240 dilution, the antibody titer is said to be 240. Thus, the serum would contain approximately 240 units of antibody per milliliter of antiserum. The titer only provides an estimate of antibody activity. For absolute amounts of antibody, quantitative precipitation or other methods must be employed.

Cross-reacting antibody  reacts with epitopes on an antigen molecule different from the one that stimulated its synthesis. The effect is attributable to shared epitopes on the two antigen molecules.

A precipitating antibody  is a precipitin. A precipitin  is an antibody that interacts with a soluble antigen to yield an aggregate of antigen and antibody molecules in a lattice framework called a precipitate. Under appropriate conditions, the majority of antibodies can act as precipitins. Antibody synthesis:  The 1012 B lymphocytes that comprise the human immune system synthesize 1020 antibody (immunoglobulin) molecules present inside and on the surface of these cells and most of all in the serum. Other species have B cell and immunoglobulin molecule numbers relative to their body weight. B cells and immunoglobulin molecules are formed and degraded throughout the human lifespan. A paratope  is the antigen-binding site of an antibody molecule, the variable (V) domain, or T cell receptor that binds to an epitope on an antigen. It is the variable or Fv region of an antibody molecule and is the site for interaction with an epitope of an antigen molecule. It is complementary for the epitope for which it is specific.  paratope is the portion of an antibody molecule where the A hypervariable regions are located. There is less than 10% variability in the light and heavy chain amino acid positions in the variable regions. However, there is 20 to 60% variability in amino acid sequence in the so-called “hot spots” located at light chain amino acid positions 29 to 34, 49 to 52, and 91 to 95, and at heavy chain positions 30 to 34, 51 to 63, 84 to 90, and 101 to 110. Great specificity is associated with this variability and is the basis of an idiotype. This variability permits recognition of multiple antigenic determinants. Intrabody  is intracellular antibody that binds key targets to inhibit tumor growth. It is postulated that this might be

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Natural autoantibodies  are polyreactive antibodies of low affinity that are synthesized by CD5+ B cells that comprise 10 to 25% of circulating B lymphocytes in normal individuals, 27 to 52% in those with rheumatoid arthritis, and less than 25% in systemic lupus erythematosus patients. Natural autoantibodies, they may appear in first degree relatives of autoimmune disease patients as well as in older individuals. They may be predictive of disease in healthy subjects. They are often present in patients with bacterial, viral, or parasitic infections and may have a protective effect. In contrast to natural autoantibodies, they may increase in disease and may lead to tissue injury. The blood group isohemagglutins are also termed natural antibodies even though they are believed to be of heterogenetic immune origin as a consequence of stimulation by microbial antigens. Cytophilic antibody  (1) An antibody that attaches to a cell surface through its Fc region. It binds to Fc receptors on the cell surface. For example, IgE molecules bind to the surface of mast cells and basophils in this manner. Murine IgG1, IgG2a, and IgG3 bind to mononuclear phagocytic cell surface Fc receptors through their Fc regions. IgG1 and IgG3 may also attach through their Fc regions to mononuclear phagocytic cell Fc receptors in humans. Immunoglobulin molecules that bind to macrophage surfaces through their Fc regions represent a type of cytophilic antibody. (2) Described in the 1960s as a globulin fraction of serum which is adsorbed to certain cells in vitro in a manner that allows them to specifically adsorb antigen. Sorkin, in 1963, suggested the possible significance of cytophilic antibody in anaphylaxis and other immunologic and/or hypersensitivity reactions. Neonatal immunity  is the resistance in the newborn attributable to maternal circulating antibodies that reached the fetus via the placenta or maternal secretory antibodies translated to the newborn in breast milk. The quality and quantity of both humoral and cellular immune responses of neonates differ from those of adults. These differences may be consequences of the lower incidences and decreased functions of immunocompetent cells, such as B cells, T cells, and antigenpresenting cells early in ontogeny. Restriction of T- and B-cell function early in ontogeny compared with their function in the adult may be due partially to limitations on the diversity of antigen-specific repertoires. Differences in responses to various types of activation by neonatal and adult lymphocytes also affect immunity. This is a type of passive immunization.

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A neonatal Fc receptor (FcRn)  is an Fc receptor specific for IgG that facilitates the transport of maternal IgG across the placenta and the neonatal intestinal epithelium. This receptor is expressed by neonatal intestinal cells that bind IgG in breast milk and facilitates its passage into the neonatal intestinal lumen. FcRn is similar to a class I molecule and is also called Brambell receptor. An adult variety of this receptor protects plasma IgG antibodies from catabolism. Cytotoxic antibody  is an antibody that combines with cell surface epitopes followed by complement fixation that leads to cell lysis or cell membrane injury without lysis. Cytotoxicity  is the fatal injury of target cells by either specific antibody and complement or specifically sensitized cytotoxic T cells, activated macrophages, or natural killer (NK) cells. Dye exclusion tests are used to assay cytotoxicity produced by specific antibody and complement. Measurement of the release of radiolabel or other cellular constituents in the supernatant of the reacting medium is used to determine effector cell-mediated cytotoxicity. Antibody humanization  is the transference of the antigenbinding part of a murine monoclonal antibody to a human antibody. Antibody-mediated suppression  is the feedback inhibition that antibody molecules exert on their own further synthesis. Cytotoxicity tests:  (1) Assays for the ability of specific antibody and complement to interrupt the integrity of a cell membrane, which permits a dye to enter and stain the cell. The relative proportion of cells stained, representing dead cells, is the basis for dye exclusion tests. See microlymphocytotoxicity. (2) Assays for the ability of specifically sensitized T lymphocytes to kill target cells whose surface epitopes are the targets of their receptors. Loss of the structural integrity of the cell membrane is signified by the release of a radioisotope such as 51Cr, which was taken up by the target cells prior to the test. The amount of isotope released into the supernatant reflects the extent of cellular injury mediated by the effector T lymphocytes. Cytotrophic antibodies  are IgE and IgG antibodies that sensitize cells by binding to Fc receptors on their surface, thereby sensitizing them for anaphylaxis. When the appropriate allergen crosslinks the Fab regions of the molecules, it leads to the degranulation of mast cells and basophils bearing IgE on their surface. Nonprecipitating antibodies:  The addition of antigen in increments to an optimal amount of antibody precipitates only approximately 78% of the amount of antibody that would be precipitated by one step addition to the antigen. This demonstrates the presence of both precipitating and nonprecipitating antibodies. Although the nonprecipitating variety

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cannot lead to the formation of insoluble antigen–antibody complexes, they can be assimilated into precipitates that correspond to their specificity. Rather than being univalent, as was once believed, they may merely have a relatively low affinity for the homologous antigen. Monogamous bivalency, which describes the combination of high-affinity antibody with two antigenic determinants on the same antigen particle, represents an alternative explanation for the failure of these molecules to precipitate with their homologous antigen. The formation of nonprecipitating antibodies, which usually represents 10 to 15% of the antibody population produced, is dependent upon such variables as heterogeneity of the antigen, characteristics of the antibody, and animal species.  he equivalence zone is narrower with native proteins of 40 T to 60 kDa and their homologous antibodies than with polysaccharide antigens or aggregated denatured proteins and their specific antibody. The equivalence zone with synthetic polypeptide antigens varies with the individual compound used. The solubility of antibody–antigen complexes and the nature of the antigen are related to these variations at the equivalence zone. The extent of precipitation is dependent upon characteristics of both the antigen and antibody. At the equivalence zone, not all antigen and antibody molecules are present in the complexes. For example, rabbit anti-BSA (bovine serum albumin) precipitates only 46% of BSA at equivalence. O phage antibody library  refers to cloned antibody variable region gene sequences that may be expressed as Fab or svFv fusion proteins with bacteriophage coat proteins. These can be exhibited on the phage surface. The phage particle contains the gene encoding a monoclonal recombinant antibody and can be selected from the library by binding of the phage to specific antigen. OKT monoclonal antibodies  are commercially available preparations used to enumerate human T cells according to their surface antigens to determine the immunophenotype. OKT designations have been replaced by CD designations. Phage display  is a technique that permits expression of the humoral immune system in vitro by phage display technology and antibody engineering. Large libraries of antibody fragments are displayed on the surface of bacteriophage particles. Phages expressing desirable antibody specificity must be selected and expanded. Favorable mutations in the genes encoding a selected antibodies specificity must be selected. Antibody fragments are selected from large libraries constructed from B cells or assembled in vitro from the genetic elements encoding antibodies. This technique is rapid and unaffected by the immunogenecity of the target antigen. Selection procedures permit the isolation of antibodies specific for membrane molecules and epitopes. Antibody fragments can be tailored to have the desired avidity, pharmacokinetic properties, and biological effector functions. Monoclonal antibodies prepared from phage display libraries

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Immunoglobulin Synthesis, Properties, Structure, and Function

formed from human V regions constitute a molecule especially amenable for immunotherapy in humans. Phage display library:  Antibody-like phage produced by cloning immunoglobulin V region genes and filamentous phage which results in their expressing antigen-binding domains on their surfaces. Antigen-binding phage can be replicated in bacteria and used like antibodies. This method can be employed to develop antibodies of any specificity. OKT4:  See CD4. A polyvalent antiserum  is an antiserum comprised of antibodies specific for multiple antigens. Antitoxin  is an antibody specific for exotoxins produced by certain microorganisms such as the causative agents of diphtheria and tetanus. Prior to the antibiotic era, antitoxins were the treatment of choice for diseases produced by the soluble toxic products of microorganisms, such as those from Corynebacterium diphtheriae and Clostridium tetani. OKT8:  See CD8. Antiantibody:  In addition to their antibody function, immunoglobulin molecules serve as excellent protein immunogens when inoculated into another species or they may become autoantigenic even in their own host. The Gm antigenic determinants in the Fc region of an IgG molecule may elicit autoantibodies, principally of the IgM class, known as rheumatoid factor in individuals with rheumatoid arthritis. Antiidiotypic antibodies, directed against the antigen-binding N-terminal variable regions of antibody molecules, represent another type of antiantibody. Rabbit antihuman IgG (the Coombs’ test reagent) is an antiantibody used extensively in clinical immunology to reveal autoantibodies on erythrocytes. Anti-immunoglobulin antibodies  are antibodies specific for immunoglobulin constant domains which render them useful for detection of bound antibody molecules in immunoassays. Antiisotype antibodies are synthesized in a different species; antiallotype antibodies are made in the same species against allotypic variants; and antiidiotype antibodies are induced against a single antibody molecule’s unique determinants. Anti-immunoglobulin antibodies  are produced by immunizing one species with immunoglobulin antibodies derived from another. Skin-fixing antibody  is an antibody such as IgE that is retained in the skin following local injection, as in passive cutaneous anaphylaxis. Antibody with this property was referred to previously as reagin before IgE was described.

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Catalytic antibodies  are not only exclusively specific for a particular ligand but are also catalytic. Approximately 100 reactions have been catalyzed by antibodies. Among these are pericyclic processes, elimination reactions, bond-forming reactions, and redox processes. Most antibody-catalyzed reactions are highly stereospecific. For efficient catalysis, it is necessary to introduce catalytic functions within the antibody-combining site properly juxtaposed to the substrate. Catalytic antibodies resemble enzymes in processing their substrates through a Michaelis M complex in which the chemical transformation takes place followed by a product dissociation. See also abzyme. A catalytic antibody  is a monoclonal antibody into whose antigen-binding site the catalytic activity of a specific biological enzyme has been introduced. This permits enzymatic catalysis of previously arranged specificity to take place. Site-directed mutagenesis, in which a catalytic residue is added to a combining site by amino acid substitution, is used to attain the specificity. Specific catalysts can be generated by other mechanisms such as alternation of enzyme sites genetically or chemical alternation of receptors with catalytic properties. Chimeric antibodies  are antibodies that have, for example, mouse Fv fragments for the Ag-binding portion of the molecule but Fc regions of human Ig which convey effector functions. Lysins  are factors such as antibodies and complement or microbial toxins that induce cell lysis. For an antibody to demonstrate this capacity, it must be able to fix complement. Cκ  is an immunoglobulin κ light chain constant region. The corresponding exon is designated as Cκ. CL  is an immunoglobulin light chain constant domain. The corresponding exon is designated CL. C𝛌  is an immunoglobulin λ light chain constant region. The corresponding exon is designated Cλ. There is more than one isotype in mouse and man. Cμ  is an immunoglobulin μ-chain constant region. The corresponding exon is designated as Cμ. Cγ  is an immunoglobulin γ chain constant region that is further subdivided into four isotypes in man that are indicated as Cγ1, Cγ2, Cγ3, and Cγ4. The corresponding exons are expressed by the same designations in italics. CH  is an immunoglobulin heavy chain’s constant region encoded by the CH gene. CH1  is an immunoglobulin heavy chain’s first constant domain encoded by the CH1 exon.

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CH2  is an immunoglobulin heavy chain’s second constant domain encoded by the CH2 exon.

in sequence among different clones and is not involved in antigen binding.

CH3  is an immunoglobulin heavy chain’s third constant domain encoded by the CH3 exon.

Cyanogen bromide  is a chemical that specifically breaks methionyl bonds. Approximately one half of the methionine residues in an IgG molecule, e.g., those in the Fc region, are cleaved by treatment with cyanogen bromide.

CH4  is an immunoglobulin heavy chain’s fourth constant domain encoded by the CH4 exon. Of the five immunoglobulin classes in man, only the μ heavy chain of IgM and the ε heavy chain of IgE possess a fourth domain. Complementarity-determining region (CDR)  refers to the hypervariable regions in an immunoglobulin molecule that form the three-dimensional cavity where an epitope binds to the antibody molecule. The heavy and light polypeptide chains each contribute three hypervariable regions to the antigen binding region of the antibody molecule. Together, they form the site for antigen binding. Likewise, the T cell receptor α and β chains each have three regions with great diversity that are analogous to the immunoglobulin’s CDRs. These hypervariable areas are sites of binding for foreign antigen and self MHC molecular complexes. Constant domain  or C domain, refers to the immunoglobulin CH and CL regions encoded by the corresponding constant exon. There is only minor variability in the amino acid content of constant domains. A globular compact structure that consists of two antiparallel twisted β sheets. There are differences in the number and the irregularity of the β strands and bilayers in variable (V) and constant (C) subunits of immunoglobulins. C domains have a tertiary structure that closely resembles that of the domains, which are comprised of a fivestrand β sheet and a four-strand β sheet packed facing one another. However, the C domain does not have a hairpin loop at the edge of one of the sheets. Thus, the C domain has seven or eight β strands rather than the nine that are found in the V domains. Refers also to the constituent domains of constant regions of T cell receptor polypeptides. Constant region  is that part of an immunoglobulin polypeptide chain that has an invariant amino acid sequence among immunoglobulin chains belonging to the same isotype and allotype. There is a minimum of two and often three to four domains in the constant region of immunoglobulin heavy polypeptide chains. The hinge region “tail end piece” (a carboxy terminal region) constitutes part of the constant region in selected classes of immunoglobulin. A few exons encode the constant region of an immunoglobulin heavy chain, and one exon encodes the constant region of an immunoglobulin light chain. The constant region is the location for the majority of isotypic and allotypic determinants. It is associated with a number of antibody functions. T cell receptor α, β, γ, and δ chains have constant regions coded for by three to four exons. MHC class I and II molecules also have segments that are constant regions in that they show little sequence variation from one allele to another. Refers also to the part of a T cell receptor (TCR) polypeptide chain that does not vary

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Distribution ratio  is the plasma immunoglobulin to whole body immunoglobulin ratio. Effector function  refers to the nonantigen-binding functions of an antibody molecule that are mediated by the constant region of heavy chains. These include Fc receptor binding, complement fixation, binding to mast cells, etc. Effector function generally results in removal of antigen from the body, such as in phagocytosis or complement-mediated lysis. The elimination of foreign entities by effector cells through such mechanisms as cytokine secretion, cytotoxicity, and antibody production. The hinge region  is an area of an immunoglobulin heavy chain situated between the first constant domain and the second constant domain (CH1 and CH2) in an immunoglobulin polypeptide chain. The high content of proline residues in this region provides considerable flexibility to this area, which enables the Fab region of an immunoglobulin molecule to combine with cell surface epitopes that it might not otherwise reach. Fab regions of an Ig molecule can rotate on the hinge region. There can be an angle up to 180 between the two Fab regions of an IgG molecule. In addition to the proline residues, there may be one or several half cysteines associated with the interchain disulfide bonds. Enzyme action by papain or pepsin occurs near the hinge region. Whereas, γ, α, and δ chains each contain a hinge region, μ and ε chains do not. The 5′ part of the CH2 exon encodes the human and mouse a-chain hinge region. Four exons encode the γ-3 chains of humans and two exons encode human δ chains. Homology unit:  A structural feature of an immunoglobulin domain. A hot spot  is a hypervariable region in DNA that encodes the variable region of an immunoglobulin molecule’s heavy (VH) and light (VL) polypeptide chains. These are also designated complementarity-determining regions (CDR). These are the areas for specific antigen binding, and they also determine the idiotype of an immunoglobulin molecule. The remaining background support structures of the heavy and light polypeptide chains are termed framework regions (FR). The κ and λ light chain hot spots are situated near amino acid residues 30, 50, and 95. Also called hypervariable regions. Humanization  refers to the genetic engineering of murine hypervariable loop specificity into human antibodies. The DNA encoding hypervariable loops of murine monoclonal antibodies or V regions selected in phage display libraries is

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Immunoglobulin Synthesis, Properties, Structure, and Function

inserted into the framework regions of human immunoglobulin genes. This technique permits the synthesis of antibodies of a particular specificity without inducing an immune response in the human subject treated with them.  o humanize an antibody means to substitute, through genetic T engineering, the CDR loops in a human antibody molecule with the corresponding murine antibody CDR sequences of a given specificity. A humanized antibody  is an engineered antibody produced through recombinant DNA technology. A humanized antibody contains the antigen-binding specificity of an antibody developed in a mouse, whereas the remainder of the molecule is of human origin. To accomplish this, hypervariable genes that encode the antigen-binding regions of a mouse antibody are transferred to the normal human gene which encodes an immunoglobulin molecule that is mostly human but expresses the antigen-binding specificity of the mouse antibody in the variable region of the molecule. This greatly diminishes any immune response to the antibody molecule itself as a foreign protein by the human host, while retaining the desired functional capacity of reacting with the specific antigen. An immunoglobulin  is a mature B cell product synthesized in response to stimulation by an antigen. Antibody molecules are immunoglobulins of defined specificity produced by plasma cells. The immunoglobulin molecule consists of heavy (H) and light (L) chains fastened together by disulfide bonds. The molecules are subdivided into classes and subclasses based on the antigenic specificity of the heavy chains. Heavy chains are designated by lower case Greek letters (μ, γ, α, δ, and ε), and immunoglobulins are designated IgM, IgG, IgA, IgD, and IgE, respectively. The three major classes are IgG, IgM, and IgA, and the two minor classes are IgD and IgE, which together comprise less than 1% of the total immunoglobulins. The two types of light chains termed (κ and λ) are present in all five immunoglobulin classes, although only one type is present in an individual molecule. IgG, IgD, and IgE have two H and two L polypeptide chains, whereas IgM and IgA consists of multimers of this basic chain structure. Disulfide bridges and noncovalent forces stabilize immunoglobulin structure. The basic monomeric unit is Y shaped, with a hinge region rich in proline and susceptible to cleavage by proteolytic enzymes. Both H and L chains have a constant region at the carboxyl terminus and a variable region at the amino terminus. The two heavy chains are alike, as are the two light chains, in any individual immunoglobulin molecule. Approximately 60% of human immunoglobulin molecules have κ light chains, and 40% have λ light chains. The five immunoglobulin classes are termed isotypes based on the heavy-chain specificity of each immunoglobulin class. Two immunoglobulin classes, IgA and IgG, have been further subdivided into subclasses based on H chain differences. The four IgG subclasses are designated

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as IgG1 through IgG4, and the two IgA subclasses are designated IgA1 and IgA 2. Digestion of IgG molecules with papain yields two Fab and one Fc fragments. Each Fab fragment has one antigen-binding site but is responsible for fixation of complement and attachment of the molecule to a cell surface. Pepsin cleaves the molecule toward the carboxyl-terminal end of the central disulfide bone, yielding an F(ab′)2 fragment and a pFc′ fragment. F(ab′)2 fragments have two antigen-binding sites. L chains have a single variable and constant domain, whereas H chains possess one variable and three to four constant domains. Secretory IgA is found in body secretions such as saliva, milk, and intestinal and bronchial secretions. IgD and IgM are present as membrane-bound immunoglobulins on B cells, where they interact with antigen to activate B cells. IgE, associated with anaphylaxis and IgG, which is the only immunoglobulin capable of crossing the placenta, is the major human immunoglobulin. mIg:  Abbreviation for membrane-bound immunoglobulin. Immunoglobulin structure:  See immunoglobulin. An immunoglobulin monomer  is the basic unit of immunoglobulin, comprised of two heavy chains and two light chains. The homology region  is a 105- to 115-amino acid residue sequence of heavy or light chains of immunoglobulins which have a primary structure that resembles other corresponding sequences of the same size. A homology region has a globular shape and an intrachain disulfide bond. The exons that encode homology regions are separated by introns. Light polypeptide chain homology regions are termed VL and CL. Heavy chain homology regions are designated VH, CH1, CH2, and CH3. N-terminus  is the amino end of a polypeptide chain bearing a free amino-NH2 group. Reagin (historical):  (1) Obsolete term for a complementfixing IgM antibody reacting in the Wassermann test for syphilis. (2) A name used previously for immunoglobulin E (IgE), the anaphylactic antibody in humans that fixes to the Fcε receptors on tissue mast cells leading to release of histamine and vasoactive amines following interaction with specific antigen (allergen). SCAB (single chain antigen-binding proteins) are  polypeptides that join the light chain variable sequence of an antibody to the antibody heavy chain variable sequence. All monoclonal antibodies are potential sources of SCABs. They are smaller and less immunogenic than the intact heavy chains with immunogenic constant regions. Their many possible uses are in imaging and treatment of cancer, in cardiovascular disease, as biosensors, and for chemical separations.

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ScFv  is a single-chain molecule comprised of both heavy and light chain variable regions fastened together by a flexible linker. Tail peptide  is an immunoglobulin heavy polypeptide chain carboxy terminus that is separate from the carboxy terminal domain. This structure is present in membraneanchored immunoglobulins. Whereas tail peptides of 20 amino acids each are present in IgM and IgA molecules that have been secreted, IgG and IgE molecules do not contain tail peptides. Telencephalin  is an immunoglobulin superfamily member with nine immunoglobulin-like domains that is expressed in the central nervous system. It is a human α1β2 ligand that has a high homology with ICAM-1 (50%) and ICAM-3 (55%) in the amino-terminal first five domains. The clustered chromosomal location for ICAM-1, ICAM-3, and telencephalin is on human chromosome 19. Their corresponding α chain receptors are located on chromosome 16. Ig  is the abbreviation for immunoglobulin. Immune serum  is an antiserum containing antibodies specific for a particular antigen or immunogen. Such antibodies may confer protective immunity. Immune serum globulin  is an injectable immunoglobulin that consists mainly of IgG extracted by cold ethanol fractionation from pooled plasma of up to 1000 human donors. It is administered as a sterile 16.5 ± 1.5% solution to patients with immunodeficiencies and as a preventive against certain viral infections including measles and hepatitis A. Immunoglobulin function  is to link an antigen to its elimination mechanism (effector system). Antibodies induce complement activation and cellular elimination mechanisms that include phagocytosis and antibody-dependent cellmediated cytotoxicity (ADCC). This type of activation usually requires antibody molecules clustered together on a cell surface rather than as free unliganded antibody. Antibodies can combine with virus particles to render them noninfectious in vitro through neutralization. IgG catabolism is regulated by the IgG concentration. All immunoglobulin classes can be expressed on B cell surfaces where they act as antigen receptors although this is mainly a function of IgM and IgD. Surface immunoglobulin has an extra C-terminal sequence compared to secreted immunoglobulin containing linker, transmembrane, and cytoplasmic segments. Cerebrospinal fluid (CSF) immunoglobulins:  In normal individuals, CSF immunoglobulins are derived from plasma by diffusion across the blood–brain barrier. The amount present is dependent on the immunoglobulin concentration in the serum, the molecular size of the immunoglobulin, and the permeability of the blood–brain barrier. IgM is normally excluded by virtue of its relatively large molecular size and

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low plasma concentration. However, in certain disease states, such as demyelinating diseases and infections of the central nervous system, immunoglobulins may be produced locally. The permeability of the blood–brain barrier is accurately reflected by the CSF total protein or albumin levels relative to those in the serum. By comparing these data, it is possible to derive information about deviation from normal. The comparative method is called Ig quotient and is calculated in various ways: 1.  CSF-IgG/albumin (normal 13.9 + 14%) 2.  CSF-IgG/total protein 3.  CSF-IgA/albumin 4.  CSF-κ/λ (ratio)  o correct for variations in the blood–brain barrier, the calT culation can be modified to give a more sensitive quotient, which is represented as

CSF IgG/serum IgG CSF albu min/serum albu min The ratio of κ to λ light chains in CSF in comparison with that of these light chains in serum is significant in that some patients with local immunoglobulin production show a change in the ratio. An increase in the IgA present in CSF appears in some viral infections of the CNS in which antiviral antibodies are also detectable. An immunoglobulin heavy chain  (Figure 7.4) is a 5-kDa to 71-kDa polypeptide chain present in immunoglobulin molecules that serves as the basis for dividing immunoglobulins into classes. The heavy chain is comprised of three to four constant domains, depending on class, and one variable domain. In addition, a hinge region is present in some chains. There is approximately 30% homology with respect to amino acid sequence among the five classes of immunoglobulin heavy chain in humans. The heavy chain of IgM is μ, of IgG is γ, of IgA is α, of IgD is δ, and of IgE is ε. A heavy chain is a principal constituent of immunoglobulin

Light chain

Heavy chain

Figure 7.4  Immunoglobulin heavy chains that are fastened to each other or to light polypeptide chains by disulfide bonds.

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molecules. Each immunoglobulin is comprised of at least one four-polypeptide chain monomer which consists of two heavy and two light polypeptide chains. The two heavy chains are identical in any one molecule as are the two light chains. A heavy chain  is an immunoglobulin polypeptide chain that designates the class of immunoglobulin. The five immunoglobulin classes are based on the heavy chains they possess and are IgM, IgG, IgA, IgD, and IgE. Each four-chain immunoglobulin molecule or each four-chain monomeric unit of IgM contains two heavy chains and two light chains. These are fastened together by disulfide bonds. At the amino terminus is the variable region of the heavy chain, designated V H. Adjacent to this is the first constant region, designated CH1 through CH3 or CH4 domains, based on immunoglobulin class. Heavy-chain antigenic determinants determine not only the immunoglobulin class but the subclass as well. Heavy chain class  refers to the immunoglobulin heavy polypeptide chain primary (antigenic) structure present in all members of a species that is different from the other heavy chain classes. Primary structural features governing immunoglobulin heavy chain class are located in the constant region. Lowercase Greek letters such as μ, γ, α, δ, and ε designate heavy chain class. H chain (heavy chain)  is a principal constituent of immunoglobulin molecules. Each immunoglobulin is comprised of at least one four-polypeptide chain monomer, which consists of two heavy and two light polypeptide chains. The two heavy chains are identical in any one molecule as are the two light chains. Heavy chain subclass:  Within an immunoglobulin heavy chain class, differences in primary structure associated with the constant region that can further distinguish these heavy chains of the same class are designated as subclasses. These differences are based on primary or antigenic structure. Heavy chain subclasses are designated as γ1, γ2, γ3, etc. Immunoglobulin heavy chain binding protein (BiP)  is a 77-kDa protein that combines with selected membrane and secretory proteins. It is believed to facilitate their passage through the endoplasmic reticulum. A light chain  (Figure  7.5) is a 22-kDa polypeptide chain found in all immunoglobulin molecules. Each four-chain immunoglobulin monomer contains two identical light polypeptide chains. They are joined to two like heavy chains by disulfide bonds. There are two types of light chains designated κ and λ. An individual immunoglobulin molecule possesses two light chains that are either κ or λ but never a mixture of the two. The types of light polypeptide chains occur in all five of the immunoglobulin classes. Each light

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L chain

Figure 7.5  Light polypeptide chains of immunoglobulins that are fastened to heavy chains through disulfide bonds and are found in all classes of immunoglobulin.

chain has an N-terminal V region which constitutes part of the antigen-binding site of the antibody molecule. The C region or constant terminal reveals no variation except for the Km and Oz allotype markers in humans. KM (formerly Inv) is the designation for the κ light chain allotype genetic markers. κ chain  is one of two types of light polypeptide chains present in immunoglobulin molecules of human and other species. κ light chains are found in approximately 60% of human immunoglobulin, whereas λ light chains are present in approximately 40% of human immunoglobulin molecules. A single immunoglobulin molecule contains either κ or λ light chains, not one of each. Light chain type  is a term for the classification of immunoglobulin light chains based on their primary or antigenic structure. Two types of light chains have been described and are designated as κ and λ. Two κ chains or two λ chains, never one of each, are present in each monomeric immunoglobulin subunit of vertebrate species. Kappa (κ)  is the designation for one of the two types of immunoglobulin light chain, with the other designated as lambda (λ). Lambda (λ)  is one of the two light polypeptide chain types found in immunoglobulin (Ig) molecules. Kappa-lambda exclusion is a means  whereby the generation of a functional κ light chain from the Igk locus on one chromosome prevents further V(D)J rearrangement at the other Igk allele, and also blocks V(D)J rearrangement of either Ig1 allele. Light chain subtype  refers to the subdivision of a type of light polypeptide chain based on its primary or antigenic structure that appears in all members of an individual species. Subtype differences distinguish light chains that share a common type. These relatively minor structural differences are located in the light chain constant region. Oz+, Oz−, Kern+, and Kern- markers represent subtypes of λ light chains in humans.

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Km allotypes:  Three Km allotypes have been described in human immunoglobulin κ light chains. They are designated Km1, Km1,2, and Km3. They are encoded by alleles of the gene that codes for the human κ light chain constant regions. Allotype differences are based on the amino acid residue at positions 153 and 191, which are in proximity to one another in a folded immunoglobulin Cκ domain. One person may have a maximum of two out of the three Km allotypes on their light chains. To fully express Km determinants, the heavy immunoglobulin chains should be present, probably to maintain appropriate three-dimensional configuration. L chain  is a 22-kDa polypeptide chain found in all immunoglobulin molecules. There are two types designated κ or λ. Each four-chain immunoglobulin monomer contains either two κ or two λ light chains. The two types of light chain never occur in one molecule under natural conditions. Refer to “light chain.” Lambda (𝛌) chain  is one of the two light polypeptide chain types found in immunoglobulin molecules. The κ light chain is the other type. Each immunoglobulin molecule contains either two λ or two κ light chains. The κ to λ light chains ratio differs among species. Approximately 60% of IgG molecules in humans are κ and 40% are λ. Mcg isotypic determinant  is a human immunoglobulin λ chain epitope that occurs on some of every person’s λ light polypeptide chains in immunoglobulin molecules. The Mcg isotypic determinant is characterized by asparagine at position 112, threonine at position 114, and lysine at position 163. Inv  is the former designation for human κ light polypeptide chain allotype. Allotypic epitopes in the immunoglobulin κ light chain constant region. Km replaced Inv.

Atlas of Immunology, Third Edition

Formation of Disulfide Bond Cys – SH + HS – Cys

Breakage of Disulfide Bond Cys – S – S– Cys

Oxidation

Reduction

Cys – S – S – Cys

Cys – SH

HS – Cys 2(ICH2COOH)

Cys – S CH2COO – Cys – S CH2COO –

Figure 7.6  Depiction of the formation of disulfide bonds from the oxidation of two sulfhydryl groups as well as the breaking of disulfide bonds through reduction leading to sulfhydryl formation.

proteins. In immunology, it refers to the loops in polypeptide chains that are linked by disulfide bonds on constant and variable regions of immunoglobulin molecule light and heavy polypeptide chains or a compact TCR chain segment comprised of amino acids around an S–S bond. An immunoglobulin domain  (Figure  7.7) is an immunoglobulin heavy or light polypeptide chain structural unit that is comprised of approximately 110 amino acid residues. Domains are loops that are linked by disulfide bonds on constant and variable regions of heavy and light chains. Immunoglobulin functions may be linked to certain domains. There is much primary and three-dimensional structural homology among immunoglobulin domains. A particular exon may encode an immunoglobulin domain.

Inv allotypes:  Original terminology for Km allotypes, which is now the preferred nomenclature. Inv allotypic determinant:  See Km allotypic determinant. Inv marker:  See Km allotypic determinant. Disulfide bonds  (Figure 7.6) are the –S–S– chemical bonds between amino acids that link polypeptide chains together. Chemical reduction may break these bonds. Disulfide bonds in immunoglobulin molecules are either intrachain or interchain. The interchain disulfide bonds include linking heavy to heavy and heavy to light. The different types of bonds in immunoglobulin molecules differ in their ease of chemical reduction. Domain  is a region of a protein or polypeptide chain that is globular and folded with 40 to 400 amino acid residues. The domain may have a spatially distinct “signature” which permits it to interact specifically with receptors or other

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S S

S S

Domains

S S

S S

Figure 7.7  Domain structure of light or heavy polypeptide chains, the subunits of immunoglobulin molecules.

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Ig domain:  See immunoglobulin domain. S antibody  is the sedimentation coefficient of immunoglobulin molecules such as IgG. 6.6 S immunoglobulins are usually referred to as 7 S immunoglobulins. C region (constant region)  is the abbreviation for the constant region carboxy terminal portion of immunoglobulin heavy or light polypeptide chain that is identical in a particular class or subclass of immunoglobulin molecules. CH designates the constant region of the heavy chain of immunoglobulin, and CL designates the constant region of the light chain of immunoglobulin. Immunoglobulin-like domain  is the 100-amino acid residue structure found in selected β sheet-rich proteins with intrachain disulfide bonds. It is found in immunoglobulins, interleukins 1 and 6, the T cell receptor, and platelet-derived growth factor. Structural regions of proteins that are similar to the immunoglobulin domain, but are present in various other proteins. C-terminus  is the carboxy terminal end of a polypeptide chain containing a free –COOH group. An immunoglobulin fold  is an immunoglobulin domain’s three-dimensional configuration. An immunoglobulin fold has a sandwich-like structure comprised of two β-pleated sheets that are nearly parallel. There are four antiparallel chain segments in one sheet and three in the other. Approximately 50% of the domain’s amino acid residues are in the β-pleated sheets. The other 50% of the amino acid residues are situated in polypeptide chain loops and in +H +H

3N

terminal segments. The turns are sites of invariant glycine residues. Hydrophobic amino acid side chains are situated between the sheets. The VH region  refers to the variable part of immunoglobulin heavy chain which is the part of a variable region encoded for by the VH gene segment (Figure 7.8). The VL region describes the variable portion of an immunoglobulin light chain. The symbol may be used to designate the VL gene encoded segment. Vκ is a variable region of an immunoglobulin κ light chain. This symbol may also be used to signify that part of a variable region encoded by the Vκ gene segment. V𝛌  is the variable region of an immunoglobulin λ light chain. The symbol may designate that part of a variable region which the Vλ gene segment encodes. VL region  is the variable region of an immunoglobulin light chain. The symbol may be used to designate the V L gene encoded segment. Hypervariable regions  (Figure 7.9) constitute a minimum of four sites of great variability which are present throughout the H and L chain V regions. They govern the antigenbinding site of an antibody molecule. Thus, grouping of these hypervariable residues into areas govern both conformation and specificity of the antigen-binding site upon folding of the protein molecule. Hypervariable residues are also responsible for variations in idiotypes between immunoglobulins produced by separate cell clones. Those parts of the variable region that are not hypervariable are termed the framework regions. Hypervariable regions are also called complementarity-determining regions (Figure 7.10 and Figure 7.11). See

NH3+

VH

NH3+

3N

H

n ai

ch

ch

ai n

H

n

L

L

ch

ai

CH1 –S –

–S

–S

–S

–

n ai ch

VL

–S–S– –S–S–

CL

H chain

H chain

CH2

–OOC

COO–

CH3

Figure 7.8  VH and VL regions on an antibody.

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31

90

55

28 51

Light chain

95

Antigen determinant

65

33

68 120

107 30

Heavy chain

Figure 7.9  Depiction of the structure of the six hypervariable regions of an antibody.

hot spot. The term refers also to those portions of the T cell receptor that constitute the antigen-binding site. Each antibody heavy chain and light chain and each TCR α chain and β chain possess three hypervariable loops, also called CDRs. Most of the variability between different antibodies or TCRs is present within these loops. HV regions:  Refers to hypervariable regions. An immunoglobulin light chain  is a 23-kDa, 214-amino acid polypeptide chain comprised of a single constant region and a single variable region that is present in all five classes of immunoglobulin molecules. The two types of light chains are designated κ and λ. They are found in association with heavy polypeptide chains and immunoglobulin molecules and are fastened to these structures through disulfide bonds.

An immunoglobulin 𝛌 chain  (Figure 7.12) is a 23-kDa 214amino acid residue polypeptide chain with a single variable region and a single constant region (Figure 7.13). The λ chains represent one of two light polypeptide chains comprising all five classes of immunoglobulin molecules. Approximately 40% of immunoglobulin light chains in humans are λ. Wide variations in percentages are observed in other species. For example, the great majority of immunoglobulin light chains in horses and dogs are λ, whereas they constitute only 5% of murine light chains. Constant region differences among λ light chains of mice and humans distinguish the molecules into four isotypes in humans. A different C gene segment encodes the separate constant regions defining each λ light chain isotype. The human λ light chain isotypes are designated Kern_Oz+, Kern+Oz-, and Mcg. Immunoglobulin heavy chain-binding protein (BiP)  is a 77-kDa protein that combines with selected membrane and secretory proteins. It is believed to facilitate their passage through the endoplasmic reticulum. An immunoglobulin κ chain  (Figure 7.14 and Figure 7.15) is a 23-kDa 214-amino acid residue polypeptide chain that is comprised of a single variable region and a single constant region. It is one of the two types of light polypeptide chain present in all five immunoglobulin classes. Approximately 60% of light immunoglobulin chains in humans are κ with wide variations of their percentages in other species. Whereas κ chains are virtually absent in immunoglobulins of dogs, they comprise the vast majority of murine immunoglobulin light chains. κ light chain allotypes in man are termed Km1, Km1,2, and Km3. A J chain  (Figure 7.16) is a 17.6-kDa polypeptide chain present in polymeric immunoglobulins that include both IgM and IgA. It links four-chain immunoglobulin monomers to produce the polymeric immunoglobulin structure. J chains are produced in plasma cells and are incorporated into IgM or IgA

Figure 7.10  Illustration of intact monoclonal antibody for canine lymphoma.

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Fab elbow bend

Fab arm waving

Arg Fab rotation

VH

CL

VL

H2N

Figure 7.11  Cartoon of an IgG molecule.

Figure 7.12  Photomicrograph of immunoglobulin 1 light “staining” by immunoperoxidase.

Val

Cys

Cys

Lys

Cys

Gly

Gln

Cys

Cys

Cys

molecules prior to their secretion. Incorporation of the J chain appears essential for transcytosis of these immunoglobulin molecules to external secretions. The J chain comprises 2 to 4% of an IgM pentamer or a secretory IgA dimer. Tryptophan is absent from both mouse and human J chains. J chains are comprised of 137-amino acid residues and a single complex N-linked oligosaccharide on asparagine. Human J chain contains three forms of the oligosaccharide which differ in sialic acid content. The J chain is fastened through disulfide bonds to penultimate cysteine residues of μ or α heavy chains. The human J chain gene is located on chromosome 4q21, whereas the mouse J chain gene is located on chromosome 5.

FC wagging

C γ3

Asp

Cys

Figure 7.15  The κ light chain showing domain structure.

Cγ1

Cγ2

H2N

Ser

Arg

Cys

Cys

An immunoglobulin class  (Figure  7.17) is a subdivision of immunoglobulin molecules based on antigenic and structural differences in the Fc regions of their heavy polypeptide chains. Immunoglobulin molecules belonging to a particular class have at least one constant region isotypic determinant in common. The different classes such as IgG, IgM, and IgA designate separate isotypes. Since the light chains of immunoglobulin molecules are one of two types, the heavy chains determine immunoglobulin class. There is about 30% homology of amino acid sequence among the five immunoglobulin heavy chain constant regions in man. Heavy chains (or isotypes) also differ in carbohydrate content. Immunization of a nonhuman species with human immunoglobulin provides antisera that may be used for class or isotype determination. Ig G is divided into four subclasses and IgA is divided into two subclasses.

Cys

Figure 7.13  Lambda light chain showing domain structure.

Disulfide bonds

β pleated sheets Cysteine Oligosaccharide

Figure 7.14  Photomicrograph of immunoglobulin κ light chain “staining” by immunoperoxidase.

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Figure 7.16  Structure of J chain that occurs in secretory IgA and IgM molecules and facilitates polymerization.

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Ig Serum concentration (mg/dl) Total lg (%) Complement fixation Principal biological effect Principal site of action

IgG

IgM

IgA

IgD

IgE

800–1700

50–190

140–420

0.3–0.40

80% of target cells killed) is employed to indicate cytotoxicity. Most of the sera used to date are multispecific, as they are obtained from multiparous females who have been sensitized during pregnancy by HLA antigens determined by their spouse. Monoclonal antibodies are being used with increasing frequency in tissue typing. This technique is useful to identify HLA-A, HLA-B, and HLA-C antigens. When purified B cell preparations and specific antibodies against B cell antigens are employed, HLA-DR and HLA-DQ antigens can be identified. A cell tray panel  (Figure 22.13) is used to detect and identify HLA antibodies. Patient serum is tested against a panel of known cells. The panel (or percent) reactive antibody (PRA) is the percent of panel cells reacting with a patient’s serum. It is expressed as a percentage of the total reactivity,

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673

Figure 22.11  A Terasaki plate consisting of depressions in a plastic plate that contains predispensed antibodies to HLA antigens of various specificities and into which are placed patient lymphocytes and rabbit complement for tissue typing. Figure 22.10  A Hamilton syringe that is used to dispense lymphocytes into Terasaki plates for tissue typing.

i.e., %PRA = (No. of positive reactions/No. of cells in panel) × 100. This percentage is a useful indicator of the proportion of HLA antibodies of the patient. The panel reactive antibody (PRA) test  is a laboratory method designed to identify the level of sensitization to potential donors in recipients awaiting an organ transplant. The recipient’s serum is screened for antibodies reactive with panels of pooled cells that express a wide spectrum of MHC molecules. PRA represents the percentage of subjects in the panel with cells that interact with the patient’s antibodies. The test is employed to detect preformed alloantibodies in a recipient that could lead to hyperacute rejection of tissue from selected allogeneic donors. PRA  is the abbreviation for panel reactive antibody. Patients may have preformed antibodies against class I or II HLA antigens. If these patients receive organs that possess the corresponding antigens, they will likely experience hyperacute or delayed rejection for class I or class II incompatibilities, respectively. In order to detect such incompatibilities before transplantation, a cross-matching procedure is performed. The conventional cross-matching

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procedure (Figure 22.14) for organ transplants involves the combination of donor lymphocytes with recipient serum. There are three major variables in the standard crossmatch procedure that predominantly affect the reactivity of the cell/sera sensitization. These include (l) incubation time and temperature; (2) wash steps after cell/sera sensitization; and (3) the use of additional reagents, such as antiglobulin in the test. Variations in these steps can cause wide variations in results. Lymphocytes can be separated into T and B cell categories for cross-match procedures that are conducted at cold (4°C), room (25°C), and warm (37°C) temperatures. These permit the identification of warm anti-T cell antibodies that are almost always associated with graft rejection. Molecular (DNA) typing:  Sequence-specific priming (SSP) is a method that employs a primer with a single mismatch in the 3′-end that cannot be employed efficiently to extend a DNA strand because the enzyme Taq polymerase, during the PCR reaction, and especially in the first PCR cycles which are very critical, does not manifest 3′5′ proofreading endonuclease activity to remove the mismatched nucleotide. If primer pairs are designed to have perfectly matched 3′-ends with only a single allele, or a single group of alleles, and the PCR reaction is initiated under stringent conditions,

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1

H

G

F

E

D

C

B

A

2 3 4 5 6 7 8 9 10 11 12

(a)

Figure 22.12  An inverted light microscope used to read Terasaki plates to determine tissue type.

+ Donor lymphocyte

TRAY POS #CENTRL 1A 1B 1C 1D 1E 1F 2F 2E 2D 2C 2B 2A 3A 3B 3C 3D 3E 3F 4F 4E

TEST 8 8 8 8 8 8 8 8 1 1 8 1 1 1 1 1 1 1 1 1

CELL ID 10571T 9891T 9884T 9898T 10356T 10990T 10367T 7109T 6606T 10567T 10988T 10359T 10549T 10361T 10570T 9899T 10352T 10547T 6688T 10568T

RACE H C B B B O C H C C C C O O O B O C C H

1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2

PANEL TYPING B C 2 8 35 7 2 44 51 1 5 2 57 82 3 6 23 45 49 6 7 23 58 72 6 24 27 37 2 6 32 8 51 7 13 64 6 8 11 18 38 7 11 37 60 3 6 24 51 55 3 25 57 62 5 6 26 39 61 1 7 26 54 62 1 3 26 60 65 4 8 30 8 58 7 30 13 46 1 6 31 35 47 4 31 50 60 3 6 32 41 61 2 7

A

BW 6 4 4 6 4 6 4 6 4 4 6 4 6 4 6 4 6 4 6 4 6 6 6 6 4 6 4 6 4 6 6 6

Figure 22.13  Cell tray panel showing positive reactions (8s) for HLA-A1 at tray positions 1A, 1B, 1C, 1D, 1E, 1F, 2F, and 2E, and a positive reaction for HLA-A24 at position 2B.

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Vital dye + Complement + (e.g. trypan bule)

Dark blue positive crossmatch

Patient’s serum White refractile negative crossmatch

(b) Figure 22.14  (a) Crossmatching procedure. (b) Example of high- resolution DRBI typing using sequence-specific primer methodology. Molecular weight ladder of known base pairs is in the far left column for base pair sizing.

a perfectly matched primer pair results in an amplification product, whereas a mismatch at the 3′-end primer pair will not provide any amplification product. A positive result, i.e., amplification, defines the specificity of the DNA sample. In this method, the PCR amplification step provides the basis for identifying polymorphism. The postamplification processing of the sample consists only of a simple agarose gel electrophoresis to detect the presence or absence of amplified product. DNA amplified fragments are visualized by ethidium bromide staining and exposure to UV light. A separate technique detects amplified product by color fluorescence. The primer pairs are selected in such a manner that

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each allele should have a unique reactivity pattern with the panel of primer pairs employed. Appropriate controls must be maintained (Figure 22.14a).

are much more likely to share one or two haplotypes than are unrelated individuals. CYNAP phenomenon:  See CYNAP antibodies.

Probe hybridization (tissue typing)  is a technique used in tissue typing for organ transplantation in which total DNA is isolated from a subject’s cells and PCR is conducted with primers that permit generic amplification of all HLA alleles. Hybridization to allele-specific probes permits the identification of sequences of interest. Direct amplicon analysis  is a technique used in HLA tissue typing for the identification of an unknown HLA allele through discovery of the specific CR primers capable of amplifying a polymorphic region of the gene. Gel electrophoresis and ethidium bromide staining are used to detect the products of amplification. CREGs  are cross-reactive groups. Public epitope-specific antibodies identify CREGs. Public refers to both similar (cross-reactive) and identical (public) epitopes shared by more than one HLA gene product. CYNAP antibodies  are cytotoxicity negative but absorption-positive antibodies that are concerned with HLA tissue typing. Most alloantibodies to public epitopes display CYNAP when tested in complement-dependent cytotoxicity assays. Most alloantisera contain public or CREG antibodies, but they act operationally as “private” antibodies because of their CYNAP phenomenon. For this reason, the relative insensitivity of standard CDC, due to CYNAP, has been useful for detecting discrete gene products. Standard CDC is not the recommended procedure to define HLA molecule binding specificities. The antiglobulin-augmented CDC (AHGCDC) more accurately defines the true binding capabilities of alloantisera than do complement-independent assays by overriding the CYNAP phenomenon. CDC is the procedure of choice for HLA antigen detection and HLA antiserum analysis. Haplotype  designates those phenotypic characteristics encoded by closely linked genes on one chromosome inherited from one parent. Each individual inherits two haplotypes, one from each parent. It frequently describes several MHC alleles on a single chromosome. Selected haplotypes are in strong linkage disequilibrium between alleles of different loci. According to Mendelian genetics, 25% of siblings will share both haplotypes. A shared haplotype  is a phenotypic characteristic shared by two siblings based on closely linked genes on one chromosome inherited from each parent. There are four different possibilities of reassortment among offspring, which leads to a particular sibling pair sharing two, one, or no haplotypes. Selected haplotypes are in strong linkage disequilibrium between alleles of different loci. According to Mendelian genetics, 25% of siblings will share both haplotypes. Siblings

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Phenotype  designates observable features of a cell or organism that are a consequence of interaction between the genotype and the environment. The phenotype represents those genetically encoded characteristics that are expressed. Phenotype may also refer to a group of organisms with the same physical appearance and the same detectable characteristics. A haploidentical transplant  is the sharing of one HLA haplotype between donor and recipient who differ in the second HLA haplotype. MHC haplotype  refers to the set of genes in a haploid genome inherited from one parent. Children of parents designated ab and cd will probably be ac, ad, bc, or bd. Ancestral haplotype  refers to a MHC haplotype that numerous families share, indicating that they probably have common ancestors. It is also called HLA supratype or common extended haplotype. Trypan blue  is a vital dye used to stain lymphoid cells, especially in the microlymphocytotoxicity test used for HLA tissue typing. Cell membranes whose integrity has been interrupted by antibody and complement permit the dye to enter and stain the cells dark blue. By contrast, the viable cells with an intact membrane exclude the dye and remain as bright circles of light in the microscope. Dead cells stain blue. Trypan blue dye exclusion test  is a test for viability of cells in culture. Living cells exclude trypan blue by active transport. When membranes have been interrupted, the dye enters the cells, staining them blue and indicating that the cell is dead. The method can be used to calculate the percent of cell lysis induced. The 2-mercaptoethanol agglutination test  is a simple test to determine whether or not an agglutinating antibody is of the IgM class. If treatment of an antibody preparation, such as a serum sample, with 2-mercaptoethanol can abolish the serum’s ability to produce agglutination of cells, then agglutination was due to IgM antibody. Agglutination induced by IgG antibody is unaffected by 2-mercaptoethanol treatment and just as effective after the treatment as it was before. Dithiothreitol (DTT) produces the same effect as 2-mercaptoethanol in this test. Small “blues”  are blue aggregates of acellular debris observed in clinical histocompatibility testing using the microlymphotoxicity test. It occurs in the wells of tissue typing trays and is due to an excess amount of trypan blue mixed with protein. This is a technical artifact.

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Serologically defined (SD) antigens  are mammalian cellular membrane epitopes that are encoded by MHC genes. Antibodies detect these epitopes. Serological determinants  are epitopes on cells that react with specific antibody and complement, leading to fatal injury of the cells. Serological determinants are to be distinguished from lymphocyte determinants, which are epitopes on the cell surface to which sensitized lymphocytes are directed, leading to cellular destruction. Although the end result is the same, antibodies and lymphocytes are directed to different epitopes on the cell surface. In a mixed-lymphocyte culture (MLC)  lymphocytes from two members of a species are combined in culture where they are maintained and incubated for 3 to 5 d. Lymphoblasts are formed as a consequence of histoincompatibility between the two individuals donating the lymphocytes. The lymphocyte antigens of these genetically dissimilar subjects each stimulate DNA synthesis by the other, which is measured by tritiated thymidine uptake that is assayed in a scintillation counter. See mixed-lymphocyte reaction (MLR). MLC:  Abbreviation for mixed-lymphocyte culture. In the mixed-lymphocyte reaction (MLR)  lymphocytes from potential donor and recipient are combined in tissue culture. Each of these lymphoid cells has the ability to respond by proliferating following stimulation by antigens of the other cell. In the one-way reaction, the donor cells are treated with mitomycin or irradiation to render them incapable of proliferation. Thus, the donor antigens stimulate the untreated responder cells. Antigenic specificities of the stimulator cells that are not present in the responder cells lead to blastogenesis of the responder lymphocytes. This leads to an increase in the synthesis of DNA and cell division. This process is followed by introduction of a measured amount of tritiated thymidine, which is incorporated into the newly synthesized DNA. In the two-way MLR, lymphoid cells from two individuals are incubated together and total proliferation is measured. The mixed-lymphocyte reaction usually measures a proliferative response and not an effector cell killing response. The test is important in bone marrow and organ transplantation to evaluate the degree of histoincompatibility between donor and recipient. Both CD4+ and CD8+ T lymphocytes proliferate and secrete cytokines in the MLR. It is also called mixed-lymphocyte culture. Mixed leukocyte reaction (MLR):  See mixed-lymphocyte reaction (MLR). Homozygous  describes containing two copies of the same allele. The homozygous typing cell (HTC) technique  is an assay that employs a stimulator cell that is homozygous at the HLA-D locus. An HTC incorporates only a minute amount

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of tritiated thymidine when combined with a homozygous cell in the MLR. This implies that the HTC shares HLA-D determinants with the other cell type. By contrast, when an HTC is combined with a nonhomozygous cell, much larger amounts of tritiated thymidine are incorporated. Many variations between these two extremes are noted in actual practice. Homozygous typing cells are frequently obtained from the progeny of marriages between cousins. Homozygous typing cells (HTCs)  are cells obtained from a subject who is homozygous at the HLA-D locus. HTCs facilitate MLR typing of the human D locus. Lymphocyte determinants  are target cell epitopes identified by lymphocytes rather than antibodies from a specifically immunized host. Cross-match testing  is an assay used in blood typing and histocompatibility testing to ascertain whether or not donor and recipient have antibodies in the serum specific for each other’s cells that might hinder successful transfusion of blood products or transplantation of organs or cells. Cross-matching reduces the changes of graft rejection by preformed antibodies against donor cell surface antigens which are usually MHC antigens. Donor lymphocytes are mixed with recipient serum, complement is added, and the preparation observed for cell lysis. Flow cytometry  can also be used to perform the crossmatching procedure. This method is highly sensitive (considerably more sensitive than the direct cytotoxicity method). Flow cross-matching is also faster and can distinguish antibodies according to class (IgG vs. IgM) and target cell specificity (T cells from B cells). It is a valuable procedure in organ and bone marrow transplantation and is particularly suitable to measuring antibodies against HLA class  I antigens on donor T cells. False positives are rare and most errors are due to low sensitivity (lower antibody concentration). Flow cross-matching has the potential to be standardized and automated. The flow cytometry cross-matching method commonly utilizes F(ab´)2 antihuman IgG conjugated to fluorescein, and anti-CD3 for T cells conjugated to phycoerythrin. A two-parameter display of anti-CD3 vs. IgG is generated. A positive flow cross-match is defined as median channel shift values >40 (Figure 22.15 and Figure 22.16).  emonstration by either serological or flow-cytometric techD niques that preformed antibodies specific for one or more HLA molecules on allogeneic donor cells are present in the blood of a transplant recipient is called a positive crossmatch. Splits  are human leukocyte antigen (HLA) subtypes (Figure  22.17). For example, the base antigen HLA-B12 can be subdivided into the splits HLA-B44 and HLA-B45. The term “split” is used to designate an HLA antigen that was first believed to be a private antigen but later

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Count

31

0

0.1

1000

Figure 22.15  Negative flow crossmatch.

was shown to be a public antigen. The former designation can be placed in parenthesis following its new designation, i.e., HLA-B44 (12). A private antigen  (Figure 22.18) is (1) an antigen confined to one MHC molecule; (2) an antigenic specificity restricted to a few individuals; (3) a tumor antigen restricted to a specific chemically induced tumor; (4) a low-frequency epitope present on red blood cells of fewer than 0.1% of the population, i.e., Pta, By, Bpa, etc.; and (5) HLA antigen encoded by one allele such as HLA-B27. A public antigen (supratypic antigen)  is an epitope which several distinct or private antigens have in common (Figure 22.18). A public antigen such as a blood group antigen is one that is present in greater than 99.9% of a population. It is detected by the indirect antiglobulin (Coombs’ test). Examples include Ve, Ge, Jr, Gya, and Oka. Antigens that occur frequently but are not public antigens include Mns, Lewis, Duffy, P, etc. In blood banking, there is a problem finding a suitable unit of blood for a transfusion to recipients who have developed antibodies against public antigens. Multilocus probes  (Figure 22.19) are used to identify multiple related sequences distributed throughout each person’s genome. Multilocus probes may reveal as many as 20 separate alleles. Because of this multiplicity of alleles, there is only a

Original Broad Specificities A2 A9 A10 A19 A28 B5 B7 B12 B14 B15 B16 B17 B21 B22 B40 B70 Cw3 DR1 DR2 DR3 DR5 DR6 DQ1 DQ3 Dw6 Dw7

Splits and Associated Antigens # A203#, A210# A23, A24, A2403# A25, A26, A34, A66 A29, A30, A31, A32, A33, A74 A68, A69 B51, B52 B703# B44, B45 B64, B65 B62, B63, B75, B76, B77 B38, B39, B3901#, B3902# B57, B58 B49, B50, B4005# B54, B55, B56 B60, B61 B71, B72 Cw9, Cw10 DR103# DR15, DR16 DR17, DR18 DR11, DR12 DR13, DR14, DR1403#, DR1404# DQ5, DQ6 DQ7, DQ8 DQ9 Dw18, Dw19 Dw11, Dw17

Figure 22.17  Splits.

remote possibility that two unrelated persons would share the same pattern, i.e., about 1 in 30 billion. There is, however, a problem in deciphering the multibanded arrrangement of minisatellite RFLPs, as it is difficult to ascertain which bands are allelic. Mutation rates of minisatellite HVRs remain to be demonstrated, but are recognized occasionally. This method can be used in resolving cases of disputed parentage.

Count

18

0

0.1

1000 Positive Flow Crossmatch

Figure 22.16  Positive flow cross-match.

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Private antigen Public antigen

Figure 22.18  Public and private antigens.

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Donor kidney Renal fossa Transplant recipient

Figure 22.20  Orthotopic graft.

An orthotopic graft  (Figure  22.20) is an organ or tissue transplant that is placed in the location that is usually occupied by that particular organ or tissue. Heterotopic  is an adjective that describes the placement of an organ or tissue graft in an anatomic site other than the one where it is normally located.

Figure 22.19  Multilocus probes.

Single locus probes (SLPs)  are probes which hybridize at only one locus. These probes identify a single locus of variable number of tandem repeats (VNTRs) and permit detection of a region of DNA repeats found in the genome only once and located at a unique site on a certain chromosome. Therefore, an individual can have only two alleles that SLPs will identify, as each cell of the body will have two copies of each chromosome, one from the mother and the other from the father. When the lengths of related alleles on homologous chromosomes are the same, there will be only a single band in the DNA typing pattern. Therefore, the use of an SLP may yield either a single- or double-band result from each individual. Single-locus markers such as the pYNH24 probe developed by White may detect loci that are highly polymorphic, exceeding 30 alleles and 95% heterozygosity. SLPs are used in resolving cases of disputed parentage. Immediate spin cross-match  is a test for incompatibility between donor erythrocytes and the recipient patient’s serum. This assay reveals ABO incompatibility in practically all cases, but is unable to identify IgG alloantibodies against erythrocyte antigens. Orthotopic  is an adjective that describes an organ or tissue transplant that has been in the site usually occupied by that organ or tissue.

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A heterotopic graft  is a tissue or organ transplanted to an anatomic site other than the one where it is usually found under natural conditions. For example, the anastomosis of the renal vasculature at an anatomical site that would situate the kidney in a place other than the renal fossa where it is customarily found. A graft  is the transplantation of a tissue or organ from one site to another within the same individual or between individuals of the same or a different species. Heart–lung transplantation  is a procedure that has proven effective for the treatment of primary respiratory disease with dysfunction of gas exchange and alveolar mechanics, together with a secondary elevation in pulmonary vascular resistance, and in primary high-resistance circulatory disorder associated with pulmonary vascular disease. A rescue graft  is a replacement graft for an original graft that failed. Privileged sites  are anatomical locations in the body that are protected from immune effector mechanisms because of the absence of normal lymphatic drainage. Antigenic substances such as tissue allografts may be placed in these sites without evoking an immune response. Privileged sites include the anterior chamber of the eye, the cheek-pouch of the Syrian hamster, and the central nervous system. Tissue allografts in these locations enjoy a period of protection from immunologic rejection, as the diffusion of antigen from graft sites to lymphoid tissues is delayed. Immune privilege alters the induction of immunity to antigens first encountered via

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privileged sites and also inhibits the expression of certain forms of alloimmunity in these same sites. Immunologically privileged sites  are certain anatomical sites within the animal body that provide an immunologically privileged environment which favors the prolonged survival of alien grafts. The potential for development of a blood and lymphatic vascular supply connecting graft and host may be a determining factor in the qualification of an anatomical site as an area which provides an environment favorable to the prolonged survival of a foreign graft. Immunologically privileged areas include (1) the anterior chamber of the eye, (2) the substantia propria of the cornea, (3) the meninges of the brain, (4) the testis, and (5) the cheek pouch of the Syrian hamster. Foreign grafts implanted in these sites show a diminished ability to induce transplantation immunity in the host. These immunologically privileged sites usually fail to protect alien grafts from the immune rejection mechanism in hosts previously or simultaneously sensitized with donor tissues. The capacity of cells expressing Fas ligand to cause deletion of activated lymphocytes provides a possible explanation for the phenomenon of immune privilege. Animals with a deficiency in either Fas ligand or the Fas receptor fail to manifest significant immune privilege. Both epithelial cells of the eye and Sertoli cells of the testes express Fas ligand. Immune privilege is a consequence not only of the lack of an inflammatory response but also from immune

consequences of the accumulation of apoptotic immune cells within a tissue. Immune cell apoptosis may be a signal to terminate inflammation. Apoptotic cell accumulation during an immune response could activate the development of cells that function to downregulate or suppress further immune activation. Thus, immunosuppression by physical barriers, hormone secretion, low numbers of dendritic cells, immune deviation, immunosuppressive cytokines, or Fas killing may facilitate the induction of immunological privilege. Immune privilege:  See immunologically privileged sites. An immunologic barrier  is an anatomical site that diminishes or protects against an immune response. This refers principally to immunologically privileged sites where grafts of tissue may survive for prolonged periods without undergoing immunologic rejection. This is based mainly on the lack of adequate lymphatic drainage in these areas. Examples include prolonged survival of foreign grafts in the brain. A semisyngeneic graft  is a graft that is ordinarily accepted from an individual of one strain into an F1 hybrid of an individual of that strain mated with an individual of a different strain (Figure 22.21). Graft facilitation  is a prolonged graft survival attributable to conditioning of the recipient with IgG antibody, which is

Takes

Rejects

1. Autograft

1. Allograft A strain

B strain

2. Isograft (Syngeneic graft) A strain

A strain

2. Semisyngeneic Graft A strain

(A × B)F1

3. Semisyngeneic Graft A strain

(A × B)F1

Figure 22.21  Semisyngeneic graft.

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believed to act as a blocking factor. It also decreases cell-mediated immunity. This phenomenon is related to immunologic enhancement of tumors by antibody and has been referred to as immunological facilitation (facilitation immunologique). Immunologic facilitation  (  facilitation immunologique) is the slightly prolonged survival of certain normal tissue allografts, e.g., skin, in mice conditioned with isoantiserum specific for the graft. Immunologic enhancement  is the prolonged survival, conversely the delayed rejection, of a tumor allograft in a host as a consequence of contact with specific antibody. Both the peripheral and central mechanisms have been postulated. In the past, coating of tumor cells with antibody was presumed to interfere with the ability of specifically reactive lymphocytes to destroy them, but today a central effect in suppressing cell-mediated immunity, perhaps through suppressor T lymphocytes, is also possible.

1.

Autograft 2. Xenograft Isograft (genetically identical)

Allograft 3.

Enhancement  is the prolonged survival, conversely the delayed rejection, of tumor or skin allografts in individuals previously immunized or conditioned by passive injection of antibody specific for graft antigens. This is termed immunological enhancement and is believed to be due to a blocking effect by the antibody. Enhancing antibodies  are blocking antibodies that favor survival of tumor or normal tissue allografts. An allograft  is an organ, tissue, or cell transplant from one individual or strain to a genetically different individual or strain within the same species. Allografts are also called homografts (Figure 22.22). An allotransplant  refers to the transplantation of an organ or tissue from one individual to another member of the same species. Fetus allograft:  Success of the haplononidentical fetus as an allograft was suggested in the 1950s by Medawar, Brent, and Billingham to rely on four possibilities. This proposal suggested that (1) the conceptus might not be immunogenic, (2) that pregnancy might alter the immune response, (3) that the uterus might be an immunologically privileged site, and (4) that the placenta might represent an effective immunological barrier between mother and fetus. Further studies have shown that transplantation privilege afforded the fetal– placental unit in pregnancy depends on intrauterine mechanisms. The pregnant uterus has been shown not to be an immunologically privileged site. Pregnancies usually are successful in maternal hosts with high levels of preexisting alloimmunity. The temporary status has focused on specialized features of fetal trophoblastic cells that facilitate transplantation protection. Fetal trophoblast protects itself from maternal cytotoxic attack by failing to express on placental villous cytotrophoblast and syncytiotrophoblast any classical

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Figure 22.22  Types of grafts.

polymorphic class I or II MHC antigens. Constitutive HLA expression is also not induced by known upregulators such as interferon y. Thus classical MHC antigens are not expressed throughout gestation. Extravillous cytotrophoblast cells selectively express HLA-G, a nonclassical class I MHC antigen which has limited genetic polymorphism. HLA-G might protect the cytotrophoblast population from MHC-nonrestricted natural killer (NK) cell attack. The trophoblast also protects itself from maternal cytotoxicity during gestation by expressing a high level of complement regulatory proteins on its surface, such as membrane cofactor protein (MCP; CD46), decay accelerating factor (DAF; CD55), and membrane attack complex inhibitory factor (CD59). The maternal immune system recognizes pregnancy, i.e., the fetal trophoblast, in a manner that results in cellular, antibody, and cytokine responses that protect the fetal allograft. CD56 positive large granular lymphocytes may be regulated by hormones in the endometrium that control their function. They have been suggested to be a form of NK cell in arrested maturation possibly due to persistent expression of HLA-G on target invasive cytotrophoblast. Contemporary studies have addressed cytokine interactions at the fetal–maternal tissue interface in pregnancy. HLA-G or other fetal trophoblast antigens have been postulated to possibly stimulate maternal lymphocytes in endometrial tissue to synthesize cytokines and growth factors that act in a paracrine manner beneficial to trophoblast growth and differentiation.

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This has been called the immunotrophism hypothesis. Other cytokines released into decidual tissue include colony stimulating factors (CSFs), tumor necrosis factor α (TNF-α), IL-6, and transforming growth factor 3 (TGF-β). Fetal syncytiotrophoblast  has numerous growth factor receptors. Thus an extensive cytokine network is preset within the uteroplacental tissue that offers both immunosuppressive and growth promoting signals. In humans, IgG is selectively transported across the placenta into the fetal circulation following combination with transporting Fcγ receptors on the placenta. This transfer takes place during the 20th to the 22nd week of gestation. Maternal HLA-specific alloantibody that is specific for the fetal HLA type is bound by nontrophoblastic cells expressing fetal HLA antigens. These include macrophages, fibroblasts, and endothelium within the villous mesenchyme of placental tissue, thereby preventing these antibodies from reaching the fetal circulation. Maternal antibodies against any other antigen of the fetus will likewise be bound within the placental tissues to a cell expressing that antigen. The placenta acts as a sponge to absorb potentially harmful antibodies. Exceptions to placental trapping of deleterious maternal IgG antibodies include maternal IgG antibodies against RhD antigen and certain maternal organ-specific autoantibodies. An allogeneic graft  is an allograft consisting of an organ, tissue, or cell transplant from a donor individual or strain to a genetically different individual or strain within the same species. Homologous  is an adjective that describes something from the same source. For example, an organ allotransplant from one member to a recipient member of the same species, i.e., renal allotransplantation in humans. Allogeneic bone marrow transplantation:  Hematopoietic cell transplants are performed in patients with hematologic malignancies, certain nonhematologic neoplasms, aplastic anemias, and certain immunodeficiency states. In allogeneic bone marrow transplantation the recipient is irradiated with lethal doses either to destroy malignant cells or to create a graft bed. The problems that arise include graft-vs.-host (GVH) disease and transplant rejection. GVH disease occurs when immunologically competent cells or their precursors are transplanted into immunologically crippled recipients. Acute GVH disease occurs within days to weeks after allogeneic bone marrow transplantation and primarily affects the immune system and epithelia of the skin, liver, and intestines. Rejection of allogeneic bone marrow transplants appears to be mediated by NK cells and T cells that survive in the irradiated host. NK cells react against allogeneic stem cells that are lacking self MHC class I molecules and therefore fail to deliver the inhibitory signal to NK cells. Host T cells react against donor MHC antigens in a manner resembling their reaction against solid tissue grafts. Hemopoietic resistance (HR):  Transplantation of allogeneic, parental, or xenogeneic bone marrow or leukemia

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cells into animals exposed to total body irradiation often results in the destruction of the transplanted cells. The mechanism causing the failure of the transplant appears similar with all three types of cells. This phenomenon, designated hemopoietic resistance (HR), has a genetic basis and mechanism different from conventional transplantation reactions against solid tumor allografts. It does not require prior sensitization and apparently involves the cooperation between NK cells and macrophages, both resistant to irradiation. The NK cells have the characteristics of null cells; macrophages play an accessory cell role. The cooperative activity seems to represent in vivo surveillance against leukemogenesis. F1 hybrid resistance  is a condition that results when two inbred mouse strains are crossed and a bone marrow graft from either of the parents is rejected by the F1 offspring. Attributable to variation in NK inhibitory receptor expression by the F1 natural (NK) cells. Homologous chromosomes  are a pair of chromosomes containing the same linear gene sequences, each derived from one parent. Immunoisolation  describes the enclosure of allogeneic tissues such as pancreatic islet cell allografts within a membrane that is semipermeable, but does not itself induce an immune response. Substances of relatively low mol wt can reach the graft through the membrane, while it remains protected from immunologic rejection by the host. Allogeneic inhibition  is the better growth of homozygous tumors when they are transplanted to homozygous syngeneic hosts of the strain of origin than when they are transplanted to F1 hybrids between the syngeneic (tumor) strain and an allogeneic strain. This is manifested as a higher frequency of tumor and shorter latency period in syngeneic hosts. The better growth of tumor in syngeneic than in heterozygous F1 hybrid hosts was initially termed syngeneic preference. When it became apparent that selective pressure against the cells in a mismatching environment produced the growth difference, the phenomenon was termed allogeneic inhibition. Syngeneic preference  is the better growth of neoplasms when they are transplanted to histocompatible recipients than when they are transplanted in histoincompatible recipients. See also allogeneic inhibition. Incompatibility  refers to dissimilarity between the antigens of a donor and recipient as in tissue allotransplantation or blood transfusions. The transplantation of a histoincompatible organ or the transfusion of incompatible blood into a recipient may induce an immune response against the antigens not shared by the recipient in injurious consequences. Homograft  is the earlier term for allograft, i.e., an organ or tissue graft from a donor to a recipient of the same species.

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Homograft reaction  is an immune reaction generated by a homograft (allograft) recipient against the graft alloantigens. Also called an allograft reaction. Homograft rejection  is an earlier term for allograft rejection, i.e., an immune response induced by histocompatibility antigens in the donor graft that are not present in the recipient. This is principally a cell-mediated type of immune response. Homotransplantation:  Homograft, i.e., allograft trans­ plantation. Heterograft:  See xenograft. Heterogeneic:  See xenogeneic. In transplantation biology, heterologous  refers to an organ or tissue transplant from one species to a recipient belonging to another species, i.e., a xenogeneic graft. It also refers to something from a foreign source. A xenograft  (Figure 22.23) is a tissue or organ graft from a member of one species, i.e., the donor, to a member of a different species, i.e., the recipient. It is also called a heterograft. Antibodies and cytotoxic T cells reject xenografts several days following transplantation. Xenogeneic  is an adjective that refers to tissues or organs transplanted from one species to a genetically different species, e.g., a baboon liver transplanted to a human. It refers to the genetic relationship of an individual from one species to a member of a different species. Xenoantigen  is a tissue antigens of one species that induce an immune response in members of a different species. It is an antigen of a xenograft, and is also called heteroantigen. Xenoantibody  is an antibody specific for xenoantigen. Xenoantibodies  are antibodies formed in one species that are specific for antigens of a separate species.

MHC-Ag TCR

CD-28

T cell proliferation

Ag

B7

CTLA41g Mouse T cell

Mouse or human antigen presenting cell

Figure 22.23  Induction of tolerance to a xenogenic tissue graft.

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Xenoreactive  refers to a T cell or antibody response to an antigen of a graft derived from another species. The T lymphocyte may recognize an intact xenogenic MHC molecule or a peptide from a xenogeneic protein bound a self MHC molecule. Xenotransplantation  is organ or tissue transplantation between members of different species. An example of transplantation of tissues or organs from one species to another is a chimpanzee heart transplanted into a human recipient. It represents a possible substitute for the shortage of human organs for clinical transplantation. Xenogeneic transplantation can involve concordant or discordant donors, according to the phylogenetic distance between the species involved. Natural preformed antibodies in a recipient specific for donor endothelial antigens that lead to hyperacute rejection of most vascularized organ transplants now occur in discordant species combinations. The immune response to a xenotransplant resembles the response to an allotransplant. However, there are greater antigenic differences between donor and host in xenotransplantation than in allotransplantation. It procedure is being explored to possibly solve an insufficient supply of human organs for clinical transplantation. Mini pigs  are a strain of diminutive pigs designed specifically to serve as organ and tissue donors for use as xenografts in humans. Their organ sizes are equivalent to those of humans. Delayed xenograft rejection  is a xenograft failure within days to weeks following transplantation. It is attributable to ischemia that is a consequence of graft vascular endothelium hyperactivation caused by binding of anti-galactose-α (1-3) galactose antibodies. It is also called acute vascular rejection. Chronic xenograft rejection  is a xenograft failure that occurs within weeks following transplantation as a consequence of cytotoxic T lymphocytes, NK cells reacting against donor endothelium. It is also called cellular xenograft rejection. Xenozoonosis  is a term that describes transmission of infection that might be the consequence of xenotransplantation. Infections resulting from xenotransplantation might involve infection of recipient cells with endogenous retroviral sequences from donor cells, giving rise themselves or after recombination with human endogenous retroviral sequences to previously unknown pathogenic viruses. Such new viruses might be pathogenic for other human beings in addition to the xenograft recipient. Zoonosis  is a term that describes the general process of cross-species infection. Xenotype  refers to molecular variations based on differences in structure and antigenic specificity. Examples would

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include membrane antigens of cells or immunoglobulins from separate species. A syngraft  is a transplant from one individual to another within the same strain. Syngrafts are also called isografts. Syngeneic  is an adjective that implies genetic identity between identical twins in humans or among members of an inbred strain of mice or other species. It is used principally to see transplants between genetically identical members of a species. Syngeneic individuals possess the same alleles at all genetic loci. An isograft  is a tissue transplant from a donor to an isogenic recipient. Grafts exchanged between members of an inbred strain of laboratory animals such as mice are syngeneic rather than isogenic. Isogeneic (isogenic)  is an adjective implying genetic identity such as identical twins. Although used as a synonym for syngeneic when referring to the genetic relationship between members of an inbred strain (of mice), the inbred animals never show the absolute identity, i.e., identical genotypes, observed in identical twins. Isologous  means derived from the same species. Also called isogeneic or syngeneic.  n antigen found in a member of a species that induces an A immune response if injected into a genetically dissimilar member of the same species is termed an isoantigen.  hese are antigens carrying identical determinants in a given T individual. Isoantigens of two individuals may or may not have identical determinants. In the latter case they are allogeneic with respect to each other and are called alloantigens.  ince the individual red blood cell antigens have the same S molecular structure and are identical in different individuals, they have been referred to in the past as isoantigens. This is only a descriptive term and should not be used, because two individuals may be allogeneic by virtue of the assortment of the antigens present on their red blood cells. An isoantigen is an antigen of an isograft. Isoantibody  is an antibody that is specific for an antigen present in other members of the species in which it occurs. Thus, it is an antibody against an isoantigen. Also called alloantibody. Isoleukoagglutinins  are antibodies in the blood sera of multiparous females and of patients receiving multiple blood transfusions that recognizes surface isoantigens of leukocytes and leads to their agglutination. Leukoagglutinin  is an antibody or other substance that induces the aggregation or agglutination of white blood cells into clumps.

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Relative sibling risk:  A comparison of the occurrence of a disease among siblings with its occurrence in the general population. A donor  is one who offers whole blood, blood products, bone marrow, or an organ to be given to another individual. Individuals who are drug addicts or test positively for certain diseases such as HIV-1 infection or hepatitis B, for example, are not suitable as donors. There are various other reasons for donor rejection not listed here. To be a blood donor, an individual must meet certain criteria which include blood pressure, temperature, hematocrit, pulse, and history. There are many reasons for donor rejection, including low hematocrit, skin lesions, surgery, drugs, or positive donor blood tests. Brain death  is the term for irreversible loss of brain function without trauma to other body systems. Artificial maintenance of the subject’s respiration can be employed to preserve organs for transplantation. A cadaveric organ  is a solid organ secured from a recently deceased donor, i.e., a cadaver, for the purpose of transplantation. An organ bank  is a site where selected tissues for transplantation, such as acellular bone fragments, corneas, and bone marrow, may be stored for relatively long periods until needed for transplantation. Several hospitals often share such a facility. Organs such as kidneys, liver, heart, lung, and pancreatic islets must be transplanted within 48 to 72 h and are not suitable for storage in an organ bank. Organ brokerage  or the selling of an organ such as a kidney from a living related donor to the transplant recipient, is practiced in certain parts of the world but is considered unethical and is illegal in the United States, as it is in violation of the National Organ Transplant Act (Public Law 98-507,3 USC). Adoptive immunity  (Figure  22.24) is the term assigned by Billingham, Brent, and Medawar (1955) to transplantation immunity induced by the passive transfer of specifically Spleen cells

Control

Donor

H-Cl Primary response Recipient

H-Cl

H-Cl

H-Cll

H-Cl

Secondary response

Primary response

Figure 22.24  Adoptive immunity.

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immune lymph node cells from an actively immunized animal to a normal (previously nonimmune) syngeneic recipient host. Adoptive immunization  is the passive transfer of immunity by the injection of lymphoid cells from a specifically immune individual to a previously nonimmune recipient host. The resulting recipient is said to have adoptive immunity. Adoptive transfer  is a synonym for adoptive immunization. The passive transfer of lymphocytes from an immunized individual to a nonimmune subject with immune system cells such as CD4+ T lymphocytes. Tumor-reactive T cells have been adoptively transferred for experimental cancer therapy. Leukocyte transfer:  See adoptive transfer. Lymphocyte transfer reaction:  See normal lymphocyte transfer reaction. Normal lymphocyte transfer reaction:  The intracutaneous injection of an individual with peripheral blood lymphocytes from a genetically dissimilar, allogeneic member of the same species leads to the development of a local, erythematous reaction that becomes most pronounced after 48 h. The size of the reaction has been claimed to give some qualitative indication of histocompatibility or histoincompatibility between a donor and recipient. This test is not used in clinical practice. A direct reaction  is a skin reaction caused by the intracutaneous injection of viable or nonviable lymphocytes into a host that has been sensitized against donor tissue antigens. This represents a type IV hypersensitivity reaction, which is classified as a delayed-type reaction mediated by T cells. Reactivity is against lymphocyte surface epitopes. A skin graft  uses skin from the same individual (autologous graft) or donor skin that is applied to areas of the body surface that have undergone third-degree burns. A patient’s keratinocytes may be cultured into confluent sheets that can be applied to the affected areas, although these may not “take” because of the absence of type IV collagen 7 S basement membrane sites for binding and fibrils to anchor the graft.

Rat

Diabetic Rat

A skin-specific histocompatibility antigen  is a murine skin minor histocompatibility antigen termed Sk that can elicit rejection of skin but not other tissues following transplantation from one parent into the other parent that has been irradiated and rendered a chimera by the previous injection of F1 spleen cells. The two parents are from different inbred strains of mice. The rate of rejection is relatively slow. Immunologic tolerance of F1 murine spleen cells to the skin epitope of the parent in which they are not in residence is abrogated following residence in the opposite parent. A split thickness graft  is a skin graft that is only 0.25 to 0.35 mm thick and consists of epidermis and a small layer of dermis. These grafts vascularize rapidly and last longer than do regular grafts. They are especially useful for skin burns, contaminated skin areas, and sites that are poorly vascularized. Thick split thickness grafts are further resistant to trauma, produce minimal contraction, and permit some amount of sensation, but graft survival is poor. Pancreatic transplantation  (Figure  22.25) is a treatment for diabetes. Either a whole pancreas or a large segment of it, obtained from cadavers, may be transplanted together with kidneys into the same diabetic patient. It is important for the patient to be clinically stable and for there to be as close a tissue (HLA antigen) match as possible. Graft survival is 50 to 80% at 1 year. Islets of Langerhans  are groups of endocrine cells within the exocrine pancreas that consist of α cells that secrete glucagon, β cells that secrete insulin, and δ  cells that secrete somatostatin. Islet cell transplantation  is an experimental method aimed at treatment of type I diabetes mellitus. The technique has been successful in rats but less so in man. It requires sufficient functioning islets from a minimum of two cadaveric donors that have been purified, cultured, and shown to produce insulin. The islet cells are administered into the portal vein. The liver serves as the host organ in the recipient who is treated with FK506 or other immunosuppressant drugs. Bone marrow  is a soft tissue within bone cavities that contains hematopoietic precursor cells and hematopoietic cells that are maturing into erythrocytes, the five types of leukocytes,

Freshly isolated Lewis islet cells inoculated into thymus, along with antilymphocyte serum.

Islet cells transplanted under renal capsule in animals with sustained intrathymic transplant and normal blood glucose.

Thymus removed. Rat still has normal blood glucose due to functional transplant of islet cells under renal capsule.

Figure 22.25  Protocol for pancreas transplant.

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and thrombocytes. Whereas red marrow is hemopoietic and is present in developing bone, ribs, vertebrae, and long bones, some of the red marrow may be replaced by fat and become yellow marrow. Bone marrow cells are stem cells from which the formed elements of the blood, including erythrocytes, leukocytes, and platelets, are derived. B lymphocyte and T lymphocyte precursors are abundant. The B lymphocytes and pluripotent stem cells in bone marrow are important for reconstitution of an irradiated host. Bone marrow transplants are useful in the treatment of aplastic anemia, leukemias, and immunodeficiencies. Patients may donate their own marrow for subsequent bone marrow autotransplantation if they are to receive intense doses of irradiation. Bone marrow cells  are stem cells from which the formed elements of the blood, including erythrocytes, leukocytes, and platelets, are derived. B lymphocyte and T lymphocyte precursors are abundant. The B lymphocytes and pluripotent stem cells in bone marrow are important for reconstitution of an irradiated host. Bone marrow transplants are useful in the treatment of aplastic anemia, leukemias, and immunodeficiencies. Patients may donate their own marrow for subsequent bone marrow autotransplantation if they are to receive intense doses of irradiation. Bone marrow transplantation  is the inoculation of a recipient with donor bone marrow, including stem cells, which serve as precursors for all mature cellular elements of the blood including lymphocytes. It is a procedure used to treat both nonneoplastic and neoplastic conditions not amenable to other forms of therapy. It has been especially used in cases of aplastic anemia, acute lymphocytic leukemia, and acute nonlymphocytic leukemia. A total of 750 ml of bone marrow are removed from the iliac crest of an HLA-matched donor. Following appropriate treatment of the marrow to remove bone spicules, the cell suspension is infused intravenously into an appropriately immunosuppressed recipient who has received whole-body irradiation and immunosuppressive drug therapy. Bone marrow cells derived from a patient during disease remission may be held frozen in liquid nitrogen for a future autologous bone marrow transplant, which permits the subject to receive his or her own cells. GVH episodes, acute graft-vs.-host disease (GVHD), or chronic GVHD may follow bone marrow transplantation in selected subjects. The immunosuppressed patients are highly susceptible to opportunistic infections.

685

This mode of therapy has improved considerably the survival rate of some leukemia patients. Immunotoxin:  The linkage of monoclonal antibody, or monoclonal antibody derivative, specific for target cell antigens with a cytotoxic substance such as the toxin ricin yields an immunotoxin. Upon parenteral injection, its antibody portion directs the immunotoxin to the target and its toxic portion destroys target cells on contact. Among its uses is the purging of T cells from hematopoietic cell preparations used for bone marrow transplantation. Immunotoxin is a substance produced by the union of a monoclonal antibody or one of its fractions to a toxic molecule such as a radioisotope, a bacterial or plant toxin, or a chemotherapeutic agent. The antibody portion is intended to direct the molecule to antigens on a target cell, such as those of a malignant tumor, and the toxic portion of the molecule is for the purpose of destroying the target cell. Contemporary methods of recombinant DNA technology have permitted the preparation of specific hybrid molecules for use in immunotoxin therapy. Immunotoxins may have difficulty reaching the intended target tumor, may be quickly metabolized, and may stimulate the development of antiimmunotoxin antibodies. Cross-linking proteins may likewise be unstable. Immunotoxins have potential for antitumor therapy and as immunosuppressive agents. Platelet-associated immunoglobulin (PAIgG)  is present in 10% of normal individuals, 50% of those with tumors, and 76% of septic patients, and may be induced by GVHD. PAIgG is present in 71% of autologous marrow graft recipients and in 50% of allogeneic marrow graft recipients. Autologous  is an adjective that refers to derivation from self. The term describes grafts or antigens taken from an individual and returned to the same subject from which they were derived. An autograft  is a graft of tissue taken from one area of the body and placed in a different site on the body of the same individual, e.g., grafts of skin from unaffected areas to burned areas in the same individual. Autologous graft  refers to the donation of tissue such as skin or bone marrow by the same individual who will subsequently receive it either at a different anatomical site, as in skin autografts for burns, or at a later date, or as in autologous bone marrow transplants.

BMT  is the abbreviation for bone marrow transplantation. Autologous bone marrow transplantation (ABMT)  is a bone marrow transplant by a donor who may, at a later date, become the recipient of the same marrow transplant. Leukemia patients in relapse may donate marrow which can be stored and readministered to them following a relapse. Leukemic cells are removed from the bone marrow which is cryopreserved until needed. Prior to reinfusion of the bone marrow, the patient receives supralethal chemoradiotherapy.

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Bone marrow chimera:  The inoculation of an irradiated recipient mouse with bone marrow from an unirradiated donor mouse which ensures that lymphocytes and other cellular elements of the blood will be of donor genetic origin. They have been useful in demonstrating lymphocyte and other blood cell development. Stem cells  have two unique biological features that include self-renewal and multilineage differentiation potential. In

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the past, stem cells were divided into two types that include the pluripotential stem cell and the committed stem cell. Pluripotential stem cells were the progenitors of many different hematopoietic cells, whereas the progeny of committed stem cells were of one cell type. “Committed stem cell” is now termed “progenitor cell.” Stem cells arise from yolk sac blood islands and usually are noncycling. They are not morphologically recognizable. Cell culture studies have yielded much information about hematopoietic precursor cells. Hematopoietic stem cells express the progenitor cell antigen CD34, which can be detected using monoclonal antibodies and by flow cytometry. A hematopoietic stem cell  is a bone marrow cell that is undifferentiated and serves as a precursor for multiple cell lineages. These cells are also demonstrable in the yolk sac and later in the liver in the fetus. HSC  is the abbreviation for hematopoietic stem cell. HSC mobilization  is the inoculation of a donor with G-CSF or GM-CSF to induce hematopoietic stem cell proliferation in the bone marrow. These cells subsequently spill over from the stimulated bone marrow into the peripheral blood from which they can be harvested for use in hematopoietic stem cell transplantation by leukapheresis. Hematopoietic stem cell (HSC) transplants  are used to reconstitute hematopoietic cell lineages and to treat neoplastic diseases. A total of 25% of allogeneic marrow transplants in 1995 were performed using hematopoietic stem cells obtained from unrelated donors. Since only 30% of patients requiring an allogeneic marrow transplant have a sibling that is HLA-genotypically identical, it became necessary to identify related or unrelated potential marrow donors. It became apparent that complete HLA compatibility between donor and recipient is not absolutely necessary to reconstitute patients immunologically. Transplantation of unrelated marrow is accompanied by an increased incidence of GVHD. Removal of mature T lymphocytes from marrow grafts decreases the severity of GVHD but often increases the incidence of graft failure and disease relapse. HLA-phenotypically identical marrow transplants among relatives are often successful. HSC transplantation provides a method to reconstitute hematopoietic cell lineages with normal cells capable of continuous self-renewal. The principal complications of HSC transplantation are GVHD, graft rejection, graft failure, prolonged immunodeficiency, toxicity from radiochemotherapy given pre- and posttransplantation, and GVHD prophylaxis. Methrotrexate and cyclosporin A are given to help prevent acute GVHD. Chronic GVHD may also be a serious complication involving the skin, gut, and liver and an associated sicca syndrome. Allogenic HSC transplantation often involves older individuals and unrelated donors. Thus, blood stem cell transplantation represents an effective method for the treatment of patients with hematologic and nonhematologic malignancies and various types of

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immunodeficiencies. The in vitro expansion of a small number of CD34+ cells stimulated by various combinations of cytokines appears to give hematopoietic reconstitution when reinfused after a high-dose therapy. Recombinant human hematopoietic growth factors (HGF) (cytokines) may be given to counteract chemotherapy treatment–related myelotoxicity. HGF increase the number of circulating progenitor and stem cells, which is important for the support of highdose therapy in autologous as well as allogeneic HSC transplantation. Both the bone marrow and umbilical cord blood serve as sources for hematopoietic stem cells. A hematopoietic cell transplant (HCT)  is the inoculation of hematopoietic stem cells from the peripheral blood, induced from the bone marrow, or occasionally from umbilical cord blood of a histocompatible donor into a recipient with an injured immune system. Solid organ transplant  is the term for the surgical transfer of a kidney, heart, lung, liver or skin from a donor to a recipient. A chimera  (Figure 22.26) is the presence in an individual of cells of more than one genotype. This can occur rarely under natural circumstances in dizygotic twins, as in cattle, who share a placenta in which the blood circulation has become fused, causing the blood cells of each twin to circulate in the other. More commonly, it refers to humans or other animals who have received a bone marrow transplant that provides a cell population consisting of donor and self cells. Tetraparental chimeras can be produced by experimental manipulation. The name chimera derives from a monster of Greek mythology that had the body of a goat, the head of a lion, and the tail of a serpent. Chimerism  is the presence of two genetically different cell populations within an animal at the same time. Hematopoietic chimerism:  A successful bone marrow trans­ plant leads to a state of hematological and/immunological

Figure 22.26  Chimera.

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chimerism in which donor type blood cells coexist permanently with host type tissues, without manifesting alloreactivity to each other. Usually incomplete or mixed hematopoietic chimerism are generated following bone marrow transplantation in which both host type and donor type blood cells can be detected in the recipient. In bone marrow transplantation, not only is immune reactivity against donor type cells an obstacle to bone marrow engraftment, there is also the problem of GVHD-mediated by donor T cells reactive against host antigens. See chimera. Full chimerism  is the state in which all of an individual’s hematopoietic cells are of donor origin. This results when a bone marrow or hematopoietic stem cell transplant is performed following myeloablative conditioning to eliminate all of the recipient’s hematopoietic cells. Myeloablative conditioning  is a type of therapy used prior to bone marrow transplantation in which a patient’s hematopoietic cells in the bone marrow are eliminated through the use of aggressive chemotherapy and total body irradiation, causing depletion of immune system cells from both the peripheral blood and the secondary lymphoid tissues. This procedure is necessary prior to hematopoietic cell transplantation or bone marrow transplantation. Mixed chimerism  occurs in a non-myeloablative conditioned hematopoietic cell transplant recipient, who has received a donor cell infusion. The recipient’s surviving hematopoietic stem cells coexist with donor hematopoietic stem cells and yield cells of the myeloid and lymphoid lineages. Non-myeloablative conditioning  is a diminished level of chemotherapy that induces only partial depletion of the bone marrow as a part of the conditioning process prior to transplantation.

Thus, the lymphocytes are genetically different from the surroundings in which they develop. These chimeric mice have yielded significant data in the investigation of lymphocyte development. Microchimerism  is the establishment in a transplant recipient of passenger donor hematopoietic cells that accompanied the solid organ transplant. Backcross  refers to a crossing of a heterozygous organism and a homozygote. The term commonly refers to the transfer of a particular gene from one background strain/stock to an inbred strain via multigenerational matings to the desired strain. Breeding an F1 hybrid with either one of the strains that produced it. Corneal transplants  (Figure 22.27) are different from most other transplants in that the cornea is a “privileged site.” These sites do not have a lymphatic drainage. The rejection rate in corneal transplants depends on vascularization; if vascularization occurs, the cornea becomes accessible to the immune system. HLA incompatibility increases the risk of rejection if the cornea becomes vascularized. The patient can be treated with topical steroids to cause local immunosuppression.  ertain anatomical sites within the animal body provide an C immunologically privileged environment which favors the prolonged survival of alien grafts. The potential for development of a blood and lymphatic vascular supply connecting graft and host may be a determining factor in the qualification of an anatomical site as an area which provides an environment favorable to the prolonged survival of a foreign

Vascularized

A donor cell infusion  is the administration of donor bone marrow or hematopoietic stem cells to the recipient of a solid organ transplant to establish chimerism and donor cell acceptance. Radiation chimera:  See irradiation chimera. An irradiation chimera  is an animal or human whose lymphoid and myeloid tissues have been destroyed by lethal irradiation and successfully repopulated with donor bone marrow cells that are genetically different. Total body irradiation (TBI)  is used on hematopoietic cell transplant recipients. It is the administration of sufficient ionizing radiation over the whole body to destroy hematopoietic cells in the bone marrow. Radiation bone marrow chimeras:  Mice that have been subjected to heavy radiation and then reconstituted with allogeneic bone marrow cells, i.e., from a different mouse strain.

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Unvascularized

Figure 22.27  Corneal transplant.

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Mitogen

T cell

La molecule

AEF

B cell

Plasma cells

Figure 22.28  Allogeneic effect factor.

graft. Immunologically privileged sites include (1) the anterior chamber of the eye, (2) the substantia propria of the cornea, (3) the meninges of the brain, (4) the testis, and (5) the cheek pouch of the Syrian hamster. Foreign grafts implanted in these sites show a diminished ability to induce transplantation immunity in the host. These immunologically privileged sites usually fail to protect alien grafts from the immune refection mechanism in hosts previously or simultaneously sensitized with donor tissues. Leptin  the antiobesity hormone, is an endothelial cell mitogen and chemoattractant, and it induces angiogenesis in a cornea implant model. Endothelial cells express OB-Rβ, the leptin receptor. The allogeneic effect  (Figure  22.28) is the synthesis of antibody by B cells against a hapten in the absence of carrier-specific T cells, provided allogeneic T lymphocytes are present. Interaction of allogeneic T cells with the MHC class II molecules of B cells causes the activated T lymphocytes to produce factors that facilitate B-cell differentiation into plasma cells without the requirement for helper T lymphocytes. There is allogeneic activation of T cells in the GVH reaction. Alloreactive  is the recognition by antibodies or T lymphocytes from one member of a species cell or tissue antigens of a genetically nonidentical member. Alloreactivity  is the stimulation of immune system T cells by non self MHC molecules attributable to antigenic differences between members of the same species. It represents the immune response to an alloantigen based on recognition of allogeneic MHC. Allogeneic disease  includes the pathologic consequences of immune reactivity of bone marrow allotransplants in immunosuppressed recipient patients as a result of GVH reactivity in genetically dissimilar members of the same species. Homologous disease:  See allogeneic disease and graft-vs.host disease (GVHD). Alloimmunization  is defined as an immune response provoked in one member or strain of a species with an alloantigen derived from a different member or strain of the same species. Examples include the immune response in man following

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transplantation of a solid organ graft such as a kidney or heart from one individual to another. Alloimmunization with red blood cell antigens in humans may lead to pathologic sequelae, such as hemolytic disease of the newborn (erythroblastosis fetalis) and in a third Rh(D)+ baby born to an Rh(D)– mother. Allogeneic (or allogenic)  is an adjective that describes genetic variations or differences among members or strains of the same species. The term refers to organ or tissue grafts between genetically dissimilar humans or unrelated members of other species. Alloantiserum  is an antiserum generated in one member or strain of a species not possessing the alloantigen (e.g., histocompatibility antigen), with which they have been challenged, that is derived from another member or strain of the same species. Allorecognition  is the detection of allelic differences manifested by cells of one member of the species by lymphocytes of another individual. It usually concerns identification of MHC-encoded differences.  he direct pathway of allorecognition is the process whereby T transplant a recipient’s T cells are stimulated by interaction of their receptors with the transplant donor’s dendritic allogeneic HLA molecules. Direct allorecognition  is the process whereby the T cells of a transplant recipient detect peptide/allo-MHC epitopes on allogeneic graft donor cell surfaces. Refer to allorecognition. An alloreactive T cell  is a T lymphocyte from one member of a species capable of responding to an allogeneic antigen from another member of the same species. A take  is the successful grafting of skin that adheres to the recipient graft site 3 to 5 d following application. This is accompanied by neovascularization as indicated by a pink appearance. Thin grafts are more likely to “take” than thicker grafts, but the thin graft must contain some dermis to be successful. The term “take” also refers to an organ allotransplant that has survived hyperacute and chronic rejection. Engraftment  is the phase during which transplanted bone marrow manufactures new blood cells. Graft rejection  (Figure 22.29) is an immunologic destruction of transplanted tissues or organs between two members or strains of a species differing at the MHC for that species (i.e., HLA in man and H-2 in the mouse). The rejection is based upon both cell-mediated and antibody-mediated immunity against cells of the graft by the histoincompatible reci­ pient. First-set rejection usually occurs within 2 weeks after transplantation. The placement of a second graft with the same antigenic specificity as the first in the same host leads

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Immunological rejection  is the destruction of an allograft or even a xenograft in a recipient host whose immune system has been activated to respond to the foreign tissue antigens.

(a) 5 days White graft 12 at 14 days First set 7 to 8 days Second set

0

2 4 6 8 10 12 Time of Rejection (in days, after transplantation) First Set Rejection (skin graft rejection)

14

(b) Figure 22.29  (a) Types of skin graft rejection. (b) Immuno­ fluorescent “staining” of C4d in peritubular capillaries.

to rejection within 1 week and is termed second-set rejection. This demonstrates the presence of immunological memory learned from the first experience with the histocompatibility antigens of the graft. When the donor and recipient differ only at minor histocompatibility loci, rejection of the transplanted tissue may be delayed, depending upon the relative strength of the minor loci in which they differ. Grafts placed in a hyperimmune individual, such as those with preformed antibodies, may undergo hyperacute or accelerated rejection. Hyperacute rejection of a kidney allograft by preformed antibodies in the recipient is characterized by formation of fibrin plugs in the vasculature as a consequence of the antibodies reacting against endothelial cells lining vessels, complement fixation, polymorphonuclear neutrophil attraction, and denuding of the vessel wall, followed by platelet accumulation and fibrin plugging. As the blood supply to the organ is interrupted, the tissue undergoes infarction and must be removed. Immunofluorescent “staining” of C4d  in peritubular capillaries of renal allograft biopsies reveals a humoral component of rejection (Figure 22.29a). First-set rejection  is an acute form of allograft rejection in a nonsensitized recipient. It is usually completed in 12 to 14 d and is mediated by type IV (delayed-type) hypersensitivity to graft antigens.

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Rejection  is an immune response to an organ allograft such as a kidney transplant. Hyperacute rejection is due to preformed antibodies and is apparent within minutes following transplantation. Antibodies reacting with endothelial cells cause complement to be fixed, which attracts polymorphonuclear neutrophils, resulting in denuding of the endothelial lining of the vascular walls. This causes platelets and fibrin plugs to block the blood flow to the transplanted organ, which becomes cyanotic and must be removed. Only a few drops of bloody urine are usually produced. Segmental thrombosis, necrosis, and fibrin thrombi form in the glomerular tufts. There is hemorrhage in the interstitium and mesangial cell swelling; IgG, IgM, and C3 may be deposited in arteriole walls. Acute rejection occurs within days to weeks following transplantation and is characterized by extensive cellular infiltration of the interstitium. These cells are largely mononuclear cells and include plasma cells, lymphocytes, immunoblasts, and macrophages, as well as some neutrophils. Tubules become separated, and the tubular epithelium undergoes necrosis. Endothelial cells are swollen and vacuolated. There is vascular edema, bleeding with inflammation, renal tubular necrosis, and sclerosed glomeruli. Chronic rejection occurs after more than 60 d following transplantation and may be characterized by structural changes such as interstitial fibrosis, sclerosed glomeruli, mesangial proliferative glomerulonephritis, crescent formation, and various other changes. Second-set rejection  is rejection of an organ or tissue graft by a host who is already immune to the histocompatibility antigens of the graft as a consequence of rejection of a previous transplant of the same antigenic specificity as the second, or as a consequence of immunization against antigens of the donor graft. The accelerated second-set rejection compared to rejection of a first graft is reminiscent of a classic secondary or booster immune response. Second-set response  is a term that describes the accelerated rejection of a second skin graft from a donor that is the same as or identical with the first donor. The accelerated rejection is seen when regrafting is performed within 12 to 80 d after rejection of the first graft. It is completed in 7 to 8 d and is due to sensitization of the recipient by the first graft. Hyperimmunized individual:  A person who has formed alloantibodies against an antigen to which the subject was previously exposed, such as a prior allograft, blood transfusion, or pregnancy. May sometimes be attributable to natural antibodies specific for antigenic determinants of pathogens but which cross-react with allogeneic donor antigens of a graft. Indirect antigen presentation:  In organ or tissue transplantation, the mechanism whereby donor allogeneic MHC

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molecules present microbial proteins. The recipient professional antigen-presenting cells process allogeneic MHC proteins. The resulting allogeneic MHC peptides are presented, in association with recipient (self) MHC molecules, to host T  lymphocytes. By contrast, recipient T cells recognize unprocessed allogeneic MHC molecules on the surface of the graft cells in direct antigen presentation. Indirect allorecognition:  Refer presentation.

to

indirect

antigen

White graft rejection  is an accelerated rejection of a second skin graft performed within 7 to 12 d after rejection of the first graft. It is characterized by lack of vascularization of the graft and its conversion to a white eschar. The characteristic changes are seen by day 5 after the second grafting procedure. The transplanted tissue is rendered white because of hyperacute rejection, such as a skin or kidney allograft. Preformed antibodies occlude arteries following surgical anastomosis, producing infarction of the tissue graft. ALG  is an abbreviation for antilymphocyte globulin. ALS (antilymphocyte serum) or ALG (antilymphocyte globulin):  See antilymphocyte serum. Antilymphocyte serum (ALS) or antilymphocyte globulin (ALG)  is an antiserum prepared by immunizing one species, such as a rabbit or horse, with lymphocytes or thymocytes from a different species, such as a human. Antibodies present in this antiserum combine with T cells and other lymphocytes in the circulation to induce immunosuppression. ALS is used in organ transplant recipients to suppress graft rejection (Figure  22.30). The globulin fraction known as ALG rather than whole antiserum produces the same immunosuppressive effect. Antithymocyte globulin (ATG):  IgG isolated from the blood serum of rabbits or horses hyperimmunized with human thymocytes is used in the treatment of aplastic anemia patients and to combat rejection in organ transplant recipients. The Antilymphocyte globulin (ALG)

Lymphocyte Cell surface

Figure 22.30  Antilymphocyte globulin (ALG).

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equine ATG contains 50mg/ml of immunoglobulin and has yielded 50% recovery of bone marrow and treated aplastic anemia patients. ATG  is an abbreviation for antithymocyte globulin. Anti-target antigen antibodies:  Antibodies used to block MHC class II molecules to prolong allograft survival or to remove Rh(D) positive cells to prevent sensitization of Rh(D) negative mothers. Lymphocyte immune globulin (injection):  Indicated in renal transplantation for the management of allograft rejection in renal allotransplant recipients. When administered with conventional therapy at the time of rejection, it increases the frequency of resolution of the acute rejection episode. May be used also in conjunction with other immunosuppressive therapy to delay the onset of the first rejection episode. Indicated also in aplastic anemia for the treatment of moderate to severe aplastic anemia patients who are unsuitable for bone marrow transplantation. Unlabeled uses include its use as an immunosuppressant in liver, bone marrow, heart and other organ transplants, treatment of multiple sclerosis, myasthenia gravis, pure red cell aplasia and scleroderma even though efficacy is fully established. Mouse immunoglobulin antibodies:  A total of 40% of human subjects may harbor heteroantibodies that include human antimouse antibodies (HAMA). HAMA in serum may induce falsely elevated results in immunoassays that involve mouse antibodies. This may represent a problem in organ transplant patients who receive mouse monoclonal antibodies such as anti-CD3, anti-CD4, and anti-IL-2R for treatment. Orthoclone OKT3  is a commercial antibody against the T cell surface marker CD3. It may be used therapeutically to diminish T cell reactivity in organ allotransplant recipients experiencing a rejection episode; OKT3 may act in concert with the complement system to induce T cells lysis, or may act as an opsonin, rendering T cells susceptible to phagocytosis. Rarely, recirculating T lymphocytes are removed in patients experiencing rejection crisis by thoracic duct drainage or extracorporeal irradiation of the blood. Plasma exchange is useful for temporary reduction in circulating antibody levels in selective diseases, such as hemolytic disease of the newborn, myasthenia gravis or Goodpasture syndrome. Immunosuppressive drugs act on all of the T and B cell maturation processes. Transplantation rejection  (Figure  22.31) is the consequence of cellular and humoral immune responses to a transplanted organ or tissue that may lead to loss of function and necessitate removal of the transplanted organ or tissue. Transplantation rejection episodes occur in many transplant recipients, but are controlled by such immunosuppressive drugs as cyclosporine, rapamycin, or FK506, or by monoclonal antibodies against T lymphocytes.

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Antigen

T cell

Kidney cells

zed

siti

S en

es c yt pho m y l

y molec

Antibod

Plasma cell Lympth node

ules

Figure 22.31  Rejection.

Host-vs.-graft disease (HVGD)  is a consequence of humoral and cell-mediated immune response of a recipient host to donor graft antigens.

Figure 22.33  Hyperacute rejection of renal allotransplant showing swelling and purplish discoloration. This is a bivalved transplanted kidney. The allograft was removed within a few hours following transplantation.

Hyperacute rejection  (Figures 22.32 to 22.37) is due to preformed antibodies and is apparent within minutes following transplantation. Antibodies reacting with endothelial cells cause complement to be fixed, which attracts polymorphonuclear neutrophils, resulting in denuding of the endothelial lining

Figure 22.34  A bivalved transplanted kidney showing hyperacute rejection. There is extensive pale cortical necrosis. This kidney was removed 5 d after transplantation.

Capillary thrombosis

Renal histology showing Hyperacute graft rejection

Figure 22.32  Schematic representation of hyperacute graft rejection.

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Figure 22.35  Microscopic view of hyperacute rejection showing a necrotic glomerulus infiltrated with numerous polymorphonuclear leukocytes. H&E stained section 25X.

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Figure 22.36  A high-power view of the same necrotic glomerulus shown in Figure 22.35. There are large numbers of polymorphonuclear leukocytes present. Extensive endothelial cell destruction is apparent. H&E stained section 50X.

Figure 22.38  Acute rejection of a renal allograft in which the capsular surface shows several hemorrhagic areas. The kidney is tremendously swollen.

of the vascular walls. This causes platelets and fibrin plugs to clock the blood flow to the transplanted organ that becomes cyanotic and must be removed. Only a few drops of bloody urine are usually produced. Segmental thrombosis, necrosis, and fibrin thrombi form in the glomerular tufts. There is hemorrhage in the interstitium, mesangial cell swelling, IgG, and IgM, and C3 may be deposited in arteriole walls.

extensive cellular infiltration of the interstitium. These cells are largely mononuclear cells and include plasma cells, lymphocytes, immunoblasts, and macrophages as well as some neutrophils. Tubules become separated and the tubular epithelium undergoes necrosis. Endothelial cells are swollen and vaculoated. There is vascular edema, bleeding with inflammation, renal tubular necrosis, and sclerosed glomeruli.

Hyperacute graft rejection (HAR)  is accelerated allograft rejection attributable to preformed antibodies in the circulation of the recipient that are specific for antigens of the donor. It is produced by complement activation induced by preformed antibodies that recognize allogeneic epitopes on the graft vasculature. These antibodies react with antigens, which may be HLA class I antigens or ABO blood group antigens, of endothelial cells lining capillaries of the donor organ. It sets in motion a process that culminates in fibrin plugging of the donor organ vessels, resulting in ischemia and loss of function and necessitating removal of the transplanted organ. It is a Type II hypersensitivity mechanism.

 cute rejection is a type of graft rejection in which T lymA phocytes, macrophages, as well as antibodies may mediate vascular and tissue injury that may commence a week following transplantation. The response to the graft includes the activation of effector T lymphocytes as well as the formation of antibodies that may mediate the process.

HAR  is the abbreviation for hyperacute graft rejection.

Acute cellular rejection  is acute graft rejection mediated by recipient cytotoxic T lymphocytes and delayed type hypersensitivity reactions against allogeneic graft cells.

Acute rejection  (Figures 22.38 to 22.44) occurs within days to weeks following transplantation and is characterized by

Figure 22.37  Microscopic view of hyperacute rejection showing necrosis of the wall of a small arteriole.

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Acute graft rejection  is the recipient host rejection of a transplanted solid organ or tissue transplant within days or weeks following transplantation. The mechanism may be by either acute cellular rejection or antibody-mediated acute humoral rejection.

Figure 22.39  Acute rejection of a bivalved kidney. The cut surface bulges and is variably hemorrhagic and shows fatty degeneration of the cortex.

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Transplantation Immunology

Figure 22.40  Microscopic view of the interstitium revealing predominantly cellular acute rejection. There is an infiltrate of variabily sized lymphocytes. There is also an infiltrate of eosinophils.

Acute humoral rejection  is a type of acute graft rejection in which antibodies are produced against allogeneic antigens in the graft, leading to vascular inflammation and neutrophil infiltration. Referred to also as delayed graft rejection or delayed vascular rejection. Characterized by C4d “staining” by immunofluorescence of peritubular capillaries in renal allotransplants undergoing acute humoral rejection. Chronic rejection  (Figures  22.45 to 22.47) occurs after more than 60 d following transplantation and may be characterized by structural changes such as interstitial fibrosis, sclerosed glomeruli, mesangial proliferative glomerulonephritis, crescent formation, and various other changes. Chronic rejection  is a type of allograft rejection that occurs during a prolonged period following transplantation and is characterized by structural changes such as fibrosis with loss of normal organ architecture. The principal pathologic change is degeneration and occlusion of arteries linking the graft to the host. This results from intimal smooth muscle cell proliferation and has been referred to as graft arteriosclerosis. It is induced by antibodies against graft HLA class I alloantigens.

Figure 22.41  Microscopic view of acute rejection showing interstitial edema. Mild lymphocytic infiltrate. In the glomerulus, there is also evidence of rejection with a thrombus at the vascular pole.

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Figure 22.42  A higher magnification of the thrombus at the hilus of the glomerulus.

Chronic graft rejection  is an anti-allograft immune response with features of fibrosis, collagen deposition and chronic graft vasculopathy, that appear several months following transplantation and lead to cessation of allograft function. Chronic graft vasculopathy  is the proliferation of smooth muscle cell arteries or arterioles of a graft induced by growth factors and cytokines released from recipient T cells activated by allorecognition that stimulate monocytes/macrophages in the graft and endothelial cells in blood vessel walls to release these substances. Narrowing of the graft vasculature leads to ischemia. Graft arteriosclerosis  is characterized by intimal smooth muscle cell proliferation that occludes graft arteries. It may occur 6 to 12 months following transplantation and leads to chronic rejection of vascularized organ grafts. It is probably attributable to a chronic immune response to alloantigens of the vessel wall. It is also termed accelerated arteriosclerosis. The graft-vs.-host reaction (GVHR)  is the reaction of a graft containing immunocompetent T cells against alloantigens of the genetically dissimilar tissues of an

Figure 22.43  A trichrome stain of a small interlobular artery showing predominantly humoral rejection. There is tremendous swelling of the intima and endothelium with some fibrin deposition and a few polymorphonuclear leukocytes.

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Figure 22.44  Immunofluorescence preparation showing humo­ ral rejection with high-intensity fluorescence of arteriolar walls and of some glomerular capillary walls. This pattern is demonstrable in antiimmunoglobulin and anticomplement stained sections.

immunosuppressed recipient. Criteria requisite for a GVHR include (1) histoincompatibility between the donor and recipient, (2) passively transferred immunologically reactive cells, and (3) a recipient host who has been either naturally immunosuppressed because of immaturity or genetic defect, or deliberately immunosuppresed by irradiation or drugs. The immunocompetent grafted cells are especially reactive against rapidly dividing cells. Target organs include the skin, gastrointestinal tract (including the gastric mucosa), and liver, as well as the lymphoid tissues. Patients often develop skin rashes and hepatosplenomegaly and may have aplasia of the bone marrow. GVHR usually develops within 7 to 30 d following the transplant or infusion of the lymphocytes. Prevention of the GVHR is an important procedural step in several forms of transplantation and may be accomplished by irradiating the transplant. The clinical course of GVHR may take a hyperacute, acute, or chronic form as seen in graft rejection. GVH:  See graft-vs.-host reaction and graft-vs.-host disease. Secondary disease  is a condition that occurs in irradiated animals whose cell population has been reconstituted with histoincompatible, immunologically competent cells derived

Figure 22.45  Renal allotransplant showing chronic rejection. The kidney is shrunken and malformed.

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Figure 22.46  Microscopic view of chronic rejection showing tubular epithelial atrophy with interstitial fibrosis and shrinkage of glomerular capillary tufts.

from allogeneic donor animals. Ionizing radiation induces immunosuppression in the recipients, rendering them incapable of rejecting the foreign cells. Thus, the recipient has two cell populations, its own and the one that has been introduced, making these animals radiation chimeras. After an initial period of recovery, the animals develop a secondary runt disease, which is usually fatal within 1 month. Posttransfusion graft-vs.-host disease  is a condition that resembles postoperative erythroderma that occurs in immunocompetent recipients of blood. There is dermatitis, fever, marked diarrhea, pancytopenia, and liver dysfunction. Graft-vs.-host disease (GVHD)  is a disease produced by the reaction of immunocompetent T lymphocytes of the donor graft that are histoincompatible with the tissues of the recipient into which they have been transplanted. It is attributable to MHC or MiHA mismatching. For the disease to occur, the recipient must be either immunologically immature, immunosuppressed by irradiation or drug therapy, or tolerant to the administered cells, and the grafted cells must also be immunocompetent. Patients develop skin rash, fever, diarrhea, weight loss, hepatosplenomegaly, and aplasia of the bone marrow. The donor lymphocytes

Figure 22.47  The wall of an artery in chronic rejection. There is obliteration of the vascular lumen with fibrous tissue. Only a slitlike lumen remains.

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Transplantation Immunology

infiltrate the skin, gastrointestinal tract, and liver. The disease may be either acute or chronic. Murine GVH disease is called “runt disease,” “secondary disease,” or “wasting disease.” Both allo- and autoimmunity associated with GVHD may follow bone marrow or hematopoietic stem cell transplantation. A total of 20 to 50% of patients receiving HLAidentical bone marrow transplants still manifest GVHD with associated weight loss, skin rash, fever, diarrhea, liver disease, and immunodeficiency. GVHD may be either acute, which is an alloimmune disease, or chronic, which consists of both allo- and autoimmune components. The conditions requisite for the GVH reaction include genetic differences between immunocompetent cells in the marrow graft and host tissues, immunoincompetence of the host, and alloimmune differences that promote proliferation of donor cells that react with host tissues. In addition to allogenic marrow and hematopoietic stem cell grafts, the transfusion of unirradiated blood products to an immunosuppressed patient or intrauterine transfusion from mother to fetus may lead to GVHD. GVHD is most common in bone marrow and hematopoietic stem cell transplantation but rare in solid organ transplants. GVH disease:  See graft-vs.-host disease. Toxic epidermal necrolysis  is a hypersensitivity reaction to certain drugs such as allopurinol, nonsteroidal antiinflammatory drugs, barbiturates, sulfonamides such as sulfmethoxazole-trimethoprim, carbamazepine, and other agents. It may closely resemble erythema multiforme. Patients develop erythema, subepidermal bullae, and open epidermal lesions. They become dehydrated, show imbalance of electrolytes, and often develop abscesses with sepsis and shock. Toxic epidermal necrolysis may also be observed in a hyperacute type of graft-vs.-host reaction, especially in some babies receiving bone marrow transplants. Graft-vs.-leukemia (GVL):  Bone marrow or stem cell transplantation as therapy for leukemia. Partial genetic incompatibility between donor and recipient is believed to facilitate elimination of residual leukemia cells by NK cells and T lymphocytes from the allogeneic transplant. This is a very desirable consequence of MiHA and MHC mismatches between recipient and donor. It is also termed graft-versustumor effect.

695

Figure 22.48  A diffuse erythematous to morbilliform rash in a child with acute graft-vs.-host disease (GVHD).

from adult chickens. Splenic lymphocytes are increased and represent a mixture of both donor and host lymphocytes. Acute graft-vs.-host reaction  the immunopathogenesis of acute GVHD, consists of recognition, recruitment, and effector phases. Epithelia of the skin (Figures  22.48 to 22.52), gastrointestinal tract (Figures 22.53 to 22.57), small intrahepatic biliary ducts, and liver (Figures 22.58 to 22.60), and the lymphoid system constitute primary targets of acute GVHD. GVHD development may differ in severity based on relative antigenic differences between donor and host and the reactivity of donor lymphocytes against non-HLA antigens of recipient tissues. The incidence and severity of GVHD has been ascribed also to HLA-B alleles, i.e., an increased GVHD incidence associated with HLA-B8 and HLA-B35. Epithelial tissues serving as targets of GVHD include keratinocytes, erythrocytes, and bile ducts, which may express Ia antigens following exposure to endogeneous interferon produced by T lymphocytes. When Ia antigens are expressed on nonlymphoid cells, they may become antigenpresenting cells for autologous antigens and aid perpetuation of autoimmunity. Cytotoxic T lymphocytes  mediate acute GVHD. While most immunohistological investigations have implicated CD8+ (cytotoxic/suppressor) lymphocytes, others have identified

Parabiotic intoxication  is the result of a surgical union of allogeneic adult animals. The course of immune reactivity can be modified to take a single direction by uniting parental and F1 animals. A hybrid recognizes parental cells as its own and does not mount an immune response against them, but alloantigens of F1 hybrid cells stimulate the parental cells leading to graft-vs.-host disease. The Simonsen phenomenon  is a graft-vs.-host reaction in chick embryos that have developed splenomegaly following inoculation of immunologically competent lymphoid cells

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Figure 22.49  Diffuse erythematous skin rash in a patient with acute graft-vs.-host reaction (GVHR).

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Figure 22.50  Histologically, there is an intense interface dermatitis with destruction of basal cells, particularly at the tips of the rete ridges, incontinence of melanin pigment, and necrosis of individual epithelial cells, referred to as apoptosis.

CD4+ (T helper lymphocytes) in human GVHD, whereas natural killer (NK) cells have been revealed as effectors of murine but not human GVHD. Following interaction between effector and target cells, cytotoxic granules from cytotoxic T or NK cells are distributed over the target cell membrane, leading to perforin-induced large pores across the membrane and nuclear lysis by deoxyribonuclease. Infection, rather than failure of the primary target organ (other than gastrointestinal bleeding), is the major cause of mortality in acute GVHD. Within the first few months posttransplant, all recipients demonstrate diminished immunoglobulin synthesis, decreased T helper lymphocytes, and increased T suppressor cells. Acute GVHD patients manifest an impaired ability to combat viral infections. They demonstrate an increased risk of cytomegalovirus (CMV) infection, especially CMV interstitial pneumonia. GVHD may also reactivate other viral diseases such as herpes simplex.

Atlas of Immunology, Third Edition

Figure 22.52  Papulosquamous rash in graft-vs.-host disease.

antibody or those in which T lymphocytes have been depleted account for most cases of BCLD, which is associated with severe GVHD. All transformed B cells in cases of BCLD have manifested the Epstein–Barr viral genome. Chronic graft-vs.-host disease (GVHD)  may occur in as many as 45% of long-term bone marrow transplant recipients. Chronic GVHD (Figure  22.61) differs both clinically and histologically from acute GVHD and resembles autoimmune connective tissue diseases. For example, chronic GVHD patients may manifest skin lesions resembling scleroderma; sicca syndrome in the eyes and mouth;

I mmunodeficiency in the form of acquired B cell lympho­ proliferative disorder (BCLD) represents another serious complication of post–bone marrow transplantation. Bone marrow transplants treated with pan-T cell monoclonal

Figure 22.51  Histological appearance of the skin in graft-vs.host disease with disruption of the basal cell layer, hyperkeratosis, and beginning sclerotic change.

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Figure 22.53 and Figure 22.54  Gastrointestinal graft-vs.host disease in which there is a diffuse process that usually involves the ileum and cecum, resulting in secretory diarrhea. Grossly there is diffuse erythema, granularity, and loss of folds, and when severe, there is undermining and sloughing of the entire mucosa, leading to fibrinopurulent clots of necrotic material. Sometimes there is frank obstruction in patients with intractable graft-vs.-host disease.

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Figure 22.58  The earliest lesions are characterized by individual enterocyte necrosis with karyorrhectic nuclear debris, the socalled exploding crypt, which progresses to a completely destroyed crypt as shown in the upper left-hand corner.

Figure 22.55  Stenotic and fibrotic segments alternating with more normal-appearing dilated segments of gut in graft-vs.-host disease.

inflammation of the oral, esophageal, and vaginal mucosa; bronchiolitis obliterans; occasionally myasthenia gravis; polymyositis; and autoantibody synthesis. Histopathologic alterations in chronic GVHD, such as chronic inflammation and fibrotic changes in involved organs, resemble changes associated with naturally occurring autoimmune disease. The skin may reveal early inflammation with subsequent fibrotic changes. Infiltration of lacrimal, salivary, and submucosal glands by lymphoplasmacytic cells leads ultimately to fibrosis. The resulting sicca syndrome, which resembles Sjögren’s syndrome, occurs in 80% of chronic GVHD patients. Drying of mucous membranes in the sicca syndrome affects the mouth, esophagus, conjunctiva, urethra, and vagina. The pathogenesis of chronic GVHD involves the interaction of alloimmunity, immune dysregulation, and resulting immunodeficiency and autoimmunity. The increased incidence of infection among chronic GVHD patients suggests immunodeficiency. The dermal fibrosis is

Figure 22.56  Sloughing of the mucosal lining of the gut in graft-vs.-host disease.

Figure 22.57  Histologically, graft-vs.-host disease in the gut begins as a patchy destructive enteritis localized to the lower third of the crypts of Lieberkuhn.

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Figure 22.59  Hepatic graft-vs.-host disease is characterized by a cholestatic hepatitis with characteristic injury and destruction of small bile ducts that resemble changes seen in rejection. In this section of early acute GVHD, there are mild portal infiltrates with striking exocytosis into bile ducts associated with individual cell necrosis and focal destruction of the bile ducts.

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Figure 22.60  This liver section from a patient with GVHD demonstrates the cholestatic changes that evolve from hepatocellular ballooning to cholangiolar cholestasis with bile microliths, which signifies prolonged GVHD.

associated with increased numbers of activated fibroblasts in the papillary dermis. T lymphocyte or mast cell cytokines may activate this fibroplasia, which leads to dermal fibrosis in chronic GVHD. OKT®3 (Orthoclone OKT®3)  is a commercial mouse monoclonal antibody against the T cell surface marker CD3. It may be used, therapeutically, to diminish T cell reactivity in organ allotransplant recipients experiencing a rejection episode. OKT3 may act in concert with the complement system to induce T cell lysis, or it may act as an opsonin, rendering T cells susceptible to phagocytosis. Venoocclusive disease (VOD)  is a serious liver complication after marrow transplantation (Figure  22.62). Histopathology of early VOD reveals concentric subendothelial widening and sublobular central venules with degeneration of surrounding pericentral hepatocytes. At this early stage, there is deposition of fibrin and Factor VIII. Late lesions of VOD show fibrous obliteration of the central venule and sinusoids by combination of type 3, 1, and even type 4 collagen. The clinical diagnosis of

Figure 22.61  Chronic GVHD of the liver with pronounced inflammation and portal fibrosis with disappearance of bile ducts.

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Figure 22.62  Venoocclusive disease (VOD) accompanying graft-vs.-host disease of the liver. On the left is early VOD with concentric subendothelial widening and sublobular central venules with degeneration of surrounding pericentral hepatocytes. There is deposition of fibrin and Factor VIII. On the right is a late lesion of VOD showing fibrous obliteration of the central venule and the sinusoids by combination of types 3, 1, and even type 4 collagen.

VOD is reasonably accurate based on the combination of jaundice, ascites, hepatomegaly, and encephalopathy in the first 2 weeks posttransplant. The incidence may be higher among older patients with diagnosis of AML or CML and with hepatitis. The mortality rate of VOD is relatively high at 32%. Post-transplant lymphoproliferative disorder (PTLD):  A group of B cell lymphomas occurring in immunosuppressed patients following organ transplantation. It is an uncommon condition occurring in 0.2% of patients within one year of transplant, with an annual incidence of 0.04% thereafter. Risk of the disease is higher in children and recipients of heart transplants. It is an uncontrolled proliferation of B lymphocytes following infection with Epstein–Barr virus. Production of IL-10, an endogenous anti-T cell cytokine, has also been implicated. In immunocompetent patients, Epstein–Barr virus causes infectious mononucleosis, characterized by proliferation of B lymphocytes, which is controlled by suppressor T cells. However, calcineurin inhibitors such as tacrolimus and cyclosporin, used as immunosuppressants in T cell function, can prevent the control of the B cell proliferation. Depletion of T cells by the use of anti-T cell antibodies in the prevention or treatment of transplant rejection further increases the risk of developing post-transplant lymphoproliferative disorder. Such antibodies include ATG, ALG, and OKT3. Polyclonal PTLD may form tumor masses and present with symptoms due to a mass effect, e.g., symptoms of bowel obstruction. Monoclonal forms of PTLD form a disseminated malignant lymphoma. It may spontaneously regress on reduction or cessation of immunosuppressant medication and can also be treated with addition of anti-viral therapy. In some cases it will progress to non-Hodgkin’s lymphoma and may be fatal.

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Tumor Immunology

Biologists have long been fascinated with possible differences between neoplastic cells (Figures  23.1 to 23.3) and their so-called normal counterparts or tissues of origin. This led to the search for antigens on tumors that are absent from normal tissues. The aim of finding such immunologic differences would be both for cancer testing and for cancer treatment purposes. This search has been met with varying degrees of success.

suppressor genes, or DNA repair genes. Carcinogens comprise the epigenetic type that does not damage DNA but causes other physiological alterations that predispose to cancer, and the genotoxic type that reacts directly with DNA or with micromolecules that then react with DNA.

A neoplasm  is any new and abnormal growth that may be either a benign or malignant tumor.

A carcinoma  is a malignant tumor composed of epithelial cells that infiltrate surrounding tissues and lead to metastases.

Cancer  is an invasive, metastatic, and highly anaplastic cellular tumor that leads to death. Neoplasms are often divided into two broad categories of carcinoma and sarcoma.

A choriocarcinoma  is an unusual malignant neoplasm of the placenta trophoblast cells in which the fetal neoplastic cells are allogeneic in the host. On rare occasions, these neoplasms have been “rejected” spontaneously by the host. Antimetabolites have been used in the treatment of choriocarcinoma.

 odification of proteins by phosphorylation or specific proM teolysis may change their covalent architecture to yield new antigenic determinants or epitopes termed neoantigens. The epitope is newly expressed on cells during development or in neoplasia. Neoantigens include tumor-associated antigens. New antigenic determinants may also emerge when a protein changes conformation or when a molecule is split, exposing previously unexpressed epitopes. A neoantigen may be produced by the union of a xenobiotic with a self protein. Carcinogenesis  is a multi-step sequence consisting of initiation, promotion, progression and malignant conversion through which a cell may progress to deregulation of cell growth resulting in malignant transformation. Initiation  is the beginning event in carcinogenesis. It occurs when a mutation that is not repaired in a cell gives it an irreversible growth advantage and makes it a target cell for subsequent malignant transformation. Malignant conversion  is stage IV of carcinogenesis. It is the progressive accumulation of mutations in neoplastic cells, rendering it a malignant tumor with total lack of growth regulation. The tumor may then become invasive and metastatic.

A complete carcinogen  is a cancer-causing agent that can promote all four stages of carcinogenesis.

Sarcomas  are malignant neoplasms that arise from connective tissue cells, including muscle, bone, or cartilage. Blastomas  are highly undifferentiated malignant tumors of children. The cells appear similar to those of a blastocyst. “Sneaking through”  is the successful growth of a sparse number of transplantable tumor cells that have been inoculated into a host in contrast to the induction of tumor immunity and lack of tumor growth in the same host if larger doses of the same cells are administered. Autochthonous  is an adjective that indicates “pertaining to self,” occuring in the same subject. Also called autologous. Spontaneous cancer  is a malignant neoplasm that arises in a laboratory animal without experimental intervention. Sporadic cancer  is a malignant neoplasm of humans induced by somatic cell rather than germ cell transformation. Refractory cancer  is a tumor that is not responsive to conventional chemotherapy or radiation treatment.

Malignant transformation  is an alteration in a cell rendering it neoplastic. A neoplasm is characterized by uncontrolled growth, invasive properties and metastatic potential. Left untreated, it can lead to death of the host.

Salvage therapy  is the term for a heroic effort to rescue a patient with refractory cancer through an innovative or experimental treatment.

A carcinogen  is any chemical or physical cancer-producing agent through mutation or deregulating oncogenes, tumor-

Remission  is the term for a disease being clinically undetectable following treatment. Remission of a hematopoietic 699

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Melanoma cell

Platelet

CD44 P-selectin

P-selectin

SLe*

SLe* CD44

Cancer cell

VLA-4 ICAM-1 VCAM-1 SLe*

SLe*

E-selectin

E-selectin

Figure 23.1  Schematic representation of a tumor cell attached through E-selectin molecules to an endothelial cell surface.

neoplasm is defined as a complete clinical response of at least 4 weeks following therapy. Spontaneous remission  is the reversal of progressive growth of the neoplasm with inadequate or no treatment. Spontaneous remission occurs only rarely. A tumor  is a neoplasm developing as a consequence of uncontrolled cell proliferation. Benign tumors are self-limiting, whereas malignant tumors may be invasive. A benign tumor  is an abnormal proliferation of cells that leads to a growth that is localized and contained within epithelial barriers. It does not usually lead to death, in contrast to a malignant tumor.

E-selectin VCAM-1 ICAM-1 LFA-1

SLe

VLA-4

Leukemia cell

Malignant  is an adjective that means leading to death, as by a malignant neoplasm. Anaplastic  is an adjective describing tumor cells that are poorly differentiated and capable of aggressive growth. Indolent  is an adjective denoting a tumor in which the transformed cells undergo relatively slow growth and progression of the neoplasm is gradual. An invasive tumor  is a neoplasm capable of accessing and obliterating the healthy architecture of adjacent organs. A primary tumor  is the original neoplasm arising from the first transformed cell. Tumor hypoxia  develops in parts of a tumor that has outgrown its blood supply, causing a diminished oxygen pressure. Sensitivity to radiation and chemotherapy become less effective as tumor cells undergo hypoxia. Tumor regression  is a decrease in size or remission of a malignant tumor as a consequence of anti-cancer therapy, or may occur spontaneously without treatment. Metastasis  is the transfer of disease from one organ or part to another not directly connected with it. For example, malignant tumors may seed anatomical sites distant from the primary tumor’s site of origin, leading to the establishment of secondary tumors.

CD44 ICAM-1

Figure 23.2  Schematic representation of a leukemia cell attached to an endothelial cell surface via adhesion molecules.

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Figure 23.3  Schematic representation of a melanoma cell attached to an endothelial cell surface through a VLA-4-VCAM-1 interaction.

Metastases  are secondary tumors that arise from daughter cells released by spread from a primary tumor to other anatomical sites.

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Malignolipin (historical)  is a substance claimed in the past to be specific for cancer and to be detectable in the patient’s blood early in the course of the disease. This is no longer considered valid. Malignolipin is comprised of fatty acids, phosphoric acid choline, and spermine. When injected into experimental animals, it can produce profound anemia, leukopoiesis, and cachexia. Tumor-suppressor genes  are genes, such as p53 and PTEN, that encode cellular proteins that prevent cells from becoming neoplastic; i.e., its absence promotes carcinogenesis. p53  is the tumor suppressor gene that is most frequently mutated in human neoplasms. It acts as a sentinel of genomic stability. It prompts arrest of the cell cycle to permit repair of DNA or facilitates apoptosis of cells in which the injury cannot be repaired. Li-Fraumeni syndrome  is an inherited susceptibility to selected tumors, usually attributable to germline mutations of p53. Cowden syndrome  is an increased propensity to develop various neoplasms as a consequence of germline mutations in the tumor suppressor gene PTEN. Oncogenes  are genes that, when deregulated, are linked to carcinogenesis. Such genes may encode positive regulators of cell growth. They may be activated by mutation or retroviral integration. They are involved in the control of cell growth and when defective in structure or expression can lead to abnormal cell proliferation resulting in tumor generation. They have the capacity to induce neoplastic transformation of cells. They are derived from either normal genes termed protooncogenes or from oncogenic RNA (oncorna) viruses. Their protein products are critical for regulation of gene expression or growth signal transduction. Translocation, gene amplification, and point mutation may lead to neoplastic transformation of protooncogene. Oncogenes may be revealed through use of viruses that induce tumors in animals or by derivation of tumor-causing genes from cancer cells. There are more than 20 protooncogenes and cellular oncogenes in the human genome. An oncogene alone cannot produce cancer. It must be accompanied by malignant transformation which involves multiple genetic steps. Oncogenes encode four types of proteins that include growth factors, receptors, intracellular transducers, and nuclear transcription factors. The oncogene theory  is a concept of carcinogenesis that assigns tumor development to latent retroviral gene activation through irradiation or carcinogens. These retroviral genes are considered to be normal constituents of the cell. Following activation, these oncogenes are presumed to govern the neoplasm through hormones that are synthesized and even the possible construction of a complete oncogenic virus. This concept states that all cells may potentially become malignant.

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Oncogenesis  is the process whereby tumors develop. Oncomouse  is a commercially developed transgenic animal into which human genes have been introduced to make the mouse more susceptible to neoplasia. This transgenic mouse is used for both medical and pharmaceutical research. A protooncogene  is a cellular gene that shows homology with a retroviral oncogene. It is found in normal mammalian DNA and governs normal proliferation and probably also differentiation of cells. Mutation or recombination with a viral genome may convert a protooncogene into an oncogene, signifying that it has become activated. Oncogenes may act in the induction and/or maintenance of a neoplasm. Protooncogenes united with control elements may induce transformation of normal fibroblasts into tumor cells. Examples of protooncogenes are c-fos, c-myc, c-myb, c-ras, etc. Alteration of the protooncogenes, leading to synthesis of an aberrant gene product, is believed to facilitate its becoming tumorigenic. An elevation in the quantity of gene product produced is also believed to be associated with protooncogenes becoming tumorigenic. Cellular oncogene:  See protooncogene. Ras  is one of a group of 21-kDa guanine nucleotide-binding proteins with intrinsic GTPase activity that participates in numerous different signal transduction pathways in a variety of cells. Ras gene mutations may be associated with tumor transformation. Ras is attracted to the plasma membrane by tyrosine phosphorylated adapter proteins during T lymphocyte activation, where GDP–GTP exchange factors are activated. GTP-Ras then activates the MAP kinase cascade that results in fos gene expression and assembly of AP-1 transcription factor. Ras:  See small G proteins. Rous sarcoma virus (RSV)  is an RNA type C oncovirus that is single stranded and produces sarcomas in chickens. It is the typical acute transforming retrovirus. Within its genome are gag, pol, env, and v-src genes; gag encodes a core protein, pol encodes reverse transcriptase, and env encodes envelope glycoprotein. V-src is an oncogene associated with the oncogenic capacity of the virus. A promoter  is (1) the DNA molecular site where RNA polymerase attaches and the point at which transcription is initiated. The promoter is frequently situated adjacent to the operator, and upstream from it is an operon. A TATA box and a promoter are both required for immunoglobulin gene transcription. (2) In tumor biology, a promoter mediates the second stage or promotion stage in the process of carcinogenesis. It may be a substance that can induce a tumor in an experimental animal that has been previously exposed to a tumor initiator. Yet the promoter alone is not carcinogenic.

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A v-myb oncogene  is a genetic component of an acute transforming retrovirus that leads to avian myeloblastosis. It represents a truncated genetic form of c-myb. Tumor promoter:  See phorbol ester(s). Amphiregulin  is a glycoprotein member of the epidermal growth factor (EGF) family of proteins. The carboxyl-terminal amino acid residues of amphiregulin positions 46–84 share much sequence homology with the EGF family of proteins. The actions of amphiregulin are wide ranging, including the stimulation of proliferation of certain tumor cell lines, fibroblasts, and various other normal cells. These actions are mediated by binding to EGF receptors possessing intrinsic tyrosine kinase activity. Tumor imaging  is an experimental and clinical medical technique employed to localize neoplastic lesions in a body using a labeled antibody or its fragment. Tumor imaging is based on the presence of an antigen expressed only on a tumor cell or at least has a significant difference in amount and/or distribution between the tumor and normal tissues. Tumor-associated antigens  (Figure  23.4) are epitopes of selected tumor cells that are also found on certain types of normal cells. They may be protein or carbohydrate molecules expressed in abnormal concentration, location, or time in a tumor cell compared with expression in a healthy differentiated cell in the tissue of origin. Normal cellular genes that have become dysregulated encode tumor-associated antigens. Examples include normal cellular proteins, differentiation antigens, tissue type-specific antigens, embryonic antigens, and idiotypic antigens. These antigens, designated as CA-125, CA-19-9, and CA195, among others, may be linked to certain tumors such as lymphomas, carcinomas, sarcomas, and melanomas, but the immune response to these tumor-associated antigens is not sufficient to mount a successful cellular or humoral immune response against the neoplasm. Three classes of tumor-associated antigens have been described. Normal cells

Neoplastic cells

Related neoplastic cells

Class I TAA TAA

Class II TAA TAA Class III

TAA

TAA

Figure 23.4  Schematic representation of tumor-associated antigens (TAA) among normal and neoplastic cells.

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Class I antigens are very specific for a certain neoplasm and are absent from normal cells. Class II antigens are found on related neoplasms from separate individuals. Class III antigens are found on malignant as well as normal cells but show increased expression in the neoplastic cells. Assays of clinical value will probably be developed for class II antigens, since they are associated with multiple neoplasms and very infrequently found in normal individuals. TAA  is the abbreviation for tumor-associated antigens. The normal cellular antigen (class of TAA)  occurs when there is overexpression of a normal macromolecule on tumor cells. It is frequently induced by gene amplification. Refer to tumor-associated antigens. Tumor antigens  are cell surface proteins on tumor cells that can induce a cell-mediated and/or humoral immune response. See also tumor-associated antigens and tumor-specific antigens. Thymicleukemia antigen (TL)  is an epitope on thymocyte membrane of TL + mice. As the T lymphocytes mature, antigen disappears but resurfaces if leukemia develops. TL antigens are specific and are normally present on the cell surface of thymocytes of certain mouse strains. They are encoded by a group of structural genes located at Tla locus, in the linkage group IX, very close to the D pole of the H-2 locus on chromosome 17. There are three structural Tl genes, one of which has two alleles. The TL antigens are numbered from 1 to 4 specifying four antigens: TL.1, TL.2, TL.3, and TL.4. Antigens TL.3 and TL.4 are mutually exclusive. Their expression is under the control of regulatory genes, apparently located at the same Tla locus. Normal mouse thymocytes belong to three phenotypic groups: Tl−, Tl.2, and TL.1, 2, 3. Development of leukemia in the mouse induces a restructuring of the TL surface antigens of thymocytes with expression of TL.1 and TL.2 in TL − cells, expression of TL.1 in TL.2 cells, and expression of TL.4 in both TL − and TL.2 cells. When normal thymic cells leave the thymus, the expression of TL antigen ceases. Thus, thymocytes are TL+ (except the TL −) and the peripheral T cells are TL −. In transplantation experiments TL+ tumor cells undergo antigenic modulation. Tumor cells exposed to homologous antibody stop expressing the antigen and thus escape lysis when subsequently exposed to the same antibody plus complement. CD10 (CALLA)  is an antigen, also referred to as common acute lymphoblastic leukemia antigen (CALLA), that has a mol wt of 100 kDa. CD10 is now known to be a neutral endopeptidase (enkephalinase). It is present on many cell types, including stem cells, lymphoid progenitors of B and T cells, renal epithelium, fibroblasts, and bile canaliculi. Prostate-specific antigen (PSA)  is a marker in serum or tissue sections for adenocarcinoma of the prostate. PSA is a 33-kDa proteolytic enzyme found exclusively in benign and

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malignant epithelium of the prostate. Men with PSA levels of 0 to 4.0 ng/ml and a nonsuspicious digital rectal examination are generally not biopsied for prostate cancer. Men with PSA levels of 10.0 ng/ml and above typically undergo a prostate biopsy. About one-half of these men will be found to have prostate cancer. Certain kinds of PSA, known as bound PSA, link themselves to other proteins in the blood. Other kinds of PSA, known as free PSA, float by themselves. Prostate cancer is more likely to be present in men who have a low percentage of free PSA relative to the total amount of PSA. This finding is especially valuable in helping to differentiate between cancer and other benign conditions, thus eliminating unnecessary biopsies among men in that diagnostic gray zone who have total PSA levels between 4.0 and 10.0 ng/ ml. The PSA molecule is smaller than prostatic acid phosphatase (PAP). In patients with prostate cancer, preoperative PSA serum levels are positively correlated with the disease. PSA is more stable and shows less diurnal variation than does PAP. PSA is increased in 95% of new cases for prostatic carcinoma compared with a 60% increase for PAP; PSA is increased in 97% of recurrent cases compared with a 66% increase of PAP. PAP may also be increased in selected cases of benign prostatic hypertrophy and prostatitis, but these elevations are less than those associated with adenocarcinoma of the prostate. It is inappropriate to use either PSA or PAP alone as a screen for asymptomatic males. TUR, urethral instrumentation, prostatic needle biopsy, prostatic infarct, or urinary retention may also result in increased PSA values. PSA is critical for the prediction of recurrent adenocarcinoma in postsurgical patients. In a minority of cases of prostate cancer, especially those confined to the prostate, serum PSA is not elevated. Refinements in PSA testing values increased its diagnostic significance. This includes rate of change of PSA values with time, i.e., PSA velocity, determination of the ratio between the serum PSA value and volume of the prostate gland, i.e., PSA density, and the measurement of free versus bound forms of circulating PSA. Free PSA levels >25% suggest a lower risk for cancer, whereas levels below 10% are bothersome. These parameters may be most useful when PSA levels range between 4 and 10 ng/ml, the so-called “gray zone.” PSA is also a useful immunocytochemical marker for primary and metastatic adenocarcinoma of the prostate. PSA  is the abbreviation for prostate-specific antigen. Capromab pendetide  is a murine monoclonal antibody specific for prostate-specific membrane antigen. When labeled with isotopic indium (111In), it is useful for immunoscintigraphy in prostate cancer patients. Oncofetal antigens  (Figure  23.5) are markers or epitopes present in fetal tissues during development but not present, or found in minute quantities, in adult tissues. These cellcoded antigens may reappear in certain neoplasms of adults due to derepression of the gene responsible for their formation. Examples include carcinoembryonic antigen (CEA),

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α Surface antigen

Embryonic cell

(gene α is functional)

α Gene α Gene repressed

Normal adult cell (repressed α gene)

α Surface antigen

α Gene derepressed

Malignant adult cell (derepressed α gene)

Figure 23.5  Oncofetal antigen.

which is found in the liver, intestine, and pancreas of the fetus, and also in both malignant and benign gastrointestinal conditions. Yet it is useful to detect recurrence of adenocarcinoma of the colon based upon demonstration of CEA in the patient’s serum; α-fetoprotein (AFP) is demonstrable in approximately 70% of hepatocellular carcinomas. A fetal or oncofetal antigen  that is expressed as a normal constituent of embryos and not in adult tissues. It is reexpressed in neoplasms of adult tissues, apparently as a result of derepression of the gene responsible for its formation. Cancer-testis antigens  are proteins normally present only on spermatogonia and spermatocytes that become tumorassociated antigens when they appear on other types of cells as a consequence of transformation. Carcinoma-associated antigens  are self antigens whose epitopes have been changed due to effects produced by certain tumors. Self antigens are transformed into a molecular structure for which the host is immunologically intolerant. Examples include the T antigen, which is an MN blood group precursor molecule exposed by the action of bacterial enzymes, and Tn antigen, which is a consequence of somatic mutation in hematopoietic stem cells caused by inhibition of galactose transfer to N-acetyl-d-galactosamine. Embryonic antigens  are protein or carbohydrate antigens synthesized during embryonic and fetal life that are either absent or formed in only minute quantities in normal adult subjects. Aberrant expression by a tumor cell can render it a tumor-associated antigen. Alpha-fetoproteins (AFP) and carcinoembryonic antigen (CEA) are fetal antigens that may be synthesized once again in large amounts in individuals with certain tumors. Their detection and level during the course of the disease and following surgery to remove a tumor reducing the substance may serve as a diagnostic and prognostic indicator of the disease process. Blood group antigens, such as the iI, which are reversed in their levels of expression in the fetus and in the adult, may show a reemergence of i antigen in adult patients with thalassemia and hypoplastic anemia. Cold autoagglutinins specific for it may be found in infectious mononucleosis patients. Common acute lymphoblastic leukemia antigen (CD10) is rarely found on peripheral blood cells of normal subjects, whereas CALLA cells coexpressing IgM and CD19 molecules may be found in fetal bone marrow

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Figure 23.6  Carcinoembryonic antigen (CEA).

and peripheral blood samples. CD10 may be expressed in children with common acute lymphoblastic leukemia. Carcinoembryonic antigen (CEA)  (Figure  23.6) is a 200-kDa membrane glycoprotein epitope that is present in the fetal gastrointestinal tract in normal conditions. However, tumor cells, such as those in colon carcinoma, may reexpress it. CEA was first described as a screen for identifying carcinoma by detecting nanogram quantities of the antigen in serum. It was later shown to be present in certain other conditions as well. CEA levels are elevated in almost one-third of patients with colorectal, liver, pancreatic, lung, breast, head and neck, cervical, bladder, medullary thyroid, and prostatic carcinoma. However, the level may be elevated also in malignant melanoma, lymphoproliferative disease, and smokers. Regrettably, CEA levels also increase in a variety of nonneoplastic disorders, including inflammatory bowel disease, pancreatitis, and cirrhosis of the liver. Nevertheless, determination of CEA levels in the serum is valuable for monitoring

the recurrence of tumors in patients whose primary neoplasm has been removed. If the patient’s CEA level reveals a 35% elevation compared with the level immediately following surgery, this may signify metastases. This oncofetal antigen is comprised of one polypeptide chain with one variable region at the amino terminus and six constant region domains. CEA belongs to the immunoglobulin superfamily. It lacks specificity for cancer, thereby limiting its diagnostic usefulness. It is detected with a mouse monoclonal antibody directed against a complex glycoprotein antigen present on many human epithelial-derived tumors. This reagent may be used to aid in the identification of cells of epithelial lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-CEA antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells show immunoreactivity. Clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. CEA  is the abbreviation for carcinoembryonic antigen. SV40 (simian virus 40)  (Figure 23.7 and Figure 23.8) is an oncogenic polyoma virus. It multiplies in cultures of rhesus monkey kidney and produces cytopathic alterations in African green monkey cell cultures. Inoculation into newborn hamsters leads to the development of sarcomas. SV40 has 5243 base pairs in its genome. It may follow either of two patterns of life cycle according to the host cell. In permissive cells, such as those from African green monkeys, the virus-infected cells are lysed, causing the escape of multiple

SV40(1)

SV40(1)

SV40(1)

SV40(2)

SV40(1)

Immunize (Inactive Tumor)

Murine Leukemia Virus (MuLV)

Challenge (Live Tumor)

Murine Leukemia Virus (MuLV) Result

No growth

No growth

Growth

Figure 23.7  SV-40.

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by the embryonic yolk sac and fetal liver and consists of a 590-amino acid residue polypeptide chain structure. It may be elevated in pregnant women bearing fetuses with open neural tube defects, central nervous system defects, gastrointestinal abnormalities, immunodeficiency syndromes, and various other abnormalities. After parturition, the high levels in fetal serum diminish to levels that cannot be detected. α-fetoprotein induces immunosuppression, which may facilitate neonatal tolerance. Based on in vitro studies, it is believed to facilitate suppressor T lymphocyte function and diminish helper T lymphocyte action. Liver cancer patients reveal significantly elevated serum levels of α-fetoprotein. In immunology, however, it is used as a marker of selected tumors such as hepatocellular carcinoma. It is detected by the avidin–biotin–peroxidase complex (ABC) immunoperoxidase technique using monoclonal antibodies. Melanomas  are malignant tumors of melanocytes in the skin. Figure 23.8  SV-40. Resolution 3.1 Å.

viral particles. Lysis does not occur in nonpermissive cells infected with the virus. By contrast, they may undergo oncogenic transformation in which SV40 DNA sequences become integrated into the genome of the host cell. Cells that have become transformed have characteristic morphological features and growth properties. SV40 may serve as a cloning vector. It is a diminutive icosahedral papovavirus that contains double-stranded DNA. It may induce progressive multifocal leukoencephalopathy. SV40 is useful for the in vitro transformation of cells as a type of “permissive” infection ultimately resulting in lysis of infected host cells.

Melanocytes  are melanin pigment-producing cells of the skin. A melanosome  is a melanocyte organelle linked to the endocytic pathway that is charged with synthesis of pigment proteins.

Oncogenic virus  (Figure 23.9) is any virus, whether DNA or RNA, that can induce malignant transformation of cells. An example of a DNA virus would be human papillomavirus, and an RNA virus would be retrovirus.

The melanoma antigen-1 gene (MAGE-1)  in humans was derived from a malignant melanoma cell line. It encodes for an epitope that a cytotoxic T lymphocyte clone specific for melanoma recognizes. This clone was isolated from a patient bearing melanoma. MAGE-1 protein is found on one-half of all melanomas and one-fourth of all breast carcinomas, but is not expressed on the majority of normal tissues. Even though MAGE-1 has not been shown to induce tumor rejection, cytotoxic T lymphocytes in melanoma patients manifest specific memory for MAGE-1 protein.

α-fetoprotein  (Figure 23.10) is a principal plasma protein in the α globulin fraction present in the fetus. It bears considerable homology with human serum albumin. It is produced

Melanoma-associated antigens (MAA)  are antigens associated with the aggressive, malignant, and metastatic tumors arising from melanocytes or melanocyte-associated nevus

RNA provirus RNA virus

Infectious RNA virus and cell membrane Leukemia

Infected cell

Steady state Tumor-specific transplantation antigen Virus-specific antigen

DNA virus

Malignancy Integrated

Figure 23.9  Oncogenic virus.

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Tumor cell

Tumor virus particle

Figure 23.10  α-fetoprotein.

Tumor specific antigen (TSA)*

*Each tumor induced by a single virus will express the same TSA on the cell surface despite the morphology of the cell

Figure 23.11  Tumor-specific antigens (TSA).

cells. Monoclonal antibodies have identified 40+ separate MAAs. They are classified as MHC molecules, cationbinding proteins, growth-factor receptors, gangliosides, high molecular weight extracellular matrix-binding molecules, and nevomelanocyte differentiation antigens. Some of the antigens are expressed on normal cells, whereas others are expressed on tumor cells. Melanoma patient blood sera often contain anti-MAA antibodies which are, regrettably, not protective. Monoclonal antibodies against MAAs aid studies on the biology of tumor progression, immunodiagnosis, and immunotherapy trials.

Tumor rejection antigen  is an antigen that is detectable when transplanted tumor cells are rejected. Also called tumor transplant antigen.

Modulation:  See antigenic modulation.

TSA  is the abbreviation for tumor-specific antigen.

 umor cells may be subject to alterations in antigenic strucT ture. Antigenic transformation refers to changes in a cell’s antigenic profile as a consequence of antigenic gain, deletion, reversion, or other process. Antigenic gain refers to nondistinctive normal tissue components that are added or increased without simultaneous deletion of other normal tissue constituents. Antigenic deletion describes antigenic determinants that have been lost or masked in the progeny of cells that usually contain them. Antigenic deletion may take place as a consequence of neoplastic transformation or mutation of parent cells resulting in the disappearance or repression of the parent cell genes. Antigenic modulation is the loss of epitopes or antigenic determinants from a cell surface following combination with an antibody. The antibodies either cause the epitope to disappear or become camouflaged by covering it. Antigenic diversion refers to the replacement of a cell’s antigenic profile by the antigens of a different normal tissue cell. Used in tumor immunology, antigenic reversion is the change in antigenic profile characteristic of an adult cell to an antigenic mosaic that previously existed in the immature or fetal cell stage of the species. Antigenic reversion may accompany neoplastic transformation.

TATA  is the abbreviation for tumor-associated transplantation antigen.

 umor cells express tumor-specific determinants or epitopes T present on tumor cells but identifiable also in varying quantities and forms on normal cells. Tumor-specific antigens (TSA) (Figure  23.11) are present on tumor cells, but not

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found on normal cells. Murine tumor-specific antigens can induce transplantation rejection in mice. They are encoded by mutated cellular genes or by viral oncogenes. Tumorspecific transplantation antigens (TSTA) (Figure 23.12) are epitopes that induce rejection of tumors transplanted among syngeneic (histocompatible) animals.

Macrophages  (Figures  23.13 and 23.14) are mononuclear phagocytic cells derived from monocytes in the blood that were produced from stem cells in the bone marrow. These cells have a powerful although nonspecific role in immune defense. These intensely phagocytic cells contain lysosomes and exert microbicidal action against microbes which they ingest. They also have effective tumoricidal activity. They may take up and degrade both protein and polysaccharide antigens and present them to T lymphocytes in the context of MHC class II molecules. They interact with both T and B lymphocytes in immune reactions. They are frequently found in areas of epithelium, mesothelium, and blood vessels. Macrophages have been referred to as adherent cells since they readily adhere to glass and plastic and may spread on these surfaces and manifest chemotaxis. They have receptors for Fc and C3b on their surfaces, stain positively for nonspecific esterase and peroxidase, and are Ia antigen positive when acting as accessory cells that present antigen to CD4+ lymphocytes in the generation of an immune response. Monocytes, which may differentiate into macrophages when they migrate into the tissues, make up 3 to 5% of leukocytes in the peripheral blood. Macrophages that are tissue-bound may be found in the lung alveoli, as microglial cells in the central nervous system, as Kupffer cells in the liver, as Langerhans

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TSTA

TSTA Methylcholanthrene (MCA)

Induces sarcoma leukemia, or carcinoma

TSTA

Normal genetically identical cells

Tumorous cells

*Each genetically identical cell develops unique antigenic specificity to MCA.

Figure 23.12  Tumor-specific transplantation antigens (TSTA).

Fc receptor

Tumor cell lysis

Tumor cell TNFα Epitope

INFγ

Macrophage

Reactive oxygen metabolites and lysosomal enzymes

IgG antibody

Figure 23.13  Macrophage-mediated tumor cell lysis is mediated by several mechanisms. Activated macrophages express Fcγ receptors that anchor IgG molecules attached to tumor cells but not normal cells, resulting in the release of lysosomal enzymes and reactive oxygen metabolites that lead to tumor cell lysis. Another mechanism of macrophage-mediated lysis includes the release of the cytokine tumor necrosis factor α that may unite with high-affinity TNFα receptors on a tumor cell surface resulting in its lysis, or the effect of TNFα on the small blood vessels and capillaries of vascularized tumors leading to hemorrhagic necrosis producing a localized Shwartzman-like reaction.

MHC class II receptor

Tumor cell epitope

T cell receptor MHC class I Tumor cell receptor

CD4+ INFγ

IL-2 CD8+

TCR

Macrophage

Tumor cell lysis

Increase in MHC class II receptors

CD4+ helper T cell

Cytotoxic T lymphocyte Perforin and granzymes (CTL) release

Figure 23.14  Macrophage-mediated tumor immunity.

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INFγ

Fc receptor

NK cell

Tumor cell lysis

Tumor cell

TNFα

IgG antibody

Perforin and granzymecontaining granules

Figure 23.15  NK cell-mediated killing of tumor cells by antibody-dependent cell-mediated cytotoxicity.

cells in the skin, as histiocytes in connective tissues, as well as macrophages in lymph nodes and peritoneum. Multiple substances are secreted by macrophages including complement components C1 through C5, factors B and D, properdin, C3b inactivators, and β-1H. They also produce monokines such as interleukin-1, acid hydrolase, proteases, lipases, and numerous other substances. Natural killer (NK) cells  (Figures 23.15 and 23.16) are lymphoid cells that recognize nonself molecular configurations

NK cell

Kill signal

Ly 49

NKR-P1 Glycoprotein

Class I MHC

Protect signal

No cytotoxicity

Normal target cell

NK cell

NKR-P1 Kill signal Glycoprotein

Ly 49 Class I MHC expression

Cytotoxicity

Virusinfected target cell

Figure 23.16  Proposed mechanism of NK cell cytotoxicity restricted to altered self cells. Kill signal is generated when the NK cell’s NKR-PI receptor interacts with membrane glycoprotein or normal and altered self cells. The kill signal can be countermanded by interaction of the NK cells Ly49 receptor with class I MHC molecules. Thus, MHC class I expression prevents NK cell killing of normal cells. Diminished class I expression on altered self cells leads to their destruction.

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with broad specificity. They attack and destroy tumor cells and certain virus-infected cells. They constitute an important part of the natural immune system, do not require prior contract with antigen, and are not MHC restricted by the MHC antigens. NK cells are lymphoid cells of the natural immune system that express cytotoxicity against various nucleated cells, including tumor cells and virus-infected cells. NK cells, killer (K) cells, or antibody-dependent, cell-mediated cytotoxicity (ADCC) cells induce lysis through the action of antibody. Immunologic memory is not involved, as previous contact with the antigen is not necessary for NK cell activity. The NK cell is approximately 15 μm in diameter and has a kidney-shaped nucleus with several, often three, large cytoplasmic granules. The cells are also called large granular lymphocytes (LGLs). In addition to their ability to kill selected tumor cells and some virus-infected cells, they also participate in ADCC by anchoring antibody to the cell surface through an Fc γ receptor. Thus, they are able to destroy antibody-coated nucleated cells. NK cells are believed to represent a significant part of the natural immune defense against spontaneously developing neoplastic cells and against infection by viruses. NK cell activity is measured by a 51Cr release assay employing the K562 erythroleukemia cell line as a target. NK cells secrete IFN-γ and fail to express antigen receptors such as immunoglobulin receptors or T-cell receptors. Cell-surface stimulatory receptors and inhibitory receptors, which recognize self-MHC molecules, regulate their activation. NK cells become activated if a target cell that expresses ligands that bind to NK activating receptors does not express MHC class I molecules to interact with NK inhibitory receptors. NK cells are sentinels of natural innate immunity. The “missing self” hypothesis  states that interaction of NK cells with cells failing to express self MHC class I antigens on their surface leads to T cell activation. Examples include neoplastic cells, virus-infected cells, and allogeneic cells. Designer lymphocytes  are lymphocytes into which genes have been introduced to increase the cell’s ability to lyse tumor cells. Tumor-infiltrating lymphocytes transfected with these types of genes have been used in experimental adoptive immunotherapy.

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Tumor cell lysis

MCH class I receptor Tumor cell

INFγ

TCR CTL differentiation Tc cell or CTL precursor

CD8+

Cytotoxic T Lymphocyte (CTL)

epitope Perforin and granzymes

Figure 23.17  Cytotoxic T lymphocyte (CTL)-mediated tumor lysis.

Cytotoxic T lymphocytes (CTLs)  (Figures 23.17 and 23.18) are specifically sensitized T lymphocytes that are usually CD8+ and recognize antigens through the T cell receptor on cells of the host infected by viruses or that have become neoplastic. CD8+ cell recognition of the target is in the context of MHC class I histocompatibility molecules. Following recognition and binding, death of the target cell occurs a few hours later. CTLs secrete lymphokines that attract other lymphocytes to the area and release serine proteases and perforins that produce ion channels in the membrane of the target, leading to cell lysis. Interleukin-2, produced by CD4+ T cells, activates cytotoxic T cell precursors. Interferon-γ generated from CTLs activates macrophages. CTLs have a significant role in the rejection of allografts and in tumor immunity. A minor population of CD4+ lymphocytes may also be cytotoxic, but they recognize target-cell antigens in the context of MHC class II molecules.

example of participation between antibody molecules and immune system cells to produce an effector function.

 umor-specific IgG antibodies may act in concert with T immune system cells to produce antitumor effects. Antibody-dependent cell-mediated cytotoxicity (ADCC) (Figure 23.19) is a reaction in which T lymphocytes and NK cells, including large granular lymphocytes, neutrophils, and macrophages, may lyse tumor cells, infectious agents, and allogeneic cells by combining through their Fc receptors with the Fc region of IgG antibodies bound through their Fab regions to target cell surface antigens. Following linkage of Fc receptors with Fc regions, destruction of the target is accomplished through released cytokines. It represents an

Heteroconjugate antibodies  (Figure  23.20) are antibodies against a tumor antigen coupled covalently to an antibody specific for a natural killer cell or cytotoxic T lymphocyte surface antigen. These antibodies facilitate binding of cytotoxic effector cells to tumor target cells. Antibodies against effector cell surface markers may also be coupled covalently with hormones that bind to receptors on tumor cells.

Antibody-directed enzyme prodrug therapy (ADEPT)  is a type of treatment in which an antibody is used to target an enzyme to a tumor and unbound reagent is allowed to clear. A nontoxic prodrug is then given, and this is activated by the enzyme to form a cytotoxic drug at the tumor site. An important part of ADEPT is bystander killing. Since the drugs are activated extracellularly by the antibody–enzyme complex, neighboring cells may also be killed by a mechanism that does not require translocation across intracellular membranes. By contrast, immunotoxins kill only the cell to which they bind. A heteroconjugate  is a hybrid of two different antibody molecules.

Immunosurveillance  refers to the policing or monitoring function of immune system cells to recognize and destroy clones of transformed cells prior to their development

MHC class I receptor Tumor cell INFγ

Epitope

CD8+T cell

Tumor cell CD8+

INFα/β TCR CD8+ Increase in MHC class I expression

Tumor cell lysis

Cytotoxic T Lymphocyte (CTL)

TCR MHC class I receptor Perforin and granzymes

Figure 23.18  CTL-mediated killing of tumor cells.

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Antibody

and molecules, and such as TGF-β and P15E, are all believed to contribute to the inefficiency of tumor immunity.

Tumor cell

Tumor cell lysis Lymphocyte

Figure 23.19  Antibody-dependent cell-mediated cytotoxicity (ADCC).

into neoplasms and to destroy tumors after they develop. Immunosurveillance is believed to be mediated by the cellular limb of the immune response. Indirect evidence in support of the concept includes (1) an increased incidence of tumors in aged individuals who have decreased immune competence, (2) increased tumor incidence in children with T cell immunodeficiencies, and (3) the development of neoplasms (lymphomas) in a significant number of organ or bone marrow transplant recipients who have been deliberately immunosuppressed. A preneoplastic clone  is a genetically altered cell clone at a stage before development into a tumor during the progression of carcinogenesis. A premalignant clone  is a genetically altered cell clone at a stage before malignant conversion during the progression of carcinogenesis. Immunoselection  is the selective survival of cells due to their diminished cell surface antigenicity. This permits these cells to escape the injurious effects of either antibodies or immune lymphoid cells. Immunological escape  is a mechanism of escape in which tumors that are immunogenic continue to grow in immunocompetent syngeneic hosts in the presence of a modest in vivo antitumor immune response. Escape mechanisms may facilitate tumors in evading a fatal tumoricidal response and render them incapable of inducing such a response. Failure of tumor antigen presentation by MHC class I molecules, and lack of costimulation and downregulation of tumor-destructive immune responses by tumor antigens, immune complexes,

Covalent bond

Cytotoxic T cell or NK cell

Tumor cell (target)

Figure 23.20  Heteroconjugate antibodies.

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Immunologic enhancement (tumor enhancement)  (Figure  23.21) describes the prolonged survival, conversely the delayed rejection, of a tumor allograft in a host as a consequence of contact with a specific antibody. Antitumor antibodies may have a paradoxical effect. Instead of eradicating a neoplasm, they may facilitate its survival and progressive growth in the host. Both the peripheral and central mechanisms have been postulated. Coating of tumor cells with antibody was presumed, in the past, to interfere with the ability of specifically reactive lymphocytes to destroy them, but today a central effect in suppressing cell-mediated immunity, perhaps through suppressor T lymphocytes, is also possible. Enhancing antibodies are blocking antibodies that favor survival of tumor or normal tissue allografts. Immunologic facilitation (facilitation immunologique)  is the slightly prolonged survival of certain normal tissue allografts, e.g., skin, in mice conditioned with isoantiserum specific for the graft. Immunotherapy  employs immunologic mechanisms to combat disease. These include nonspecific stimulation of the immune response with BCG immunotherapy in treating certain types of cancer, and the IL-2/LAK cell adoptive immunotherapy technique for treating selected tumors. Biological response modifiers (BRM)  are a wide spectrum of molecules, such as cytokines, that alter the immune response. They include substances such as interleukins, interferons, hematopoietic colony-stimulating factors, tumor necrosis factor, B  lymphocyte growth and differentiating factors, lymphotoxins, and macrophage-activating and chemotactic factors, as well as macrophage-inhibitory, eosinophils chemotactic, and osteoclast-activating factors, etc. BRM may modulate the immune system of the host to augment antirecombinant DNA technology and are available commercially. An example is α interferon used in the therapy of hairy cell leukemia. Interferon α (IFN-α)  is an immunomodulatory 189 amino acid residue glycoproteins synthesized by macrophages and B cells that are able to prevent the replication of viruses, are antiproliferative, and are pyrogenic, inducing fever. IFN-α stimulates natural killer cells and induces expression of class I MHC antigens. It also has an immunoregulatory effect through alteration of antibody responsiveness. The 14 genes that encode IFN-α are positioned on the short arm of chromosome 9 in man. Polyribonucleotides, as well as RNA or DNA viruses, may induce IFN-α secretion. Recombinant IFN-α has been prepared and used in the treatment of hairy cell leukemia, Kaposi’s sarcoma, chronic myeloid leukemia, human papilloma virus-related lesions, renal cell carcinoma, chronic hepatitis, and other selected conditions. Patients may experience severe flu-like symptoms as long as the drug

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Tumor Immunology

C3Hf/He(H-2k) fibrosarcoma

IgG2 antibody against C3Hf/He(H-2k) fibrosarcoma

TS (H-2S) mouse

Progressive growth of tumor allograft

Lymphocyte Fc receptor site

Central

Fibrosarcoma epitope Peripheral

Figure 23.21  Immunologic enhancement (tumor enhancement).

is administered. They also have malaise, headache, depression, supraventricular tachycardia, and may possibly develop congestive heart failure. Bone marrow suppression has been reported in some patients. Immunoscintigraphy  (Figures  23.22 and 23.23) is the formation of two-dimensional images of the distribution of radioactivity in tissues following the administration of antibodies labeled with a radionuclide that are specific for tissue antigens. A scintillation camera is used to record the images. Immunolymphoscintigraphy is a method used to determine

Tumor Localization/Detection

the presence of tumor metastasis to lymph nodes. Antibody fragments or monoclonal antibodies against specific tumor antigens are radiolabeled and then detected by scintigraphy. Radioimmunoscintigraphy  is the use of radiolabeled antibodies to localize tumors or other lesions through use of radioactivity scanning following injection in vivo. Arcitumomab  is a murine F(ab')2 fragment of an anti-carcinoembryonic antigen (CEA) antibody labeled with technetium 99m (99mTc). It is used for imaging patients with metastatic colorectal cancer by immunoscintigraphy to evaluate spread of the disease. The use of the F(ab')2 fragment diminishes the immunogenicity permitting it to be used more than once. Immunolymphoscintigraphy  is a method to determine the presence of tumor metastasis to lymph nodes. Antibody fragments or monoclonal antibodies against specific tumor antigens are radiolabeled and then detected by scintigraphy. An immunoconjugate  is a chimeric protein product of the linkage of a whole monoclonal antibody or one of its structural derivatives to another molecule either chemically or at the DNA level. Refer to immunocytokine, immunoradioisotope, and immunotoxin.

IV

125I-Labeled

antitumor monoclonal antibody

External gamma-ray imaging

Figure 23.22  Immunoscintigraphy.

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An immunocytokine  is an immunoconjugate in which a monoclonal antibody or one of its derivatives is bound to a cytokine. It is designed to deliver anti-tumor cytokines to the tumor site during cancer treatment. An immunoradioisotope  is an immunoconjugate used in radioimmunotherapy in which a monoclonal antibody or monoclonal antibody derivative is bound to a radioisotope capable of destroying a tumor cell on contact. An immunotoxin  (Figure  23.24) is produced by linking a monoclonal antibody, or monoclonal antibody derivative,

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2d

Figure 23.25  Abrin–A.

5d

 mong the uses of immunotoxin is the purging of T cells A from hematopoietic cell preparations used for bone marrow transplantation. Immunotoxins have potential for antitumor therapy and as immunosuppressive agents.

7d

Figure 23.23  Immunoscintigraphy (nude mouse) with a 131Ilabeled monoclonal antibody. The mouse shown bears a human colon carcinoma in its left flank. The scintigrams were recorded 2, 5, and 7 days postinjection. While the second picture shows mainly the blood pool and little of the tumor, the tumor is the major imaged spot in the body after 5 days; after 7 days, only the tumor is recognizable.

specific for target cell antigens with a cytotoxic substance such as the toxin ricin. Upon parenteral injection, its antibody portion directs the immunotoxin to the target and its toxic portion destroys target cells on contact. An immunotoxin may also be a monoclonal antibody or one of its fractions linked to a toxic molecule such as a radioisotope, a bacterial or plant toxin, or a chemotherapeutic agent. The antibody portion is intended to direct the molecule to antigens on a target cell such as those of a malignant tumor and the toxic portion of the molecule is for the purpose of destroying the target cell. Contemporary methods of recombinant DNA technology have permitted the preparation of specific hybrid molecules for use in immunotoxin therapy. Immunotoxins may have difficulty reaching the intended target tumor, may be quickly metabolized, and may stimulate the development of antiimmunotoxin antibodies. Cross-linking proteins may likewise be unstable. Epitopes

Immunotoxin molecule Antibody specific for tumor cell Antibody/toxin complex

Tumor cell

Figure 23.24  Immunotoxin.

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Ricin  is a toxic protein found in seeds of Ricinus communis (castor bean) plants. It is a heterodimer comprised of a 30-kDa α chain, which mediates cytotoxicity, and a 30-kDa β chain, which interacts with cell surface galactose residues that facilitate passage of molecules into cells in endocytic vesicles. Ricin inhibits protein synthesis by linkage of a dissociated α chain in the cytosol to ribosomes. The ricin heterodimer or its α chain conjugated to a specific antibody serves as an immunotoxin. Ricinus communis:  See ricin. Abrin  (Figure 23.25) and ricin are examples of immunotoxins. Abrin is a powerful toxin and lectin used in immunological research by Paul Ehrlich (circa 1900). It is extracted from the seeds of the jequirity plant and causes agglutination of erythrocytes. Magic bullet  is a term coined by Paul Ehrlich in 1900 to describe what he considered to be the affinity of a drug for a particular target. He developed “606” (salvarsan), an arsenical preparation, to treat syphilis. In immunology, it describes a substance that could be directed to a target by a specific antibody and injure the target once it arrives. Monoclonal antibodies have been linked to toxins such as diphtheria toxin, or ricin, as well as to cytokines for use as magic bullets. Adoptive immunotherapy  (Figures  23.26 and 23.27) is the experimental treatment of terminal cancer patients with metastatic tumors unresponsive to other modes of therapy by the inoculation of lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) together with IL-2. This mode of therapy has shown some success in approximately one-tenth of treated individuals with melanoma or renal cell carcinoma.

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IL-2

Cytotoxic T lymphocyte (CTL)

Tc cell or CTL precursor CTL differentiation

MHC class I receptor

Tumor cell lysis

Tumor cell

TCR

Epitope CD8+

IL-2

Perforin and granzymes

Fc receptor

NK cell

Perforin and granzymes

Tumor cell lysis

Tumor cell

TNFα

IgG antibody

Figure 23.26  Interleukin-2 (IL-2) immunotherapy.

Lymphokine-activated killer (LAK) cells  are lymphoid cells derived from normal or tumor patients cultured in medium with recombinant IL-2, which become capable of lysing NK-resistant tumor cells as revealed by 51Cr-release cytotoxicity assays. These cells are also referred to as lymphokineCytotoxic T lymphocyte (CTL)

activated killer cells. Most LAK activity is derived from NK cells. The large granular lymphocytes (LGL) contain all LAK precursor activity and all active NK cells. In accord with the phenotype of precursor cells, LAK effector cells are also granular lymphocytes expressing markers associated with Tumor cell lysis

Perforin and granzymes Tumor cell

IL-12

Epitope CD8+

IL-12

NK cell

Fc receptor

Perforin and granzymes

MHC class I receptor

Tumor cell lysis

IgG antibody Tumor cell

TNFα

IgG antibody

Figure 23.27  Interleukin-12 (IL-12) immunotherapy.

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human NK cells. The asialo Gm1+ population, known to be expressed by murine NK cells, contains most LAK precursor activity. Essentially all LAK activity resides in the LGL population in the rat. LAK cell and IL-2 immunotherapy has been employed in human cancer patients with a variety of histological tumor types when conventional therapy has been unsuccessful. Approximately one-fourth of LAK- and IL-2treated patients manifested significant responses, and some individuals experienced complete remission. Serious side effects include fluid retention and pulmonary edema attributable to the administered IL-2. LAK cells  are lymphokine-activated killer cells. IL-2/LAK cells  are interleukin-2/lymphokine-activated killer cells. NK cells, which express only the p70 and not the p55 receptor for IL-2, are incubated with IL-2, converting them into an activated form referred to as LAK cells. The IL-2/LAK combination has been used to treat cancer patients through adoptive immunotherapy, which has been successful in inducing transient regression of tumors in selected cases of melanoma, colorectal carcinoma, non-Hodgkin’s lymphoma, and renal cell carcinoma, as well as regression of metastases in the liver and lung of some patients. There may be transient defective chemotaxis of neutrophils, and patients often develop “capillary leak syndrome,” producing pulmonary edema. Patients may also develop congestive heart failure. Tumor immunity:  Numerous experimentally induced tumors in mice express numerous specific transplantation antigens which can induce an immune response that leads to destruction of neoplastic cells in vivo. Lymphocytes play a critical role in the immunological destruction of many antigenic tumors. Both cell-mediated and antibody-mediated immune responses to human neoplasms have been identified and their targets characterized in an effort to develop clinically useful immunotherapy. Tumor-infiltrating lymphocytes (TIL)  are cytotoxic T lymphocytes within a tumor mass. These T lymphocytes are isolated from the tumor they are infiltrating. They are cultured with high concentrations of IL-2, leading to expansion of these activated T lymphocytes in vitro. TILs are very effective in destroying tumor cells and have proven much more effective than LAK cells in experimental models. TILs have 50 to 100 times the antitumor activity produced by LAK cells. TILs have been isolated and grown from multiple resected human tumors, including those from kidney, breast, colon, and melanoma. In contrast to the non-B–non-T LAK cells, TILs nevertheless are generated from T lymphocytes and phenotypically resemble cytotoxic T lymphocytes. TILs from malignant melanoma exhibit specific cytolytic activity against cells of the tumor from which they were extracted, whereas LAK cells have a broad range of specificity. TILs appear unable to lyse cells of melanomas from patients other than those in whom the tumor originated. TILs may be tagged in order that they may be identified later.

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TIL  is the abbreviation for tumor-infiltrating lymphocytes. Reverse immunology  is a process that involves computerized algorithms to predict the likelihood of a particular mutation resulting in a strong antigen. Several of the mutant proteins and peptides have been used to examine the possibility of inducing tumor-specific immunity. Hybrid resistance  is the resistance of members of an F1 generation of animals to growth of a transplantable neoplasm from either one of the parent strains. Concomitant immunity  is resistance to a tumor that has been transplanted into a host already bearing that tumor. Immunity to the reinoculated neoplasm does not inhibit growth of the primary tumor. CA-15-3  is an antibody specific for an antigen frequently present in the blood serum of metastatic breast carcinoma patients. CA-19-9  is a tumor-associated antigen found on the Lewis A blood group antigen that is sialylated or in mucin-containing tissues. In individuals whose serum levels exceed 37 U/ml, 72% have carcinoma of the pancreas. In individuals whose levels exceed 1000 U/ml, 95% have pancreatic cancer. Anti-CA-19-9 monoclonal antibody is useful to detect the recurrence of pancreatic cancer following surgery and to distinguish between neoplastic and benign conditions of the pancreas. However, it is not useful for pancreatic cancer screening. CA-125  is a mucinous ovarian carcinoma cell surface glycoprotein detectable in the patient’s blood serum. Increasing serum concentrations portend a grave prognosis. It may also be found in the blood sera of patients with other adenocarcinomas such as breast, gastrointestinal tract, uterine cervix, and endometrium. CALLA  is common acute lymphoblastic leukemia antigen. Also known as CD10. Calcitonin  is a hormone that influences calcium ion transport. Immunoperoxidase staining demonstrates calcitonin in thyroid parafollicular or C cells. It serves as a marker characteristic of medullary thyroid carcinoma and APUD neoplasms. Lung and gastrointestinal tumors may also form calcitonin. Blocking factors  are agents such as immune complexes in the serum of tumor-bearing hosts that interfere with the capacity of immune lymphoid cells to mediate cytotoxicity of tumor target cells. Antimalignin antibodies  are specific for the 10-kDa protein malignin comprised of 89 amino acids. These antibodies are claimed to be increased in cancer patients without respect

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Macrophage

Tumor cell lysis

Tumor cell

TNF

-α

Mast cell Helper T cell

F-β

TN

Tc cell

Figure 23.28  Tumor necrosis factor (TNF)-mediated immune reaction.

to tumor cell type. It has been further claimed that antibody levels are related to survival. These claims will require additional confirmation and proof to be accepted as fact. The Winn assay  is a method to determine the ability of lymphoid cells to inhibit the growth of transplantable tumors in vivo. Following incubation of the lymphoid cells and tumor cells in vitro, the mixture is injected into the skin of

X-irradiated mice. Growth of the transplanted cells is followed. T lymphocytes that are specifically immune to the tumor cells will inhibit tumor growth and provide information related to tumor immunity. Tumor necrosis factor α (TNF-α)  (Figures  23.28 and 23.29) is a cytotoxic monokine produced by macrophages stimulated with bacterial endotoxin. TNF-α participates Inhibits tumor cell proliferation

Tumor cell TNF-α

Cytolysis of tumor cell

TNF-β SLex

SLea

E-selectin TNF-α

Damage to vascular Vascular endothelial cell

Inhibits oxygen and blood flow to the tumor

Figure 23.29  Tumor necrosis immunotherapy.

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in inflammation, wound healing, and remodeling of tissue. TNF-α, which is also called cachectin, can induce septic shock and cachexia. It is a cytokine comprised of 157 amino acid residues. It is produced by numerous types of cells including monocytes, macrophages, T lymphocytes, B lymphocytes, NK cells, and other types of cells stimulated by endotoxin or other microbial products. The genes encoding TNF-α and TNF-β (lymphotoxin) are located on the short arm of chromosome 6 in man in the MHC region. High levels of TNF-α are detectable in the blood circulation very soon following administration of endotoxins or microorganisms. The administration of recombinant TNF-α induces shock, organ failure, and hemorrhagic necrosis of tissues in experimental animals including rodents, dogs, sheep, and rabbits, closely resembling the effects of lethal endotoxemia. TNF-α is produced during the first 3 d of wound healing. It facilitates leukocyte recruitment, induces angiogenesis, and promotes fibroblast proliferation. It can combine with receptors on selected tumor cells and induce their lysis. TNF mediates the antitumor action of murine natural cytotoxic (NC) cells, which distinguishes their function from that of NK and cytotoxic T cells. TNF-α was termed “cachectin” because of its ability to induce wasting and anemia when administered on a chronic basis to experimental animals. Thus it mimics the action in cancer patients and in those with chronic infection

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with HIV or other pathogenic microorganisms. It can induce anorexia which may lead to death from malnutrition. Chachexia  is a body wasting attributable to unregulated cellular catabolism. Elevated levels of tumor necrosis factor cause cachexia in patients with malignant tumors. Tumor necrosis factor-β (TNF-β)  is a 25-kDa protein synthesized by activated lymphocytes. It can kill tumor cells in culture, induce expression of genes, stimulate proliferation of fibroblasts, and can mimic most of the actions of TNF-α (cachectin). It participates in inflammation and graft rejection and was previously termed “lymphotoxin.” TNF-β and TNF-α have approximately equivalent affinity for TNF receptors. Both 55-kDa and 80-kDa TNF receptors bind TNF-β. TNF-β has diverse effects that include killing of some cells and causing proliferation of others. It is the mediator whereby cytolytic T cells, natural killer cells, lymphokine-activated killer cells, and “helper-killer” T cells induce fatal injury to their targets. TNF-β and TNF-α have been suggested to play a role in AIDS, possibly contributing to its pathogenesis. Tumor necrosis factor receptor  is a receptor for tumor necrosis factor that is comprised of 461 amino acid residues and which possesses an extracellular domain that is rich in cysteine.

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Immunity against Microorganisms

Natural immunity:  Entry of a pathogenic organism into a susceptible host is followed by invasion and colonization of tissues, circumvention of the host immune response, and injury or decreased function of host tissues. Microbial immunity consists of several factors. Natural and acquired immune mechanisms facilitate the body’s resistance against microorganisms. Microbes vary in the lymphocyte responsiveness and effector mechanisms they elicit. The skill with which pathogenic microorganisms resist the host’s immune defense mechanisms governs their survival and pathogenicity. Paradoxically, the host response to a pathogenic microorganism, rather than the microbe itself, may induce injury to host tissues. Factors that determine the outcome of man’s encounter with pathogenic microorganisms include the microbe’s virulence and the size of the infecting dose on the one hand and specific defense mechanisms of the host on the other. An infection  is a consequence of the adherence and penetration by a pathogen into the host body through successful avoidance of innate host defense mechanisms, allowing the infectious agent to reproduce. Incubation time  is the term used to describe the interlude between an initial infection and disease onset. An abortive infection  is a condition in which a pathogen is able to enter the body but is unable to propagate. I f a pathogenic microorganism proliferates even in the presence of an immune response against it, it is called a productive infection. In this type of infection, pathogens employ evasion strategies, which are methods imposed by a pathogen to circumvent or compromise a host’s immune response. A persistent infection  is the prolonged existence of a pathogenic microorganism in the host body, which may be throughout life. Infections of this type may be latent or induced chronic disease.  he long-term presence of a pathogenic microorganism that T remains uninfectious and fails to produce clinical symptoms is a latent infection. I t is a consequence of viral genome integration into host cell DNA or by altered expression of viral genes. Reactation is the resumption of replication of the infectious agent following reactivation of a latent infection, which leads to a productive infection that results in disease symptoms.

A chronic disease  is a malady, such as a persistent infection, characterized by persistent or recurring symptoms. An emerging infectious disease  is an infection potentially capable of impacting the world population, since it is induced by a pathogenic microorganism that has recently emerged or one that is undergoing alteration. A pandemic  is a worldwide eruption of an infectious disease. Pathogenicity  refers to the capacity of a microorganism to induce disease. Factors that contribute to pathogenicity include toxin production, activation of host inflammatory responses, and perturbation of host cell metabolism. If host defenses are decreased significantly, as in the immunocompromised host, opportunistic infections, produced by microorganisms that are not normally pathogenic for the individual, may result. There are multiple causes for diminished host resistance that include accidentally or surgically induced trauma to the mucous membranes or skin, localized lesions, leukocyte defects, complement defects, or defective B or T cell responses. Various drugs such as antibiotics may also alter the normal flora of the body. Microbes that produce opportunistic infections generally are of low virulence, i.e., their level of pathogenicity is low. Fever:  An increase in the body temperature above normal. Attributable to cytokines released during infection. Septicemia:  The presence and persistence of pathogenic microorganisms or their toxins in the blood. Also termed blood poisoning. Virulence  is the pathogenicity of a microorganism to invade host tissues as indicated by the severity of the disease it causes.  oth nonspecific constitutional factors and specific immune B mechanisms provide host resistance. Nonspecific resistance mechanisms protect against body surface colonization by microorganisms with pathogenic potential, thereby blocking their penetration of underlying tissues. Reservoir  is the term for a host or carrier that harbors pathogenic microorganisms without being injured itself, and serves as a source of infection for others. Also termed reservoir host or reservoir of infection. 717

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A carrier (person)  is an individual with a latent infection who can serve unknowingly as a source of infection to other persons. An animal reservoir  is the term for an intermediate animal host for a pathogenic microorganism whose primary host is another species. Protective immunity  consists of both natural, nonspecific immune mechanisms as well as actively acquired specific immunity that results in the defense of a host against a particular pathogenic microorganism. Protective immunity may be induced either by active immunization with a vaccine prepared from antigens of a pathogenic microorganism or by experiencing either a subclinical or clinical infection with the pathogenic microorganism. The skin, as well as mucous membranes of various anatomical regions such as the conjunctiva, nose, mouth, intestinal tract, and lower genital tract, have a normal commensal flora. Microbial properties, host factors, and exogenous factors determine the nature of colonization. The ability of a microorganism to adhere to mucosa or epithelial cells is a significant factor. Microbes in the normal flora may compete with pathogenic microorganisms for receptors on cell surfaces. Fibronectin on epithelial cells may bind Staphylococcus aureus and group A hemolytic streptococci. Microbes in the commensal flora may also synthesize bacteriocins that inhibit other bacteria. They may also compete with them for nutrient substances. Thus, the normal flora serves as an effective mechanism for inducing colonization resistance. This can be interrupted by the use of broad-spectrum antibiotics resulting in colonization of the surface by pathogenic microorganisms. Gram-negative bacteremia may even result in an immunocompromised host. Another consequence of antibiotic therapy may be overgrowth of yeast or of Clostridium difficile, a toxin-producing Gram-positive bacillus that is an anaerobic and antibiotic resistant that can lead to diarrhea and colitis. Host age, hormones, nutrition, and diseases such as diabetes mellitus or malignancy may influence the normal flora. For example, the vaginal flora is sparse in both prepubertal and post-menopausal females, but is rich in acidophilic lactobacilli during the child-bearing years of life. Lactobacilli convert glycogen to lactic acid, yielding a pH of 4 to 5, which inhibits many potential pathogens that might otherwise colonize the vaginal mucosa. Microorganisms in the normal flora of various anatomical regions may induce natural antibodies that would be active against potential pathogenic microorganisms bearing cross-reacting antigens. Secretory immunoglobulin A (SIgA)  can interfere with the attachment of bacteria to host cells by coating the microbes. It may also neutralize their exotoxins, inhibit their motility, and agglutinate them. It is not involved in opsonization or lysis of bacteria through complement. SIgA’s ability to prevent adherence of such microorganisms as Vibrio cholerae, Giardia lamblia, and selected respiratory viruses to

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Eyes • Washing of tears • Lysozyme

Respiratory tract • Mucus • Cilated epithelium • Alveolar macrophages Genitourinary tract • Washing of urine • Acidity of urine • Lysozyme • Vaginal lactic acid

Skin • Anatomic barrier • Antimicrobial secretions Digestive tract • Stomach acidity • Normal flora

Figure 24.1  External defense barriers of the human body.

mucosal surfaces represents a significant defense mechanism. Whereas gastric acidity can destroy most microorganisms, Mycobacterium tuberculosis and enteroviruses are not destroyed by it. Gram-negative bacteria may colonize the stomach and small intestine in subjects with achlorhydria. Unconjugated bile may prevent bacterial growth in the small gut. Intestinal peristalsis also guards against overgrowth of microorganisms in blind loops. The skin and mucous membrane serve as mechanical barriers  to the entrance of microorganisms (Figures  24.1 and 24.2). The papilloma virus and a few other infection agents may penetrate the skin, but most microorganisms are excluded by it. Free fatty acids from sebaceous glands and lactic acid present in perspiration together with an acid Intact skin Mucus Motion of cilia Coughing/sneezing Cell shedding Flushing of microbes by tears, saliva, urine, perspiration, other body fluids Emesis and diarrhea aid microbial elimination Figure 24.2  Mechanical barriers against infection.

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pH of 5 to 6 and the dryness of the skin are unfavorable to microorganisms. Staphylococcus aureus may colonize hair follicles and sweat glands to produce furuncles, carbuncles, and abscesses. Pseudomonas aeruginosa may infect skin injured by burns. Injury to the gastric mucosa by irradiation or cytotoxic drugs may culminate in infection by the normal flora of the intestine.

This substance, which is found widely among vertebrates, invertebrates, plants, and bacteria, has been sequenced and its three-dimensional structure determined. It is widely distributed in such normal cells as histiocytes, leukocytes, including neutrophil granules, and monocytes. By immunoperoxidase staining, this marker identifies histiocytes and neoplasms associated with them.

 ysozyme and lactoferrin are both antimicrobial substances L found in surface secretions of the mucosa. Lysozyme induces lysis of bacterial cells through breaking the linkage connecting N-acetyl muraminic acid and N-acetyglucosamine in the walls of Gram-positive bacterial cells. Lactoferrin interrupts metabolism of bacterial iron.

I nhaled microorganisms in dust or droplets greater than t μm adhere to the mucosa lining the upper respiratory tract and are swept upward by cilia to the posterior pharynx, followed by expectoration or swallowing; this is called directional flow. Particles less than 5 μm reach the alveoli and are phagocytized by alveolar macrophages. Cigarette smoke or other pollutants, as well as bacterial or viral infection such as pertussis and influenza, may diminish the sweeping action of cilia, thereby rendering the subject susceptible to secondary bacterial pneumonia. Intubation and tracheostomy may also decrease normal resistance mechanisms, leading to infection. Tears and blinking actions protect the eyes causing microorganisms to be diluted and flushed out through the nasolacrimal duct into the nasopharynx. The antibacterial action of tears is attributable to lysozyme.

Lysozyme  can induce lysis of some Gram-positive bacterial cell walls but not Gram-negative bacteria unless antibody and complement are also present. It accentuates complement activity. Lactoferrin, a protein that binds iron, competes with microorganisms for this substance. By chelating iron, lactoferrin deprives microbes of the free iron they require for growth. Neutrophil secondary granules also contain lysozyme and lactoferrin. Beta lysin is a thrombocytederived antibacterial protein that is effective mainly against Gram-positive bacteria. It is released when blood platelets are disrupted, as occurs during clotting. β lysin acts as a nonantibody humoral substance that contributes to nonspecific immunity (Figure 24.3).  ysozyme (muraminidase) is a cationic, low molecular weight L enzyme found in egg white, tears, nasal secretions, body fluids, lysosomal granules, on skin, and in lesser amounts in serum that leads to hydrolysis of the β-1,4 glycosidic bond that joins N-acetylmuramic acid with N-acetylglucosamine in bacterial cell wall mucopeptide. This causes osmotic lysis of the bacterial cell. Lysozyme is effective principally against Gram-positive cocci, but it may facilitate the effect of antibodies and complement on Gram-negative microorganisms. Factor Lysozyme Lactoferrin, Transferrin Lactoperoxidase Beta-lysin Chemotactic factors Properdin Interferons Defensins

 ven though urine can support bacterial growth in the bladE der, the acid pH of urine and voiding serve as defensive mechanisms against infection. Ascending infection that is discouraged by the longer male urethra is more common in females with a shorter urethra. Urinary stasis in subjects with posterior urethral valves, prostatic hypertrophy, or calculi facilitates infections. An opsonin  is a substance that adheres to the surface of a microorganism and makes it more attractive or delectable to a phagocyte. Opsonins facilitate or enhance phagocytosis of microbes, which constitutes a cornerstone of constituitive defense against infection. Both nonimmune and immune substances may serve as opsonins. C3b, produced during

Function Catalyzes hydrolysis of cell wall mucopeptide Binds iron and competes with microorganisms for it May be inhibitory to many microorganisms Effective mainly against Gram-positive bacteria Induce reorientation and directed migration of PMNs, monocytes, and other cells Activates complement in the absence of antibody-antigen complex Act as immunomodulators to increase the activities of macrophages Block cell transport

Source Tears, saliva, nasal secretions, body fluids, lysosomal granules Specific granules of PMNs Milk and saliva Thrombocytes, normal serum Bacterial substances and products of cell injury and denatured proteins Normal plasma Leukocytes, fibroblasts, natural killer cells, T cells Polymorphonuclear granules

Figure 24.3  Nonspecific humoral defense mechanisms.

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complement activation, forms a covalent bond with the bacterial cell surface thereby rendering it susceptible to phagocytosis by C3b receptor-bearing neutrophils, monocytes, and macrophages. Adherence of opsonized bacteria to the phagocyte cell surface facilitates phagocytosis. Leukocyte receptors for C3b are termed CR1, CR2, CR3, and CR4. Among the pediatric population, CR3 is associated with increased susceptibility to bacterial infections, a condition termed leukocyte adhesion deficiency. Opsonins  bind to bacteria, erythrocytes, or other particles to increase their susceptibility to phagocytosis. They are host proteins that coat a pathogenic microorganism or macromolecule to make it bind more readily to phagocyte receptors, thereby enhancing phagocytosis. Opsonins include antibodies such as IgG3, IgG1, and IgG2 that are specific for epitopes on the particle surface. Following interaction, the Fc region of the antibody becomes anchored to Fc receptors on phagocyte surfaces, thereby facilitating phagocytosis of the particles. In contrast to these so-called heat-stable antibody opsonins are the heat-labile products of complement activation such as C3b or C3bi, which are linked to particles by transacylation with the C3 thiolester. C3b combines with complement receptor 1 and C3bi combines with complement receptor 3 on phagocytic cells. Besides immunoglobulins of the above mentioned isotypes, and complement proteins, other opsonic complement intermediates include iC3b and C4b. Opsonins facilitate phagocytosis of particulate antigens by neutrophils or macrophages. Other substances that act as opsonins include the basement membrane constituent, fibronectin (Figure 24.4). Paneth cells:  Narrow, pyramidal, or columnar epithelial cells, with a round or oval nucleus near the base of the cell, present in the fundus of the crypts of Lieberkühn. They contain large secretory granules that may contain peptidase. They produce anti-microbial proteins. Fibronectin  is an adhesion-promoting dimeric glycoprotein of relatively high mol wt found abundantly in the connective tissue and basement membrane. The tetrapeptide Arg-GlyAsp-Ser facilitates cell adhesion to fibrin; Clq; collagens; heparin; and type I-, II-, III-, V-, and VI-sulfated proteoglycans. Fibronectin is also present in plasma and on normal cell surfaces. Approximately 20 separate fibronectin chains are known. They are produced from the fibronectin gene by alternative splicing of the RNA transcript. Fibronectin is comprised of two 250-kDa subunits joined near their carboxy terminal ends by disulfide bonds. The amino acid residues in the subunits vary in number from 2145 to 2445. Fibronectin is important in contact inhibition, cell movement in embryos, cell-substrate adhesion, inflammation, and wound healing. It may also serve as an opsonin and function as an adhesion molecule in cellular interactions. Fibronectin may also react with complement components. Intensive care patients often lose fibronectin from their pharynx causing alteration of the normal flora with colonization by coliforms.

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Atlas of Immunology, Third Edition Inefficient phagocytosis Fc receptor

Bacteria Macrophage

Opsonization Antibacterial Ab (opsonin)

Efficient phagocytosis

Figure 24.4  Opsonization.

PMNs (polymorphonuclear neutrophils)  sometimes called the soldiers of the body, are first to arrive at areas of invading and rapidly multiplying bacteria. They contain both primary or azurophilic granules and secondary or specific granules that serve as reservoirs for the digestive and hydrolytic enzymes such as lysozyme (Figure  24.5) before they are delivered to the phagosome. Frequently, the PMNs die after ingesting and destroying the invading microorganisms. Macrophages that serve as scavengers ingesting debris left by neutrophils killed by the microorganisms they phagocytized, are resilient and survive. A secondary granule  is a structure in the cytoplasm of polymorphonuclear leukocytes that contains vitamin B12binding protein, lysozyme, and lactoferrin in neutrophils. Cationic peptides are present in eosinophil secondary granules. Histamine, platelet-activating factor, and heparin are present in the secondary granules of basophils. Phagocytic cells  are polymorphonuclear neutrophils and eosinophils as well as macrophages (the mononuclear phagocytes) which have a critical role in defending the host against

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Immunity against Microorganisms

Microorganism Extracellular bacteria Parasites Intracellular microorganisms (i.e., mycobacteria or fungi) Viruses

Cell Type Polymorphonuclear neutrophils (PMNs) Eosinophils Macrophages Lymphocytes, NK cells

Figure 24.5  Substances associated with neutrophils.

microbial infection (Figure  24.6). Polymorphonuclear neutrophils  and occasionally eosinophils appear first in areas of acute inflammation followed later on by macrophages. Chemotactic factors, including N-formyl-methionyl-leucylphenylalanine (f-Met-Leu-Phe) are released by actively multiplying bacteria. This is a powerful attractant for PMNs whose membranes have a specific receptor for it. Different types of infectious agents may stimulate different types of cellular response. When particles greater than 1 mm become attached and engulfed by a cell, the process is known as phagocytosis. Various factors present in the serum and known as opsonins coat microorganisms or other particles and make them more delectable to phagocyte cells. These include nonspecific substances such as complement component C3b, as well as specific antibodies located in the IgG or IgG3 fractions. Capsules enable microorganisms such as pneumococci and Hemophilus to resist phagocytosis. Azurophil granules (Primary granules) Bacterial permeabilityinducing protein (BPI) Cathespin G Cationic antimicrobial protein (CAP) 57 Cationic antimicrobial protein (CAP) 37 Defensins: HP1 HP2 HP3 Elastase Lysozyme Myeloperoxidase

Specific granules (Secondary granules)

Bacterial chemotaxin receptors

Collagenase C5a receptors Gelatinase Lactoferrin Lysozyme NADPH Vitamin B12-binding protein Figure 24.6  Nonspecific cellular defense.

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Mononuclear phagocytes  are mononuclear cells with pronounced phagocytic ability that are distributed extensively in lymphoid and other organs. “Mononuclear phagocyte system” should be used in place of the previously popular “reticulo-endothelial system” to describe this group of cells. Mononuclear phagocytes originate from stem cells in the bone marrow that first differentiate into monocytes that appear in the blood for approximately 24 h or more with final differentiation into macrophages in the tissues. Macrophages usually occupy perivascular areas. Liver macrophages are termed Kupffer cells, whereas those in the lung are alveolar macrophages. The microglia represent macrophages of the central nervous system, whereas histiocytes represent macrophages of connective tissue. Tissue stem cells are monocytes that have wandered from the blood into the tissues and may differentiate into macrophages. Mononuclear phagocytes have a variety of surface receptors that enable them to bind carbohydrates or such protein molecules as C3 via complement receptor 1 and complement receptor 3, and IgG and IgE through Fcγ and Fcε receptors. The surface expression of MHC class II molecules enables both monocytes and macrophages to serve as antigenpresenting cells to CD4+ T lymphocytes. Mononuclear phagocytes secrete a rich array of molecular substances with various functions. A few of these include interleukin-1; tumor necrosis factor α; interleukin-6; C2, C3, C4, and factor B complement proteins; prostaglandins; leukotrienes; and other substances.  ononuclear phagocytes include monocytes in the blood and M macrophages in the tissues which have cell surface receptors for Fcγ and C3b. They are also able to phagocytize microorganisms coated with opsonins and kill many but not all microorganisms during the process. Some microorganisms, such as mycobacteria, survive and multiply with macrophages which may serve as a reservoir or transport mechanism to help them reach other areas of the body. Other intracellular microorganisms that are not killed by nonimmune macrophages include Listeria monocytogenes, Brucella species, Legionella pneumophilia, Cryptococcus neoformans, Toxoplasma gondii, and Pneumocystis carinii. However, the development of cell-mediated immunity with the production of gamma interferon by lymphocytes is able to activate these macrophages to enable them to kill the intracellular pathogens. Activated macrophages produce interleukin-1 and tumor necrosis factor alpha which promote inflammation. Phagocytosis  (Figure 24.7) is the uptake of particulate material, such as bacteria, by endocytosis. Particle ligands unite with numerous receptors on the surface of the phagocyte in a “zippering” effect and cause polymerization of actin, invagination of the plasma membrane and sequestration of the particle into an intracellular vesicle termed a phagosome. Microbes taken up in this manner are killed by reactive oxygen intermediates and reactive nitrogen intermediate species inside the phagosome, which joins the endocytic processing pathway where it matures incrementally to generate a phagolysosome. It is an important clearance mechanism for the removal and disposition of foreign agents and particles or

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Fused granule (azurophil) membrane

IgG receptor

C3b receptor

Primary granule Secondary granule

Cationic antimicrobial proteins Fused granule (specific) membrane

Acidic phagolysosome contains short-chain fatty acids

Figure 24.7  Steps of phagocytic endocytosis.

damaged cells. Macrophages, monocytes, and polymorphonuclear cells are phagocytic cells. In special circumstances, other cells such as fibroblasts may show phagocytic properties; these are called facultative phagocytes.  hagocytosis may involve nonimmunologic or immunologic P mechanisms. Nonimmunologic phagocytosis refers to the ingestion of inert particles such as latex beads, or of other particles that have been modified by chemical treatment or 1.

coated with protein. Details of the recognition process of such particles are not known. Damaged cells are also phagocytized by nonimmunologic mechanisms. It is believed that in the latter case, damaged cells are also coated with immunoglobulin or other proteins which facilitate their recognition. Phagocytosis involves several steps  attachment, internalization, and digestion (Figure 24.8). The initiation of ingestion is known as the “zipper mechanism.” After attachment, the

PMN Plasma membrane

IgG

3. Bacterium

C3b

Fused granule (azurophil) membrane

IgG receptor C3b receptor

Specific (secondary) granule Cationic microbial proteins Azurophilic (primary) granule

Acidic phagolysosome contains short-chain fatty acids 2.

Fused granule (specific) membrane Lactoferrin Lysozyme Collagenase Vitamin B–binding protein

Figure 24.8  Phagocytosis.

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particle is engulfed within a fragment or plasma membrane and forms a phagocytic vacuole. This vacuole fuses with the primary lysosomes to form the phagolysosome in which the lysosomal enzymes are discharged and the enclosed material is digested. Remnants of indigestible material can be recognized subsequently as residual bodies. The process is associated with stimulation of phagocyte metabolism.  hagocytic dysfunction may be due to either extrinsic or P intrinsic defects. The extrinsic variety encompasses opsonin deficiencies secondary to antibody or complement factor deficiencies, suppression of phagocytic cell numbers by immunosuppressive agents, corticosteroid-induced interference with phagocytic function, decreased neutrophils through antineutrophil autoantibody; and abnormal neutrophil chemotaxis as a consequence of complement deficiency or abnormal complement components. Intrinsic phagocytic dysfunction is related to deficiencies in enzymatic deficiencies that participate in the metabolic pathway leading to bacterial cell killing. These intrinsic disorders include chronic granulomatous disease, characterized by defects in the respiratory burst pathway, myeloperoxidase deficiency, and glucose-6-phosphate dehydrogenase deficiency (G6PD). Consequences of phagocytic dysfunction include increased susceptibility to bacterial infections but not to viral or protozoal infections. Selected phagocytic function disorders may be associated with severe fungal infections. Severe bacterial infections associated with phagocytic dysfunction range from mild skin infections to fatal systemic infections.  hagocytosis may involve nonimmunologic or immunologic P mechanisms. Nonimmunologic phagocytosis refers to the ingestion of inert particles such as latex particles or of other particles that have been modified by chemical treatment or coated with protein. Damaged cells are also phagocytized by nonimmunologic mechanisms. Damaged cells may become coated with immunoglobulin or other proteins which facilitate their recognition.  hagocytosis of microorganisms involves several steps: P attachment, internalization, and digestion. After attachment, the particle is engulfed within a membrane fragment and a phagocytic vacuole is formed. The vacuole fuses with the primary lysosome to form the phagolysosome, in which the lysosomal enzymes are discharged and the enclosed material is digested. Remnants of indigestible material can be recognized subsequently as residual bodies. Polymorphonuclear neutrophils (PMNs), eosinophils, and macrophages play an important role in defending the host against microbial infection. PMNs and occasional eosinophils appear first in response to acute inflammation, followed later by macrophages. Chemotactic factors are released by actively multiplying microbes. These chemotactic factors are powerful attractants for phagocytic cells which have specific membrane receptors for the factors. Certain pyogenic bacteria may be destroyed soon after phagocytosis as a result of oxidative reactions. However, certain intracellular microorganisms

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such as Mycobacteria or Listeria are not killed merely by ingestion and may remain viable unless there is adequate cell-mediated immunity induced by γ interferon activation of macrophages.  hagocytic dysfunction may be due to either extrinsic or P intrinsic defects. The extrinsic variety encompasses opsonin deficiencies secondary to antibody or complement factor deficiencies, suppression of phagocytic cell numbers by immunosuppressive agents, corticosteroid-induced interference with phagocytic function, neutropenia, or abnormal neutrophil chemotaxis. Intrinsic phagocytic dysfunction is related to deficiencies in enzymatic killing of engulfed microorganisms. Examples of the intrinsic disorders include chronic granulomatous disease, myeloperoxidase deficiency, and glucose-6-phosphate dehydrogenase deficiency. Consequences of phagocytic dysfunction include increased susceptibility to bacterial infections but not to viral or protozoal infections. Selected phagocytic function disorders may be associated with severe fungal infections. Severe bacterial infections associated with phagocytic dysfunction range from mild skin infections to fatal systemic infections. Chemotaxis  is the process whereby chemical substances direct cell movement and orientation. The orientation and movement of cells in the direction of a chemical’s concentration gradient is positive chemotaxis, whereas movement away from the concentration gradient is termed negative chemotaxis. Substances that induce chemotaxis are referred to as chemotaxins and are often small molecules, such as C5a, formyl peptides, lymphokines, bacterial products, leukotriene B4, etc., that induce positive chemotaxis of polymorphonuclear neutrophils, eosinophils, and monocytes. These cells move into inflammatory agents by chemotaxis. A dual chamber device called a Boyden chamber is used to measure chemotaxis, in which phagocytic cells in culture are separated from a chemotactic substance by a membrane. The number of cells on the filter separating the cell chamber from the chemotaxis chamber reflect the chemotactic influence of the chemical substance for the cells. Chemotaxis  is locomotion of cells that may be stimulated by the presence of certain substances in their environment. This locomotion may be random in direction, i.e., it is not oriented with respect to the stimulus although there is a direct cause/effect relationship between stimulus and response. In contrast, the directed locomotion implies an orientation of cell movement with respect to the inducing stimulus. The latter form of cell movement is called chemotaxis and may be positive, in which the stimulus acts as an attractant, or negative, in which the stimulus acts as a repellent.  ubstances that may stimulate random cell locomotion are S called cytotoxigens; those that stimulate directed migration are called cytotoxins or chemotactic factors. The main element in the effect of chemotactic factors is the presence of a concentration gradient that determines the direction of cell

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migration. Under these circumstances a chemotactic signal is provided to the cells under consideration. In the absence of such a gradient, chemotactic factors enhance the random migration. CD230  is a 35-kDa molecule with broad antigenic expression. It is a large membrane prion protein that occurs normally in neurons of the human brain and is thought to be involved in synaptic transmission. In prion diseases, such as Creutzfeld-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE), altered forms of the normal cellular protein (PrPc) can occur upon contact with an infectious prion protein (PrPsc) from another host. The altered PrPsc form differs from the host-encoded PrPc in its conformational structure, but the sequence of both forms is reported to be the same. The altered PrPsc form is resistant to proteolytic degradation and accumulates in cytoplasmic vesicles of diseased individuals, forming lesions, vacuoles, and amyloid deposits. Also referred to as a prion protein.  hagocytes may kill microorganisms they have ingested by P either of two separate mechanisms. One of these, oxygendependent killing, is activated by a powerful oxidative burst that culminates in the formation of hydrogen peroxide and other antimicrobial substances (Figure 24.9). In addition to this oxygen-dependent killing mechanism, phagocytized intracellular microbes may be the targets of toxic substances released from granules into the phagosome, leading to microbial cell death by an oxygen-independent mechanism.  or oxygen-dependent killing of microbes, membranes of F specific granules and phagosomes fuse. This permits interaction of NADPH oxidase with cytochrome b. With the aid of quinone, this combination reduces oxygen to superoxide anion, O2. In the presence of a catalyst superoxide dismutase, superoxidase ion is converted to hydrogen peroxide.

– Cl

–

Cl

–

Cl

–

Cl

– Cl

IgG

Bacterium –

Cl

–

C3b

Cl

PMN plasma membrane

OH–

C3b receptor IgG receptor

H2O2 H2O2

O–2

OH– O–2 H2O2

O–2 OH–

Oxidase NADPH

Figure 24.9  Formation of bactericide and hydrogen peroxide catalyzed by NADPH oxidase.

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 he clinical relevance of this process is illustrated by chronic T granulomatous disease (CGD) in children who fail to form superoxide anions. They have diminished cytochrome b. Even though phagocytosis is normal, they have impaired ability to oxidize NADPH and destroy bacteria through the oxidative pathway. The oxidative mechanism kills microbes through a complex process. Hydrogen peroxide together with myeloperoxidase transforms chloride ions into hypochlorous ions that kill microorganisms. Azurophil granule fusion releases myeloperoxidase to the phagolysosome. Some microorganisms such as pneumococci may themselves form hydrogen peroxide. Natural killer (NK) cells  (Figures 24.10 and 24.11) attack and destroy tumor cells and certain virus-infected cells. They constitute an important part of the natural immune system, do not require prior contact with antigen, and are not MHC restricted by the major histocompatibility complex (MHC) antigens. NK cells are lymphoid cells of the natural immune system that express cytotoxicity against various nucleated cells including tumor cells and virus-infected cells. NK cells, killer (K) cells, or antibody-dependent cell-mediated cytotoxicity (ADCC) cells induce lysis through the action of antibody. Immunologic memory is not involved, as previous contact with the antigen is not necessary for NK cell activity. The NK cell is approximately 15 μm in diameter and has a kidney-shaped nucleus with several, often three, large cytoplasmic granules. The cells are also called large granular lymphocytes (LGL). In addition to their ability to kill selected tumor cells and some virus-infected cells, they also participate in ADCC by anchoring antibody to the cell surface through an Fcγ receptor. Thus, they are able to destroy antibody-coated nucleated cells. NK cells are believed to represent a significant part of the natural immune defense against spontaneously developing neoplastic cells and against infection by viruses. NK cell activity is measured by a 51Cr release assay employing the K562 erythroleukemia cell-line as a target. NK cells secrete IFN-γ and fail to express antigen receptors such as immunoglobulin receptors or T  cell receptors. Cell surface stimulatory receptors and inhibitory receptors that recognize self MHC molecules regulate their activation.  lthough not phagocytic, natural killer (NK) cells attack A and destroy certain virus-infected cells. They constitute a part of the natural immune system, do not require prior contact with antigen, and are not MHC restricted. On contacting a virus-infected cell, NK cells produce perforin that leads to the formation of pores in the infected cell membrane leading to osmotic lysis. Interferon enhances NK cell activity. These cells appear to be large granular lymphocytes and are significant in antiviral defense and in surveillance against the development of neoplasia. NK cells lyse certain virusinfected cells without MHC restriction. Questions remain concerning the phenotype of NK cells, even though several monoclonal antibodies reactive with them are available. The natural immune system, in which the NK cells are key

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INFγ

FC receptor

NK cell

Tumor cell lysis

Tumor cell

IgG antibody

TNFα Perforin and granzyme containing granules

Figure 24.10  NK cell-mediated killing of tumor cells by antibody-dependent cell-mediated cytotoxicity.

participants, does not involve memory. It does not require sensitization and cannot be enhanced by specific antigens. Other nonmemory cells include polymorphonuclear leukocytes and macrophages (Figure  24.12), which are important in early defense against infectious agents and possibly tumors. NK cells are able to lyse selected tumor target cells without prior sensitization and in the absence of antibody or complement. NK and cytotoxic T cells have been shown to share similar lytic mechanisms. Both cell types have

NK cell

Kill NKR-P1 signal Glycoprotein

Ly 49 Protect Class I signal MHC

No cytotoxicity

Normal target cell

NK cell

Kill NKR-P1 signal Glycoprotein Virusinfected traget cell

Ly 49 Class I MHC expression

Cytotoxicity

Figure 24.11  Proposed mechanism of NK cell cytotoxicity restricted to altered self cells. Kill signal is generated when the NK cell’s NKR-PI receptor interacts with membrane glycoprotein of normal and altered self cells. The kill signal can be countermanded by interaction of the NK cells Ly49 receptor with class I MHC molecules. Thus, MHC class I expression prevents NK cell killing of normal cells. Diminished class I expression on altered self cells leads to their destruction.

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granules that contain perforin or C9-related protein which lyse target cells without antibody or complement.  K activity is measured by a chromium release assay, N employing the K562 erythroleukemia cell line as a target. Whereas NK cells mediate their effect in the absence of antibody or complement, killer (K) cells or ADCC (antibodydependent cell-mediated cytotoxicity) cells induce lysis through the action of antibody. With the demonstration of Fc receptors on their surface, NK cells may actually be the killer (K) cells responsible for ADCC activity through attached IgG antibody. They mediate their classic effects via cell-surface receptors for antigen.  ther than NK or ADCC cells, circulating monocytes or O macrophages also mediate cell lysis through antibody molecules. Cytotoxic T cells (CTL) apparently recognize specific target cells through interaction with MHC antigens on the cell surface. Whereas either helper or killer T cells are directed to MHC proteins, NK cells apparently do not recognize MHC determinants. NK cell activity is located in the low-density population of lymphocytes which have large granules in their cytoplasm, i.e., large granular lymphocytes (LGL). Even though NK cells are lethal to tumor cells in vitro, very little data exists about their in vivo activity. Studies in mice suggest NK cells to be important in protection against selected virus infections. NK cells are also believed to play a regulatory role in the immune system, encompassing downregulation of antibody responses.

Natural killer (NK) cells Antibody-dependent cytotoxic cells K cells NK cells Lymphokine-activated killer (LAK) cells Tumor-infiltrating lymphocytes (TILS) Figure 24.12  Lymphoid cells participating in nonspecific immunity.

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Leukocyte activation:  The first step in activation is adhesion through surface receptors on the cell. Stimulus recognition is also mediated through membrane-bound receptors. An inducible endothelial–leukocyte adhesion molecule that provides a mechanism for leukocyte-vessel wall adhesion has been described. Studies on H2O2 secretion by PMNs exposed to macrophage and lymphocyte products suggest that surface adherent leukocytes undergo a large prolonged respiratory burst. Recombinant TNF-alpha delays H2O2 release, demonstrating that soluble factors from macrophages and lymphocytes can affect adherent PMNs with respect to cytotoxic potential. Studies on regulation of neutrophil activation by platelets reveal that platelet-derived growth factor (PDGF) does not alter the resting level of superoxide generation but inhibits the rate and extent of f-Met-Leu-Phe-induced oxidative burst. Intracellular Ca++ increases upregulation of ligandindependent cell surface expression of f-Met-Leu-Phe receptors in neutrophils, whereas phorbol myristate acetate (PMA) activates downregulation of these receptors. A pertussis toxin-sensitive GTP-binding protein regulates monocyte phagocytic function.  omplement receptor 3 (CR3) facilitates the ability of C phagocytes to bind and ingest opsonized particles. There is a relatively large family of homologous adhesion-promoting receptor proteins, including leukocyte proteins, that identify the sequence Arg-Gly-Asp. Molecules found to be powerful stimulators of PMN activity include recombinant IFN-γ, granulocyte–macrophage colony-stimulating factor, TNF, and lymphotoxin. Investigations of storage sites for the several protein receptors have revealed a mobile intracellular storage compartment in human neutrophils. Chemotactic stimuli, such as f-Met-Leu-Phe, may cause translocation of granules acting as storage sites to the cell surface, which could be a requisite for neutrophil adhesion and chemotaxis.  ephosphorylation pathways for inositol triphosphate isomers D culminate in the elevation of intracellular Ca++ and protein kinase C activation. NADPH oxidase, which utilizes hexose monophosphate shunt-generated NADPH, catalyzes the respiratory burst. Both Ca++ and protein kinase C play a key role in the activated pathway. Activated human neutrophils manifest an elevated expression of complement decay-accelerating factor, which protects erythrocytes from injury by autologous complement. Transduction of decay-accelerating factors to the cell surface following stimulation by chemoattractants may be significant in protecting PMNs from complementmediated injury. This type of process would permit PMNs to manifest unreserved function in sites of inflammation.  urface adherent leukocytes undergo a large prolonged respiS ratory burst. NADPH oxidase, which utilizes hexose monophosphate shunt-generated NADPH, catalyzes the respiratory burst. Both Ca2+ and protein kinase C play a key role in the activation pathway. Complement receptor 3 (CR3) facilitates

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the ability of phagocytes to bind and ingest opsonized particles. Molecules found to be powerful stimulators of PMN activity include recombinant IFN-γ, granulocyte–macrophage colony-stimulating factor, TNF, and lymphotoxin.  he K cells (killer cells), also called null cells, have lymT phocyte-like morphology but functional characteristics different from those of B and T  cells. They are involved in a particular form of immune response, the antibody-dependent cellular cytotoxicity (ADCC), killing target cells coated with IgG antibodies. A K cell is an Fc-bearing killer cell that has an effector function in mediating antibody-dependent cellmediated cytotoxicity. An IgG antibody molecule binds through its Fc region to the K cell’s Fc receptor. Following contact with a target cell bearing antigenic determinants on its surface for which the Fab regions of the antibody molecule attached to the K cell are specific, the lymphocyte-like K cell releases lymphokines that destroy the target. This represents a type of immune effector function in which cells and antibody participate. Besides K  cells, other cells that mediate antibody-dependent cell-mediated cytotoxicity include NK cells, cytotoxic T cells, neutrophils, and macrophages.  umor specific IgG antibodies may act in concert with T immune system cells to produce antitumor effects. Antibody-dependent cell-mediated cytotoxicity (ADCC) (Figure  24.13) is a reaction in which T lymphocytes, NK cells, including large granular lymphocytes, neutrophils, and macrophages may lyse tumor cells, infectious agents, and allogeneic cells by combining through their Fc receptors with the Fc region of IgG antibodies bound through their Fab regions to target cell surface antigens. Following linkage of Fc receptors with Fc regions, destruction of the target is accomplished through released cytokines. It represents an example of participation between antibody molecules and immune system cells to produce an effector function. Humans have innate immunity against extracellular bacteria. Neutrophil (PMN), monocyte, and tissue macrophage phagocytosis leads to rapid microbicidal action against ingested microbes from the extracellular environment. The capacity of a microorganism to resist phagocytosis and digestion in phagocytic cells is a principal feature of its virulence. Complement activation represents a significant mechanism for ridding the body of invading microorganisms. A peptidoglycan layer (Figure 24.14) in the cell walls Antibody

Tumor cell Tumor cell lysis

Lymphocyte

Figure 24.13  ADCC.

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GlcNAc

CH2OH O OH

CH2OH O

O

MA

O

MDP

OH

HO

NCOCH2OH H

NAc H CH3C CO

L-Ala

D-Glu

H NHCHCO-NHCHCONH2 CH2 CH3 CH2

D-Ala

CO-NHCH-CO-NHCH-COOH CH2 CH3 CH2 Meso-DAP CH2 R-H-N-C-CONH2 H

Figure 24.14  Peptidoglycan (murein).

of Gram-positive bacteria as well as lipopolysaccharide, or LPS (Figure 24.15), in the cell walls of Gram-negative bacteria is able to activate the alternative pathway of complement without antibody. Also associated with LPS is flagellar antigen and somatic antigen (Figure 24.16). Flagellar antigens, or H antigens  are epitopes on flagella of enteric bacteria that are motile and Gram-negative. H is from the German word hauch, which means breath, and refers to the production of a film on agar plates that resembles breathing on glass. In the Kaufmann–White classification scheme for Salmonella, H antigens serve as the basis for the division of microorganisms into Phase I and Phase II, depending on the flagellin. Phase variation may result in a switch to production of the other type that is genetically controlled. A somatic antigen, or O antigen  is a lipopolysaccharideprotein antigen of enteric microorganisms which is used for their serological classification. O antigens of the Proteus species serve as the basis for the Weil-Felix reaction which is employed to classify Rickettsia. O antigens of Shigella permit them to be subdivided into 40 serotypes. The exterior oligosaccharide repeating unit side chain is responsible for specificity and is joined to lipid A to form lipopolysaccharide Cross-Section of Bacterial Cell Wall

and to lipid B. The O antigen is the most variable part of the lipopolysaccharide molecule.  3b may deposit on LPS where it is safe from the inactiC vating effects of factors H and I. LPS may also activate the classical complement pathway in the absence of antibody by combining with Clq. The C3b that results from activation of complement serves as an opsonin when linked to the bacterial surface, making the bacterial cell more attractive to phagocytes. The membrane attack complex (MAC) induces lysis of bacterial cells. Complement reaction products play an active role in inflammation through the attraction and stimulation of leukocytes. Lipopolysaccharides or endotoxins  (Figure  24.17) induce macrophages and selected other cells such as endothelial cells of vessels to synthesize cyto­k ines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-8 (IL-8) molecules that participate in inflammation (Figure 24.18). Monokines and cytokines from macrophages activate nonspecific inflammation and facilitate lymphocyte activation by bacterial epitopes. PMNs and monocytes adhere to the endothelium of vessels in areas of infection through the action of cytokines. These inflammatory cells migrate, accumulate in local areas, and become activated, enabling them to destroy the microorganisms. Local tissue injury may

Capsular polysaccharide Lipopolysaccharide (endotoxin) Outer membrane

Flagella antigen

Peptidoglycan Inner membrane

Figure 24.15  Cross-section of Gram-negative bacterial cell wall.

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Figure 24.16  Bacterial cell.

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FA

O

O

CH2

CH2

O

O

O HO P O O EtN KDO KDO

FA

NH

O

C

O

CH2 HC OH

O FA

O NH C

O

O

O P O P OH O

O

O

CH2 HC O

FA(CH2)10

FA(CH2)10

CH3

CH3

FA

Oligosaccharide repeats Core polysaccharide

Lipid A

O side chain

Figure 24.17  Lipopolysaccharide or endotoxin.

be an unintended consequence of these resisting processes. Fever and the formation of acute-phase reactants may also be consequences of cytokine action. Some cytokines may facilitate specific immune mechanisms by stimulating both T and B cells. Excessive cytokine synthesis may lead to pathologic sequelae during infection by extracellular microorganisms. Gram-negative bacterial infection can lead to disseminated intravascular coagulation (DIC) and vascular collapse known also as endotoxin shock or septic shock in which it is mediated mainly by TNF. Flagellar antigens  are epitopes of flagella in an organelle that renders some bacteria motile. Also called H antigens. Cell differentiation factors CSF Cytotoxic factors TNF-α

Cachectin

Hydrolytic enzymes Collagenase Lipase Phosphatase Endogenous pyrogen IL-1

Alpha interferon Plasma proteins Coagulation factors Oxygen metabolites H2O2 Superoxide anion Arachidonic acid metabolites Prostaglandins Thromboxanes Leukotrienes Complement components C1 to C5 Properdin Factors B, D, I, H

Figure 24.18  Secreted products of macrophages that have a protective effect on the body.

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Flagellin  is a protein that is a principal constituent of bacterial flagella. It consists of 25- to 60-kDa monomers that are arranged into helical chains which wind around a central hollow core. Polymeric flagellin is an excellent thymus-independent antigen. Mutations may occur in the central part of a flagellin monomer that has a variable sequence. A somatic antigen  is an antigen such as the O antigen which is part of a bacterial cell’s structure. Endotoxin  is a Gram-negative bacterial cell wall lipopolysaccharide (LPS) that is heat stable and causes neutrophils to release pyrogens. It may produce endotoxin or hemorrhagic shock and modify resistance against infection. Endotoxins comprise an integral constituent of the outer membrane of Gram-negative microorganisms. They are significant virulence factors and induce injury in a number of ways. Toxicity is associated with the molecule’s lipid A fraction, which is comprised of a β-1,6-glucosaminyl-glucosamine disaccharide substituted with phosphate groups and fatty acids. Lipopolysaccharide (LPS) has multiple biological properties that include ability to induce fever, lethal action, initiation of both complement and blood coagulation cascades, mitogenic effect on B lymphocytes; the ability to stimulate production of cytokines such as tumor necrosis factor and interleukin-1; and the ability to clot Limulus amebocyte lysate. Cytokines induced by endotoxins cause fever, increased capillary permeability, and possible endotoxic shock. Relatively large amounts of lipopolysaccharide released from Gram-negative bacteria during Gramnegative septicemia may produce endotoxin shock. Endotoxin shock  is the circulatory and metabolic collapse that occurs following exposure to excessive quantities

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of cytokines, especially IL-1 and TNF, introduced into the blood circulation by macrophages following Gram-negative bacterial infection or their products, such as lipopolysaccharide. It is characterized by falling blood pressure and disseminated intravascular coagulation (DIC) following exposure to relatively large amounts of endotoxin produced during bacterial sepsis with Escherichia coli, Pseudomonas aeruginosa, or meningococci. DIC leads to the formation of thrombi in small blood vessel, leading to such devastating consequences as bilateral cortical necrosis of the kidneys and blockage of the blood supply to the brain, lungs, and adrenals. When DIC affects the adrenal glands, as in certain meningococcal infections, infarction leads to adrenal insufficiency and death. This is the Waterhouse–Friderichsen syndrome. Endotoxin shock is also referred to as septic shock. An enterotoxin  is a bacterial toxin that is heat stable and causes intestinal injury. A mitogen  is a substance, often derived from plants, that nonspecifically stimulates DNA synthesis and induces blast transformation and cell division by mitosis. The mitogen-binding site on lymphocytes is distinct from the antigen-binding site of the B cell receptor or of the T cell receptor. Lectins, representing plant-derived mitogens or phytomitogens, have been widely used in both experimental and clinical immunology to evaluate T and B lymphocyte function in vitro. Phytohemagglutinin (PHA) is principally a human and mouse T cell mitogen, as is concanavalin A (Con A). By contrast, lipopolysaccharide (LPS) induces B lymphocyte transformation in mice, but not in humans. Staphylococcal protein A is the mitogen used to induce human B lymphocyte transformation. Pokeweed mitogen (PWM) transforms B cells of both humans and mice, as well as their T cells. Natural immunity against viruses  occurs when virusinfected host cells sensitize type I interferon. This blocks virus replication. NK cells, which are not MHC restricted, provide early antiviral effects following infection. Type I interferon accentuates their action. Both complement and phagocytosis play significant roles in removal of extracellular viruses.  hagocytosis is the chief mechanism of innate immunity P against intracellular bacteria whereby intracellular pathogenic microorganisms should be eliminated. However, this is frustrated by the resistance of many intracellular microbes to intracellular dissolution. Thus, the natural immune mechanism of phagocytosis is of little use in controlling infection by intracellular microorganisms. Bacteria of this category may persist in the tissues leading to chronic infection. I nnate immunity against intracellular bacteria  is frustrated by the resistance of many intracellular microbes to intracellular dissolution. Thus, the natural immune mechanism of phagocytosis is of little use in controlling infection by intracellular microorganisms. Bacteria of this category may persist in the tissues, leading to chronic infection.

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 arasitic protozoa and helminths are adept at survival within P the host through successful resistance of host innate immune mechanisms against parasites. Whereas parasitic stages isolated from invertebrates may be lysed through activation of the alternate complement pathway, parasites isolated from humans or other veterbrate hosts are often susceptible to complement lysis. This could be attributable to either disappearance of surface molecules that activates complement or adherence to the surface of decay-accelerating factor (DAF) or other regulatory proteins.  echanisms whereby mature adult schistosomes are able to M evade the immune response of the host include low surface antigenicity, disguise, host molecule mimicry, surface antigen sequestration and shedding, and reduced surface antigenicity, as well as other evasions mechanisms. Reducing surface antigenicity by host molecule masking, shedding, or sequestration of antigen represents a successful mechanism for parasites to escape the immune system. Whereas macrophages may ingest protozoa, numerous pathogenic parasites may resist intracellular killing or even replicate within the phagocyte. The outer coat of helminths helps to protect against intracellular killing by neutrophils or macrophages.  pecific immune responses can be mounted against paraS sites. Parasites such as protozoa and helminths elicit a variety of immune responses. Helminthic infections including Nippostrongylus schistosomes and filaria evoke titers of IgE that exceed those induced by other infectious agents. Helminths specifically stimulate Cd4+ helper t lymphocytes that form IL-4 and IL-5. ADCC involving eosinophils and IgE antibody is believed to be effective in immunity against helminths since the major basic protein in eosinophil granules is toxic to helminths. Coating helminths with IgE-specific antibody followed by eosinophil attachment through the Fc regions leads to ADCC by eosinophils. Innate immune mechanisms against parasites:  Whereas parasitic stages isolated from invertebrates may be lysed through activation of the alternate complement pathway, parasites isolated from humans or other veterbrate hosts are often susceptible to complement lysis. This could be attributable to either disappearance of surface molecules that activates complement or adherence to the surface of decayaccelerating factor (DAF) or other regulatory proteins. Acquired immunity  can develop after previous contact with the organism through infection (overt or subclinical) or by deliberate immunization with a vaccine prepared from the etiologic agent. It generates protective resistance against an infectious agent as a consequence of infection with a specific microorganism or as a result of deliberate immunization. Refer also to primary immune response, secondary immune response, and adaptive immunity. Acute-phase serum  is a serum sample drawn from a patient with an infectious disease during the acute phase.

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An adaptive immune response  is the response of B and T lymphocytes to a specific antigen and the development of immunological memory. The response involves clonal selection of lymphocytes that respond to a specific antigen. Also called acquired immune response. Adaptive immunity  is protection from an infectious disease agent as a consequence of clinical or subclinical infection with that agent or by deliberate immunization against that agent with products from it. This type of immunity is mediated by B and T lymphocytes following exposure to specific antigen. It is characterized by specificity, immunological memory, and self/nonself recognition. This type of immunity is in contrast to natural or innate immunity. Cardiolipin  is diphosphatidyl glycerol, a phospholipid, extracted from beef heart as the principal antigen in the Wasserman complement fixation test for syphilis used earlier in the century. Cecropin  is an antibacterial protein derived from immunized cecropia moth pupae. It is also found in butterflies. Cecropin is a basic protein that induces prompt lysis of selected Gramnegative and Gram-positive bacteria. Coagglutination  refers to the interaction of IgG antibodies with the surface of protein A–containing Staphylococcus aureus microorganisms through their Fc regions, followed by interaction of the Fab regions of these same antibody molecules with surface antigens of bacteria for which they are specific. Thus, when the appropriate reagents are all present, coagglutination will take place in which the Y-shaped antibody molecule will serve as the bridge between staphylococci and the coagglutinated microorganism for which it is specific. Concomitant immunity  is the continued survival of adult worms from a primary infection while the host is demonstrably resistant to reinfection by a secondary challenge of fresh cercariae. 1. In tumor immunology, resistance to a tumor that has been transplanted into a host already bearing that tumor. Immunity to the reinoculated neoplasm does not inhibit growth of the primary tumor. 2. Resistance to reinfection of a host currently infected with that parasite. Convalescent serum  is a patient’s blood serum sample obtained 2 to 3 weeks following the beginning of a disease. The finding of an antibody titer to a pathogenic microorganism that is higher than the titer of a serum sample taken earlier in the disease is considered to signify infection produced by that particular microorganism. For example, a fourfold or greater elevation in antibody titer would represent presumptive evidence that a particular virus, for example, had induced the infection in question.

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Effector mechanisms  refer to the means whereby post innate and adaptive immune responses destroy and eliminate pathogens from the body. The effector phase  is that part of an immune response following recognition and activation phases during which a foreign antigen, such as a microbe, is inactivated or destroyed. The effector response  is an event that follows antigen recognition and binding by antibody, such as complement-mediated lysis. Immunological memory:  The effectiveness of protective immunity against an infectious agent depends on the ability of the immune system to retain a memory of the original infective agent in order to provide an enhanced immune response on reexposure to the same agent. This usually prevents overt infection and prevents a fatal outcome. However, not all consequences of immune memory are beneficial. A second infection with the dengue virus may be more severe than the first. Another detrimental effect of immunity is sensitization to an allergen, leading to a hypersensitivity reaction. During a primary immune response to an infectious agent, each antigen-specific lymphocyte clone activated produces numerous memory lymphocytes of identical specificity and greater affinity. A second attack by the same pathogen leads to a secondary immune response when memory lymphocytes are activated, which leads to more rapid and efficient elimination of the invading pathogen. Immunologic memory is specific for a particular antigen and is long lasting. Immunological memory is governed by many factors with both B  cells and T cells contributing to it. It depends upon interactions between memory T cells and memory B cells. Systemic inflammatory response syndrome (SIRS):  The systemic effects of disseminated bacterial infection. Mild and severe forms have been described. The mild form of SIRS is characterized by fever, neutrophilia, and an increase in acute-phase reactants. LPS or other bacterial products may stimulate these changes that are mediated by innate immune system cytokines. Severe SIRS is characterized by disseminated intravascular coagulation, adult respiratory distress syndrome, and septic shock. Innate or constitutive defense system:  Humans are confronted with a host of microorganisms with the potential to induce serious or fatal infections. Yet, nature has provided appropriate molecules, cells, and receptors that can protect against these microbes. Many of these defenses are general or nonspecific and do not require previous exposure to the offending pathogen (or closely related organism). These important mechanisms constitute the innate or constitutive defense system. Another important defense system is acquired immunity, which can develop after previous contact with the organism through infection (overt or subclinical).

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Naturally acquired immunity  describes the protection provided by previous exposure to a pathogenic microorganism or antigenically related organism. In contrast, artificially acquired immunity  develops as a result of immunization with vaccines—either with attenuated organisms or with killed organisms or subunit components. Toxoids provide excellent immunity against the effects of microorganisms such as Corynebacterium diphtheriae and Clostridium tetani that produce powerful exotoxins. Active immunization with appropriate booster injections leads to the development of IgG which provides immunity of long duration. Acquired immunity depends upon antibodies and T cells. Passive immunity  involves the transfer of resistance against an infectious disease agent from an immune individual to a previously susceptible recipient. Natural passive immunity describes the transfer of IgG antibodies across the placenta from mother to child. IgA secretory antibodies may also be passively transferred from mother to child in breast milk. Antitoxins generated to protect against diphtheria or tetanus toxins represent a second example of passive humoral immunity, as used in the past. The transfer of specifically sensitized lymphoid cells from an immune to a previously nonimmune recipient is termed adoptive immunization. The passive transfer of antibodies in immune serum can be used for the temporary protection of individuals exposed to certain infectious disease agents who may be injected with hyperimmune globulin. No immunological memory is established. Artificially acquired passive immunity  describes the transfer of immunoglobulins from an immune individual to a nonimmune, susceptible recipient. Passive immunity of this type is more often used for prophylaxis than for therapy. It provides immediate protection of the recipient for relatively short periods (few weeks). Human sera are preferred for passive immunization to avoid serum sickness induced by foreign serum proteins. Specific immune response to extracellular bacteria:  Antibodies are the primary agents that protect the body against extracellular bacteria. Microbial cell wall polysaccharides serve as thymus-independent antigens that stimulate specific IgM antibody responses. Cytokine production may even permit switching from IgM to IgG production. Protein antigens of extracellular bacteria primarily stimulate activate CD4+ T cells. Toxins of extracellular bacteria may activate multiple CD4+ T lymphocytes. When a bacterial toxin stimulates an entire family of T lymphocytes that express products of a certain family of vβT lymphocyte receptor genes, it is referred to as a superantigen. Immune stimulation of this type may lead to the production of abundant quantities of cytokines that lead to pathologic sequelae. The resistance mechanism against extracellular bacteria regrettably may include two reactions that produce tissue injury: acute

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inflammation and endotoxin shock. In addition, late in the course of a bacterial infection, pathogenic antibodies may appear. The multiple lymphocytes clones stimulated by either bacterial endotoxins or superantigens may lead to the production of autoimmunity through overriding specific T cell bypass mechanisms. Autoreactive lymphocytes may also be activated during this process. Bacteria  are prokaryotic microorganisms, found throughout nature, that are responsible for many infectious diseases of humans and other animals. There are two main types of bacteria. Gram-positive bacteria are microorganisms with thick cell walls that contain peptidoglycan and lipoteichoic and teichoic acids that stain purple in the Gram staining technique, while Gram-negative bacteria are microorganisms with thin cell walls that contain peptidoglycan and lipopolysaccharide (LPS) that stain red in the Gram staining technique. Bacterial immunity:  Bacteria produce disease by toxicity, invasiveness, or immunopathology, or combinations of these three mechanisms. Immune mechanisms may require the development of a neutralizing antitoxin or mechanisms to destroy the microorganism. The animal body provides both nonspecific and specific defenses. Nonspecific defenses include natural barriers such as the skin, acidity in the gut and vagina, mucosal coverings, etc. Those microorganisms not excluded may be recognized by acute-phase proteins, formol peptide receptors, receptors for bacterial cell-wall components, complement, and receptors that promote cytokine release. Other factors influence the TH1/TH2 balance of the T  cell response. Cytokines play a protective role during nonspecific recognition and early defense. Bacteria may interact with complement leading to three types of protective function. Antibodies are important in neutralizing bacterial toxins. Secretory IgA can inhibit the binding of bacteria to epithelial cells. This antibody may also sensitize bacterial Opsonic-promote ingestion and killing by phagocytic cells (IgG) Block attachment (IgA) Neutralize toxins. Agglutinate bacteria—may aid in clearing Render motile organisms nonmotile Abs only rarely affect metabolism or growth of bacteria (Mycoplasma) Abs, combining with antigens of the bacterial surface, activate the complement cascade, thus inducing an inflammatory response and bringing fresh phagocytes and serum Abs into the site Abs, combining with antigens of the bacterial surface, activate the complement cascade, and through the final sequences the membrane attack complex (MAC) is formed involving C5b-C9 Figure 24.19  Antimicrobial actions of antibodies.

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cells and render them susceptible to injury by complement. Phagocytic cells are important antibacterial defenses. Monocytes and polymorphonuclear cells have both oxygendependent and oxygen-independent antimicrobial mechanisms. Oxygen-independent killing may be accomplished by exposure to lysozyme and neutroproteases. Cell-mediated immunity mediated by T lymphocytes is another important antibacterial mechanism. They function through the release of lymphokines that have various types of consequences. Cytotoxic T cells and NK cells also have an important role in antimicrobial immunity. Mechanisms of immunopathology include septisemic shock in the adult respiratory distress syndrome; the Shwartzman reaction; the Koch phenomenon and necrotizing T  cell-dependent granulomas; and heat shock proteins and the possible development of autoimmunity. Thus, the immune response to bacteria is varied and complex but effective in the animal organism with an intact immune response.  n infection or bacterial allergy is a hypersensitivity, espeA cially of the delayed T  cell type, that develops in subjects infected with certain microorganisms, such as Mycobacterium tuberculosis or certain pathogenic fungi. Bacteriolysin  is an agent such as an antibody or other substance that lyses bacteria. Bacteriolysis  refers to the disruption of bacterial cells by such agents as antibody and complement or lysozyme, causing the cells to release their contents. Capsule swelling reaction:  Pneumococcus swelling reaction. See Quellung reaction. Cat scratch disease  or regional lymphadenitis, is common usually in children following a cat scratch. The condition is induced by a small Gram-negative bacillus. Erythematous papules may appear on the hands or forearms at the site of the injury. Patients may develop fever, malaise, swelling of the parotid gland, lymphadenapathy that is regional or generalized, maculopapular rash, anorexia, splenic enlargement, and encephalopathy. There may be hyperplasia of lymphoid tissues, formation of granulomas, and abscesses. A positive skin test together with the history establishes the diagnosis. Gentamycin and ciprofloxacin have been used in treatment. Chancre immunity  describes the resistance to reinfection with Treponema pallidum that develops 3 months following a syphilis infection that is untreated. Cholera toxin  is a Vibro cholerae enterotoxin comprised of five B subunits that are cell-binding 11.6-kDa structures that encircle a 27-kDa catalase that conveys ADP-ribose to G protein, leading to continual adenyl cyclase activation. Other toxins that resemble cholera toxin in function include diphtheria toxin, exotoxin A, and pertussis toxin.

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Circulatory system infections:  The principal infection involving the circulatory system with immunologic sequelae is infective endocarditis. In acute endocarditis attributable to Staphylococcus aureus, there is high fever and, if untreated, a fatal rapid destruction of the heart valves. In subacute endocarditis there is a more indolent course and immunologic complications follow. Emboli break off from the infected heart valve. The principal causative microorganisms are streptococci, which include viridans streptococci and the more antibiotic resistant enterococci. Antibody levels, measured by immunoblotting, are greatly increased in enterococcal or streptococcal endocarditis and these are species specific. Corynebacterium diphtheriae immunity:  Toxins produced by all strains of this organism are identical immunologically, which means that antitoxins may neutralize them equally. A single toxoid is used for effective immunization. There is no type-specific immunity. Immunization does not protect against the infection but against the systemic and local effects of the toxin. A high level of immunity is conferred but it is not complete. Dapsone  (diaminodiphenyl sulfone) is a sulfa drug that has been used in the treatment of leprosy. It has also shown efficacy for prophylaxis of malaria and for therapy of dermatitis herpetiformis. DDS syndrome  refers to a hypersensitivity reaction that occurs in 1 in 5000 leprosy patients who have been treated with dapsone (DDS, 4,4′-diaminodiphenyl sulfone), a drug that prevents folate synthesis by inhibiting the paminobenzoic acid condensation reaction. Patients develop hemolysis, agranulocytosis, and hypoalbuminemia, as well as exfoliative dermatitis and life-threatening hepatitis. Encapsulated bacteria  are surrounded by a thick carbohydrate coating or capsule that protects microorganisms such as pneumococci from phagocytosis. Infection-producing encapsulated bacteria cannot be effectively phagocytized and destroyed unless they are first coated with an opsonizing antibody, formed in an adaptive immune response, and complement. End-binders  are selected anticarbohydrate-specific antibodies that bind the ends of oligosaccharide antigens, in contrast to those that bind the sides of these molecules. The Fernandez reaction  is an early (24 to 48-h) tuberculin-like delayed-type hypersensitivity reaction to lepromin observed in tuberculoid leprosy; a skin test for leprosy. FTA-ABS (fluorescence treponema antibody absorption)  is a serological test for syphilis that is very sensitive, i.e., approaching 100% in the diagnosis of secondary, tertiary, congenital, and neurosyphilis. It is an assay for specific antibodies to Treponema pallidum in the serum of patients

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Immunity against Microorganisms

suspected to have syphilis. Before combining the patient’s serum with killed T. pallidum microorganisms fixed on a slide, the serum is first absorbed with an extract of Reiter’s treponemes to remove group-specific antibodies. After washing, the specimen is covered with fluorescein-labeled antihuman globulin. This is followed by examination by fluorescence microscopy equipped with ultraviolet light. Demonstration of positive fluorescence of the target microorganisms reveals specific antibody present in the patient’s serum. The greater specificity and sensitivity of this test makes it preferable to the previously used FTA-200 assay. H antigens  are epitopes on flagella of enteric bacteria that are motile and Gram-negative. H is from the German word hauch, which means breath, and refers to the production of a film on agar plates that resembles breathing on glass. In the Kaufmann–White classification scheme for Salmonella, H antigens serve as the basis for the division of microorganisms into phase I and phase II, depending on the flagellins they contain. A single cell synthesizes only one type of flagellin. Phase variation may result in a switch to production of the other type that is genetically controlled. Halogenation  refers to halogen binding to the cell wall of a microorganism with resulting injury to the microbe. Hib (Hemophilus influenzae type b)  is a microorganism that induces infection mostly in infants less than 5 years of age. Approximately 1000 deaths out of 20,000 annual cases are recorded. A polysaccharide vaccine (Hib Vac) was of only marginal efficacy and poorly immunogenic. By contrast, anti-Hib vaccine that contains capsular polysaccharide of Hib bound covalently to a carrier protein such as polyribosylribitol-diphtheria toxoid (PRP-D) induces a very high level of protection that reached 94% in one cohort of Finnish infants. PRP-tetanus toxoid has induced 75% protection. PRP-diphtheria toxoid vaccine has been claimed to be 88% effective. Homozygote  describes an organism whose genotype is characterized by two identical alleles of a gene. Intimin  is a bacterial membrane protein expressed on enteropathogenic Escherichia coli and can bind to both α4β1 and α4β7 integrins. Intracellular pathogens  are microorganisms, including viruses and bacteria, that grow within cells. Extracellular pathogens  are pathogenic microorganisms that reproduce in the interstitial fluid, blood or lumens of the respiratory, urogenital and gastrointestinal tracts rather than entering host cells. Invasive pathogens  are pathogenic microorganisms that successfully gains access to body even though defense mechanisms are intact.

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Jarisch-Herxheimer  reaction is a systemic reaction associated with fever, lymphadenopathy, skin rash, and headaches that follows the injection of penicillin into patients with syphilis. It is apparently produced by the release of significant quantities of toxic or antigenic substances from multiple Treponema pallidum microorganisms. K antigens  are surface epitopes of Gram-negative microorganisms. They are either proteins (fimbriae) or acid polysaccharides found on the surface of Klebsiella and Escherichia coli microorganisms. K antigens are exterior to somatic O antigens. They are labile to heat and cross-react with the capsules of other microorganisms such as Hemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis. K antigens may be linked to virulent strains of microorganisms that induce urinary tract infections. Anti-K antibodies are only weakly protective. Lyme disease  is a condition first described in Lyme, Connecticut where an epidemic of juvenile rheumatoid arthritis (Still’s disease) was found to be due to Borrelia burgdorferi. It is the most frequent zoonosis in the United States with concentration along the eastern coast. Insect vectors include the deer tick (Ixodes dammini), white-footed mouse tick (I.  pacificus), wood tick (I. ricinus), and lone star tick (Amblyomma americanum). Deer and field mice are the hosts. In stage I, a rash termed erythema chronicum migrans occurs. The rash begins as a single reddish papule and plaque that expands centrifugally to as much as 20 cm. This is accompanied by induration at the periphery with central clearing that may persist months to years. The vessels contain IgM and C3 deposits. Stage II is the cardiovascular stage, which may be accompanied by pericarditis, myocarditis, transient atrial ventricular block, and ventricular dysfunction. Neurological symptoms also ensue and include Bell’s palsy, meningoencephalitis, optic atrophy, and polyneuritis. Stage III is characterized by migratory polyarthritis. The diagnosis requires the demonstration of IgG antibodies against the causative agent by Western immunoblotting. Lyme disease is treated with the anti­ biotics tetracycline, penicillin, and erythromycin. Borrelia burgdorferi, the causative agent of this chronic infection, is a spirochete that may evade the immune response. Mycoplasma immunity:  High-titer cold agglutinin autoantibodies against sialo-oligosaccharide of the Ii antigen type are sometimes found during Mycoplasma pneumoniae infections. The first line of defense against these microorganisms is phagocytosis. Yet mycoplasmas can survive neutrophil phagocytosis if specific antibodies are not present. Secretory IgA is significant in preventing localized colonization, yet systemic antibodies protect from primary infection and secondary spread from localized colonization. Mycoplasmas can evade the humoral immune response by undergoing antigenic variation of surface antigens. T cells also appear to play a role in immunity to mycoplasma, which needs to be further investigated.

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Organism-specific antibody index (OSAI)  is the ratio of organism-specific IgG to total IgG in cerebrospinal fluid compared to the ratio of organism-specific IgG in serum to the total serum IgG. This is illustrated in the following formula: I f the index is greater than 1, signifying a greater quantity of organism-specific immunoglobulin in CSF than in the blood serum, this implies that organism-specific IgG is being synthesized in the intra-blood–brain barrier (IBBB) and suggests that the specific organism of interest is producing an infection of the central nervous system. Similar indices can be calculated for IgM and IgA antibody classes. A pathogen  is an agent such as a microorganism that can produce disease through infection of the host. Pathogen-associated molecular pattern (PAMP)  are repetitive motifs of molecules such as lipopolysaccharide, peptidoglycan, lipoteichoic acids, and mannans that are broadly expressed by microbial pathogens not found on host tissues. The immune system’s pattern recognition receptors (PRRs) make use of them in differentiating between pathogen antigens and self antigens. PAMP  is the abbreviation for pathogen-associated molecular pattern. Pattern recognition receptors (PRRs)  bind to pathogenassociated molecular patterns (PAMPs). They are natural or innate immune system receptors which recognize molecular patterns that comprise frequently encountered structures produced by microorganisms. These receptors enhance natural immune responses against microbes. CD14 receptors on macrophages that bind bacterial endotoxin to activate macrophages, and the mannose receptor on phagocytes that bind microbial glycoproteins or glycolipids, are examples of pattern recognition receptors. These plasma membrane or endocytic vesicle membrane-bound pattern recognition molecules have a broad distribution pattern. Toll-like receptors, NK activatory receptors, the γδ T cell receptor and the NKT semi-invariant TCR also belong to this category of molecules. Binding of PRRs leads to pro-inflammatory cytokine expression. Encapsulation  is the reaction of leukocytes to foreign material that cannot be phagocytized because of its large size. Multiple layers of flattened leukocytes form a wall surrounding the foreign body and isolate it within the tissues. This type of reaction occurs in invertebrates, including annelids, mollusks, and arthropods. In higher invertebrates, it is mediated by pattern recognition receptors (PRRs), whereby pathogens too large to be phagocytized are encircled by numerous phagocytic cells. Phagocyte reactive oxygen intermediates and lysosomal enzymes then destroy the encapsulated pathogens. In vertebrates, macrophages surround the foreign body, a granuloma is formed, and fibroblasts subsequently appear. A fibrous capsule is formed.

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Protein M (M antigen)  is Group A streptococcal protein found on fimbriae. It is antiphagocytic and facilitates virulence of streptococci. A protoplast  is a bacterial cell from which the cell wall has been removed. It includes the cell protoplasm and the cytoplasmic membrane. Lysozyme digestion of Gram-positive bacteria that contain a peptidoglycan cell wall yields protoplasts that require hypertonic media for survival, and they do not usually multiply. The hypertonic solution protects them from lysis. Protoplasts can be produced from Gram-positive bacteria also by treatment with penicillin or other antibiotics which inhibit synthesis of the cell wall. Gram-negative bacteria have a cell wall comprised of a thin peptidoglycan layer enclosed by an exterior membrane of lipopolysaccharide. Protoplasts prepared from Gram-negative bacteria are frequently termed spheroplasts. PRP antigen  is polyribosyl-ribitol capsular polysaccharide. An antiphagocytic cell wall constituent of Hemophilus influenzae that provides this microorganism with an effective mechanism to induce disease. Type-specific antibodies that facilitate immunization are a requisite for protective immunity against H. influenzae. Children less than 2 years old are poor producers of anti-PRP antibodies, making them more susceptible to the infection. Pyogenic bacteria  are microorganisms such as Gram-positive staphylococci and streptococci that induce predominantly polymorphonuclear leukocytes inflammatory responses leading to the formation of pus at sites of infection. A pyogenic infection  is infection associated with the generation of pus. Microorganisms that are well known for their pus-inducing or pyogenic potential include Streptococcal pyogenes, Staphylococcus aureus, Streptococcus pneumoniae, and Hemophilus influenzae. Antibody-deficient patients and those having defective phagocytic cell capacity show increased susceptibility to pyogenic infections. Patients with complement deficiency such as C3 deficiency, factor I deficiency, etc., are also prone to developing pyogenic infections. Pyogenic microorganisms  stimulate a large polymorphonuclear leukocyte response to their presence in tissues. Pyrogen  is a substance that induces fever. It may be either endogenously produced, such as interleukin-1 released from macrophages and monocytes, or it may be an endotoxin associated with Gram-negative bacteria produced exogenously that induces fever. Phosphocholine antibodies  are synthesized during selected bacterial infections especially by Streptococcus (S), but also by Mycloplasma, Proteus, Trichinella, and Neisseria, in addition to helminithic parasites. CD5+ B cells form IgM antibodies that have a limited idiotype spectrum VH/VL gene

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usage and provide protective immunity from infection. These antibodies are a cross-react with phophatidycholine, pneumo­ ccoci, DsDNA, and sphingomellin. Phosphocholine antibody affinity diminishes with age. Pili  are structures that facilitate adhesion of bacteria to host cells and are therefore direct determinants of virulence. The Quellung phenomenon  refers to the swelling of the pneumococcus capsule following exposure to antibodies against pneumococci. See Quellung reaction. A Quellung reaction  is the swelling of bacterial capsules when the microorganisms are incubated with species-specific antiserum. Examples of bacteria in which this can be observed include Streptococcus pneumoniae, Hemophilus influenzae, Neisseria, and Klebsiella species. The combination of a drop of antiserum with a drop of material from a patient containing an encapsulated microorganism and the addition of a small loopful of 0.3% methylene blue produces the Quellung reaction. The microorganisms are stained blue and are encircled by a clear halo that resembles swelling but is in fact antigen–antibody complex produced at the surface of the organism. The reaction is due to an alteration of the refractory index. Scarlet fever  is a condition associated with production of erythrogenic toxin by group A hemolytic streptococci associated with pharyngitis. Patients develop a strawberry-red tongue and generalized erythematous blanching areas that do not occur on the palms, the soles of the feet, or in the mouth. Patients may also develop Pastia’s lines, which are petechiae in a linear pattern. Septic shock:  Hypotension, with a systolic blood pressure of less than 90 mmHg or a decrease in the systolic pressure baseline of more than 40 mmHg, in individuals with sepsis. It may be induced by the systemic release of TNF-α following bacterial infection of the blood, frequently with Gram-negative bacteria. Vascular collapse, disseminated intravascular coagulation, and metabolic disorders occur. Septic shock results from the effects of bacterial LPS and cyto­k ines, including TNF, IL-12, and IL-1. Also termed endotoxin shock. Seroconversion  is the first appearance of specific antibodies against a causative agent in the blood either during the course of an infection or following immunization. Serotype  refers to the use of specific antibodies to classify bacterial subtypes based on variations in the surface epitopes of the microorganism. Serotyping has long been used to classify Salmonella, streptococci, Shigella, and many other bacteria. May also describe human alloantigens such as HLA and blood group antigens. Shigella immunity:  The host protective immune response to Shigella infection is poorly understood. Since M cells of

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the gut take up the microorganisms, secretory IgA immune responsiveness has been postulated to be protective in Shigellosis, but it has been hard to establish. The immunity induced is type specific with reinfection occurring only within different Shigella species or serotypes. This is believed to be associated with an immune response to lipopolysaccharide determinants. Shigella may destroy antigen-presenting cells in the host following systemic exposure to Shigella antigens and toxins before an immune response can be established. Serum IgG has no protective effect. Oral vaccines with attenuated Shigella induce type-specific protection. Previous Shigella infection leads to specific IgA secretion in breast milk. Antibodies develop early against somatic Shigella antigens. Shiga toxin is a multimeric protein comprised of a single enzymatically active A subunit and 5B subunits needed for toxin binding. It is synthesized only by Shigella dysenteriae type I strains. It is an important virulence factor in the pathogenesis of hemolytic–uremic syndrome, which may be a complication of infection. Shiga toxin induces IgM antibody responses but the IgG response is lacking. However, IgG can be raised against Shiga toxin in animal models. Protection against Shigella has been associated with the humoral response to LPS or plasmid-encoded protein antigens. T cells also become activated in vivo during Shigella infection. There may be some correlation between T cell activation and the severity of the disease. Local cytokine synthesis is also significant in Shigellosis. Increased levels of IL-1, IL-6, TNF, and IFNγ have been found in stool specimens and plasma of infected patients. Both TH1 and TH2 cytokines are present in Shigellosis. There may be both a humoral and a cytotoxic defense mechanism during infection. High-serum antitoxin titers failed to protect monkeys against intestinal disease following challenge with live Shigella dysentariae. Postinfection reactive arthritis may also occur. Heat-killed whole cell Shigella vaccines used in the past failed to give protection. Although mucosal secretory IgA is to prevent bacterial attachment to the mucosa and to neutralize toxins, it has not been proved that either mechanism is significant in Shigellosis. A spheroplast  is a Gram-negative bacterial protoplast that contains outer membrane remains. Staphylococcal enterotoxins (SEs)  are bacterial cell constituents that cause food poisoning and activate numerous T lymphocytes by binding to MHC class II molecules and the Vβ domain of selected T cell receptors, which qualifies staphylococcal enterotoxins to be classified as superantigens. SE  is the abbreviation for staphylococcal enterotoxins. Staphylococcus immunity:  Most individuals synthesize antibodies reactive with staphylococcal antigens that include the cell wall-associated teichoic acid and the extracellular protein α-hemolysin. These antibodies are not protective against staphylococcal infections. They may be of some use in diagnosis, but it is necessary to show a significant increase

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in antibody titer. Yet high titers of antibodies against TSST, enterotoxins, and exfoliatins are protective. These toxins are immunomodulatory, mitogenic, and induce cytokine synthesis. No effective staphylococcal vaccine has been developed. Streptobacillus immunity:  Even though an immune res­ ponse is induced in subjects infected with S. moniliformis, both the exact mechanisms and relative contribution of antibody remain to be determined. In mouse experiments, antibody confers only partial immunity to challenge with the microorganism. Inactivated S. moniliformis vaccines induce only partial protection against challenge. Streptococcus immunity:  The most effective host resistance against pneumococcal (Streptococcus penumoniae) infection is by synthesizing IgG or IgM that interacts with capsular polysaccharide which opsonizes the bacteria for ingestion and killing by professional phagocytes. Anticapsular antibody develops following colonization or infection. Most individuals have low resistance as reflected by their lack of antibody to most of the commonly infecting pneumococcal serotypes even though normal adults may have sufficient antibody against phosphocholine-containing epitopes of pneumococcal cell wall. This antibody does not appear to be protective in human subjects since reaction between this antibody and the bacterial cell wall occurs beneath the capsule. The complement fragments and bound Fc are inaccessible to phagocytic cells. Vaccines to prevent streptococcal infections are hindered by the systemic local and systemic reactions that follow administration of large does of M protein given in an effort to induce type-specific antibody responses. This may be attributable to M proteins serving as superantigens. Heart reactive epitopes have been removed from M proteins, which has made possible immunization with purified M protein preparations to induce type-specific opsonic antibodies that do not cross-react with heart antibodies. Immunization protocols have also included attempts to stimulate antibodies against lipoteichoic acid, the adherence constituent of streptococci. But these efforts have been limited by the poor immunogenicity of this component. Vaccination with capsular polysaccharides is effective in preventing pneumococcal infection. Streptococcal M protein  is a cell-wall protein of virulent Streptococcus pyogenes microorganisms which interferes with phagocytosis and also serves as a nephritogenic factor. Superinfection “immunity”  refers to the inability of two related organisms, for example, plasmids, to invade a host cell at the same time. Toxic shock syndrome  is an acute systemic toxic reaction, that is potentially lethal, in which the patient manifests shock, skin exfoliation, conjunctivitis, and diarrhea that results from the excessive production of cytokines by CD4 + T  cells activated by the bacterial superantigen toxic shock syndrome toxin-1 (TSST-1), secreted by Staphylococcus

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aureus. It has been linked to the improper use of feminine hygiene products. Toxin-1 (TSST-1)  is a toxic bacterial product secreted by Staphylococcus aureus that is implicated in toxic shock syndrome. Transferrin  is a protein that combines with and competes for iron with bacteria. Tetanus  is a disease in which the exotoxin of Clostridium tetani produces tonic muscle spasm and hyperreflexia, leading to tris (lock jaw), generalized muscle spasms, spasm of the glottis, arching of the back, seizures, respiratory spasms, and paralysis. Tetanus toxin is a neurotoxin. The disease occurs 1 to 2 weeks after tetanus spores are introduced into deep wounds that provide anaerobic growth conditions. Tetanus toxin  is the exotoxin synthesized by Clostridium tetani. It acts on the nervous system, interrupting neuromuscular transmission and preventing synaptic inhibition in the spinal cord. It binds to a nerve cell membrane glycolipid, i.e., disialosyl ganglioside. The effects of tetanus toxin are countered by specific antitoxin. Treponema immunity:  Infection with Treponema pallidum induces both cellular and humoral immune responses. The humoral response is characterized by the synthesis of both phospholipid and treponemal antibodies that are detected in the serological diagnosis of syphilis. Flocculation tests have long been used to detect phospholipid or cardiolipin antibodies. They make use of a cardiolipin–lecithin–cholesterol antigen in the VDRL test. Cardiolipin F antibody specific for host cell mitochondrial cardiolipin (autoantibody) is also associated with syphilis but is not used in diagnosis. Treponemal antibodies that appear after infection together with cardiolipin antibodies are detectable by immunofluorescence. Treponemal antibodies are still detectable in the host for many years. Autoantibodies against tissue phospholipids and other tissue components also occur in T. pallidum-infected hosts. The cell-mediated immune response consists of macrophage activation and induction of CD8+ T cells and CD4+ T cells. The cell-mediated immunity appears to be more important than the humoral response in the development of immunity. The VDRL (Venereal Disease Research Laboratory) test  is a reaginic screening assay for syphilis. VDRL antigen is combined with heat-inactivated serum from the patient, and the combination is observed for flocculation by light microscopy after 4 min. Reaginic assays are helpful as screening tests for early syphilis and are usually positive in secondary syphilis, although the results are more variable in tertiary syphilis. VDRL is negative in approximately half of the neurosyphilis cases. Reaginic tests for syphilis may be biologically false-positive in such conditions as malaria, lupus erythematosus, and acute infections. Biologic falsepositive VDRL tests may also be seen in some cases of

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hepatitis, infectious mononucleosis, rheumatoid arthritis, or even pregnancy. A biological false-positive reaction  results when a positive serological test for syphilis, such as in the VDRL (Venereal Disease Research Laboratory), is produced by the serum of an individual who is not infected with Treponema pallidium. It is attributable to antibodies reactive with antigens of tissues such as the heart from which cardiolipin antigen used in the test is derived. The blood sera of patients with selected autoimmune diseases, including SLE, may contain antibodies that give a biological false-positive test for syphilis. BFPR  is an abbreviation for biological false-positive reaction. Vibrio cholerae immunity:  Vibrio cholerae induces disease by production of virulence factors and toxins. Protective antigens have been identified and progress has been made in vaccine development. Mucosal immunity is mediated by secretory IgA antibodies as necessary for protection against cholera. The V. cholerae is noninvasive. Thus, antibodies block adherence and inhibit colonization. They either neutralize the toxin or inhibit their binding to specific receptors. Intestinal immune response begins in the lymphoid follicle where the mononuclear cells are responsible for phagocytic uptake of antigen from the intestine. Live microorganisms are more effective immunogens than are killed ones. An initial infection with cholera yields long-lasting immunity. The most important protective antigen is the O antigen of the LPS. Antibodies to TCP are protective but are biotype-restricted as a consequence of biotype-specific antigenic variation in the major structural subunit. The other protective antigen is the B subunit of CT. The major epidemic strain now appears to be 0139 which increases the significance of anti-LPS immunity since this new strain has the virulence of the original 01 El Tor strains. Immunity against non-LPS antigens is far less important than to the specific LPS type. Non-LPS antigens are less immunogenic than is the LPS antigen. Virulence genes  govern the expression of multiple other genes with changing environmental conditions such as temperature, pH, osmolarity, etc. An example is the toxR gene of Vibrio cholerae that coordinates 14 other genes of this microorganism. Yersinia immunity:  Both humoral and cell-mediated immune responses develop in Yersiniosis. The antibody is directed mainly to lipopolysaccharide. Antibodies in patients with Yersinia infection are specific for numerous epitopes. The antibodies persist for months and in some cases even years. Reactive arthritis, a frequent postinfection complication of Yersiniosis, is strongly associated with HLA-B27 antigen. The immune response against the infectious agents is believed to have a significant role in the pathogenesis of Yersinia-triggered reactive arthritis. No effective vaccine is available for Yersinia infection.

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Diphtheria toxin  is a 62-kDa protein exotoxin synthesized and secreted by Corynebacterium diphtheriae. The exotoxin, which is distributed in the blood, induces neuropathy and myocarditis in humans. Tryptic enzymes nick the single-chain diphtheria toxin. Thiols reduce the toxin to produce two fragments. The 40-kDa B fragment gains access to cells through their membranes, permitting the 21-kDa A fragment to enter. Whereas the B fragment is not toxic, the A fragment is toxic and it inactivates elongation factor-2, thereby blocking eukaryocytic protein synthesis. Guinea pigs are especially sensitive to diphtheria toxin, which causes necrosis at injection sites, hemorrhage of the adrenals, and other pathologic consequences. Animal tests developed earlier in the century consisted of intradermal inoculation of C. diphtheriae suspensions into the skin of guinea pigs that were unprotected, compared to a control guinea pig that had been pretreated with passive administration of diphtheria antitoxin for protection. In later years, toxin generation was demonstrated in vitro by placing filter paper impregnated with antitoxin at right angles to streaks of C. diphtheriae microorganisms growing on media in Petri plates. Formalin treatment or storage converts the labile diphtheria toxin into toxoid. It is a secretory product of the bacterium Corynebacterium diphtheriae, the etiologic agent of diphtheria, which produces symptoms of the disease. Immunization requires the use of an inactive form of the toxin termed diphtheria toxoid. Refer to diphtheria toxin and diphtheria toxoid. In a Moloney test  diphtheria toxoid is injected intradermally and the skin response is observed to determine whether or not the subject is hypersensitive to diphtheria prophylactic substances. Bacteroides immunity:  Bacteroides fragilis has a capsular polysaccharide that serves as a virulence factor which led to use of this polysaccharide as a vaccine in rats. The vaccine led to excellent antibody levels but did not prevent abscess formation. In the rat model, the findings strongly point to the role of T cells in the immune response to B. fragilis in abscess prevention. Murine studies confirmed the rat findings and demonstrated that immune T cells are antigen specific. Abscess formation and prevention in response to B. fragilis is mediated by T cell mechanisms. Abscess formation requires a precursor-type T cell. Prevention of abscess requires T cells belonging to the suppressor phenotype which communicate via a small polypeptide factor. These cells are antigen-specific but not MHC-restricted. Bacterial immunoglobulin-binding proteins  are molecules expressed by selected microorganisms that interact with immunoglobulin at sites other than their antigen-combining regions. Thus, the antibody’s ability to bind its homologous antigen is not impaired. Gram-positive bacteria express six types of IgG-binding protein, including protein A (type I) which is expressed on Staphylococcus aureus. Most human group A steptococci express type II receptors and exhibit

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great variability in immunoglobulin-binding capacity. Protein G, the type III IgG binding protein, is expressed by most human group C and G streptococcal isolates. Type IV, V, and VI immunoglobulin-binding proteins require further investigation. Bactericidin  is an agent such as an antibody or nonantibody substance in blood plasma which destroys bacteria. Bacillus anthracis immunity:  Protective immunity against anthrax is induced only by the antigen designated PA. The B. anthracis polypeptide capsule is only weakly immunogenic and is not believed to contribute to naturally acquired immunity or to induce protection against anthrax. The Ca _ /tx+ strains of B. anthracis are protective. One of these strains is used to prepare a very successful live-spore animal vaccine. It is considered unsuitable in the West to use in humans because it retains some residual virulence. Human vaccine developed half a century ago consists of aluminum hydroxide-adsorbed cell-free filtrates of cultures of a noncapsulating, nonproteolytic derivative of strain V770. Several doses are required. An immune response to PA but not to LF or EF is critical for protection. Immunization with strains synthesizing either PA or EF or PA and LF resulted in higher antibody responses to EF or LF, respectively. There is a synergistic relationship between PA and LF or EF in immunoprotection. Some cellular immune mechanisms are believed to be significant in the induction of protective immunity. PA has been shown with monoclonal antibodies to have at least three nonoverlapping antigenic regions. It is anticipated that the elucidation of protective motifs on PA may lead to the development of a subunit-based vaccine. Passive immunity in the treatment of anthrax has been unsuccessful. Klebsiella immunity:  Most healthy adults have high levels of natural resistance against Klebsiella pneumoniae. The organism classically induces lung infection leading to a massive confluent lobar consolidation with polymorphonuclear leukocytes, edema, and abscess formation with extensive cavity formation. Type-specific antibodies to the carbohydrate capsule are critical to recovery. These antibodies that appear within the first two weeks following infection serve as opsonins to facilitate the killing of this microorganism by phagocytes. The immunity to K. pneumoniae is long lasting. This microorganism is believed to have a part in the development of ankylosing spondylitis (AS) since these individuals develop high levels of serum IgA against the microbe. AS is associated with HLA-B27 antigen. The mechanism has been claimed to be either molecular mimicry in which antibody against this antigen will bind to self-antigenic determinants leading to destructive immunity or that Klebsiella plasmids might encode the formation of a bacterial modifying factor that reacts with HLA-B27, rendering it susceptible to immune attack. The microbe’s main virulence factor is the capsule that helps it to resist phagocytosis. Although there have been reports of several enterotoxins, the main pathologic effect is associated with the production of an endotoxin.

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The extracellular toxic complex ETC contains endotoxin, capsule, and protein that is lethal when injected into mice, leading to pathologic changes that resemble those produced in K. pneumoniae lobar pneumonia in humans. Vaccines are aimed at developing antibodies against capsule polysaccharides which are able to prevent experimental K. pneumoniae sepsis. An effective vaccine must contain capsular polysaccharide from 25 different serotypes. Bordetella pertussis  is the etiologic agent of whooping cough in children. Killed B. pertussis microorganisms are administered in a vaccine together with diphtheria toxoid and tetanus toxoid as DPT. The endotoxin of B. pertussis has an adjuvant effect that can facilitate antibody synthesis. Bordetella immunity:  Bordetella pertussis produces the respiratory disease whooping cough or pertussis. Both humoral and cell-mediated immune responses follow infection. Neutralizing antibodies are believed to be the principal protective mechanism against infection with B. pertussis. Antibodies are formed against B. pertussis antigens that include PT, FHA, PRN, and fimbriae, which are associated with protection against pertussis. Pertussis infection can lead to long-lasting immunity against subsequent pertussis. Patients who have recovered from the infection develop anti-B. pertussis immunoglobulin A in serum and saliva, pointing to the role of mucosal antibodies. Cell-mediated immunity is also believed to be significant since TH1 cells specific for the microorganism occur in persons following either infection or vaccination. High-titer IgG antibodies may clear bacteria in respiratory infections revealed by animal studies, but cell-mediated immunity is necessary to completely eliminate the microorganism from mouse lungs. In humans, immunity that follows infection may prevent respiratory colonization, whereas immunity that follows vaccination may protect against toxin-mediated disease. B. pertussis antigens suppress the host response to pertussis both in vitro and in vivo. The whole-cell vaccine comprised of killed whole virulent B. pertussis has been replaced with acellular preparation combined with diphtheria and tetanus toxoid in the currently used DTaP vaccine. It provides fewer and milder side effects than the whole-cell vaccine preparation and is more effective in inducing serum antibody responses and protection from pertussis. Borrelia immunity:  Relapsing fever and Lyme disease are produced by members of this genus. Borrelia burgdorferi sensu lato spirochetes are the causative agents of Lyme disease. These microorganisms are covered by a slime layer comprised of self-molecules that block immune recognition. The slime layer acting as a capsule prevents phagocytosis, but when incubated with specific antibody in complement, the microbe is killed. The principal surface proteins in the exterior cell membrane include A, B, C, D, E, and F that are designated as Outer surface proteins (Osp). The proteins are heterogeneous but their function remains to be determined. Spirochetes that cause Lyme disease upregulate or

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downregulate Osp A and Osp C during the course of human infection. Other antigens in the outer membrane of B. burgdorferi sensu lato include 16-, 27-, 55-, 60-, 66-, and 83-kDa proteins. Osp A is the principal candidate for a Lyme disease vaccine. Soon after infection with B. burgdorferi sensu lato, the microorganisms become refractory to the action of bactericidal antibodies. Brucella immunity:  The immune response to Brucella infection is marked by early IgM synthesis followed by a switch to IgG and IgA. IgE is also detected. IgM persists for an unusually long time, possibly responding to the T cell independent antigen LPS. Antibodies are important for the serodiagnosis of Brucella infection. Antibody’s only protective role is probably as a preexisting mucosal antibody that decreases initial infection. Cell-mediated immune responses confer protective immunity. Brucellae are facultative intra­ cellular bacteria that are controlled by macrophage activation and granuloma formation to isolate the infectious agents. Both CD4+ and CD8+ T lymphocytes participate in the experimental infection in mice. The CD4+ T cells synthesize interferon γ, and the CD8+ T cells lyse ineffective macrophages. IL-12 is important in controlling the differentiation of T cells and natural killer cells to produce IFN-γ, which facilitates cellmediated immunity. IL-1, TNF-α, IL-6, M-CSF, and G-CSF are all produced during experimental infection. Minute granulomata comprised of epithelioid cells, neutrophils, mononuclear leukocytes, and giant cells are produced in the tissues of humans infected with Brucella species. Hepatosplenomegaly is a common clinical feature. Delayed-type hypersensitivity, which is a correlate of cell-mediated immunity, induces immunopathological changes. In addition to the granulomata, there is development of a severe generalized delayedtype hypersensitivity response that mimics many features of the infection itself. Diagnosis depends on testing for antibodies in the serum. Most of the antibodies are directed against LPS. The agglutination test is the one most widely used but is being replaced by ELISA. Whereas B. abortus strain 19 is employed to immunize cattle and B. melitensis stain Rev 1 is used to immunize sheep and goats, human vaccination has been used essentially only in Russia. Other preventive measures include pasteurization of milk products. Campylobacter immunity:  Circulating antibodies develop rapidly in patients with Campylobacter enteritis. These antibodies fix complement, are bactericidal, and agglutinate. Following an initial but short-lived IgM response, there is a rapid IgA response that peaks 14 d after onset of symptoms but declines by the fifth week. IgG antibodies appear by the tenth day after infection and are present for several months. Antibodies are believed to limit the infection. Serologic tests for diagnosis depend on an acid-extractable surface antigen that consists mainly of flagellin which is the immunodominant surface antigen. Antiflagellin antibodies appear early during an infection and are believed to be protective. MOMP is also immunogenic. All of these antibodies have specificity for homologous and heterologous strains. LPSs induce

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variable antibody responses. Half of the cases of C. enteritis develop during Guillian–Barré syndrome. These patients develop increased circulating IgG and IgM that bind to GM1 and GD1 ganglioside epitope and which cross-react with LPS of certain serotypes of C. Jejuni. Definitive studies of the T cell response to Campylobacter remain to be performed. Clostridium immunity:  Clostridia produce disease by releasing exotoxins. They may produce more than one toxin and each one is immunologically unique. For example, each of the five types of Clostridium perfringens produces a different toxin. Clostridia enter the host by many routes to produce disease. Clostridium perfringens gains access through traumatic or surgical wounds to produce gas gangrene and wound infections. The microorganism is aided by a poor blood supply in the area of the wound. The clostridia divide and produce toxins that cause disease. When antibiotics upset the normal bowel florae, C. difficile may multiply and induce colitis. The toxins released from this organism act on intestinal epithelial cells and produce diarrhea and chronic inflammation. C. botulinum does not grow in the host but forms toxins in contaminated food that when ingested lead to the disease. Most clostridial diseases do not induce protective immunity because the amount of toxin required to produce disease is less than that need to induce an immunologic response. Even though systemic immunity does not follow an episode of the disease, tetanus toxoid can induce immunity that may last for 5 years. Tetanus immunoglobulin is also valuable for passive immunization in suspected cases. Antibotulinum toxin antibody is available for laboratory workers. Intravenous administration of gammaglobulin containing high titers of antibody to C. difficile toxin has been useful in the therapy of patients with relapsing C. difficile diarrhea. Escherichia coli immunity:  IgM and IgG antibodies are formed against O, H, and K antigens of E. coli of the diarrhea-producing strains in infants. Secretory IgA specific for E. coli diminishes the adherence of diarrhea-producing E. coli in the intestines of infants. Secretory IgA in breast milk also offers significant protection of infants through passive immunity. Secretory IgA may be specific for LT enterotoxins and for colonization factor antigens. Francisella immunity:  The causative agent of tularemia, Francisella tularensis, may induce two forms of the disease, ulceroglandular tularemia which is borne by vectors or induced by contact with infected animals, and respiratory tularemia which is caused by inhalation of contaminated dust. A powerful antibody response occurs during infection with this microorganism and it is detectable by agglutination, ELISA, or other techniques. The antibodies appear at the end of the second or during the third week of infection. They persist for several years following recovery. IgM antibodies do not appear before IgG antibodies and may even be present for years following recovery. Cell-mediated immunity in tularemia is demonstrated by a delayed-type hypersensitivity

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test or by in vitro activation of T cells. Cell-mediated immunity against F. tularensis is a requisite for host protection. The microorganism’s capsule protects it against lysis by complement and affords resistance to intracellular killing by polymorphonuclear leukocytes. Attenuated strains of the microorganism, termed F. tularensis LVS are easily killed by polymorphonuclear leukocytes and are susceptible to hydrochloric acid and hydrogen peroxide produced as a result of the oxidative burst. No significant toxins are produced by F. tularensis. T-cell mediated immunity can prevent fulminating disease. Infection or vaccination with live attenuated bacteria can induce host protection against tularemia. Fusobacterium immunity:  Fusobacterium nucleatum adheres to lymphocytes through lectin-like ligands to facilitate other activity or exert an immunosuppressive effect on them. The latter enhances the pathogenicity of the microorganism. Patients with periodontal disease, peritonsillar cellulitis and abscesses, infectious mononucleosis, and acute streptococcal, nonstreptococcal, and recurrent tonsillitis manifest increased levels of antibodies against protein antigens of F. nucleatum. Antibodies against these outer membrane proteins may point to a pathogenic role for this microorganism in these infections. Delayed-type hypersensitivity to F. necrophorum has been induced in mice, and leukotoxin-specific antibodies have been found in cattle. Natural infection or vaccination with a toxoid does not always protect against reinfection with this microorganism. It is believed to be a weak immunogenic pathogen. Haemophilus immunity:  Serum bactericidal activity to Haemophilus influenzae serotype b(Hib) is associated with protection from infection. This effect is mediated by antibody to the capsule. Antibody to the PRP polysaccharide capsule protects against invasive infection. Two-year-olds have only low titers of anticapsular antibodies and are susceptible to reinfection and episodes of Hib meningitis. NTHI strains of the organism demonstrate antigenic heterogeneity of surface antigens among strains. Infection does not induce protective immunity from infection by NTHI strains following otitis media attributable to an NTHI strain. Serum bactericidal antibody develops and this is associated with protection. The immune response is strain specific, which renders the child susceptible to infection with other strains of NTHI. The immune response is strain specific, with reactivity directed to the immunodominant surface epitopes on the P2 OMP. H. influenzae LOS is a principal toxin against which humoral immune responses are directed. The mucosal immune response to NTHI remains to be defined. Conjugate vaccines developed to prevent invasive infections by Hib have been very successful in preventing the infectious disease. High titers of serum IgG are specific for Hib capsular polysaccharide are associated with increased bactericidal and protective activity. Linking the PRP capsular polysaccharide to protein carriers has yielded a conjugate vaccine which is effective in protecting infants against invasive Hib infections.

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Legionella immunity:  Immunity against Legionella pneumophila, a facultative intracellular pathogen that induces Legionnaires’ disease in humans, depends on cellular immune mechanisms, including the release of IFNγ. TH1 CD+ T cells play a significant role in the development of acquired immunity in mice. Acquired immunity to Legionella pneumophila is believed to be a consequence of both humoral and cellular immune responses that facilitate enhanced uptake of the microorganisms by activated mononuclear phagocytic cells. Frei test  is a tuberculin type of delayed hypersensitivity skin test employed to reveal delayed-type hypersensitivity in lymphogranuloma venereum patients. Following intradermal injection of lymphogranuloma venereum virus, an erythematous and indurated papule develops after 4 d. Lepra cells  are foamy macrophages that contain clusters of Mycobacterium leprae microorganisms that are not degraded because cell-mediated immunity has been lost. These are found in lepromatous leprosy, but are not observed in tuberculoid leprosy. Lepromatous leprosy  is a chronic granulomatous disease induced by Mycobacterium leprae. The condition is contagious and is also known as Hansen’s disease. A second form of leprosy is termed tuberculoid, which is a more benign and stable form of leprosy. Both lepromatous and tuberculoid leprosy infect the peripheral nervous system. Leptospira immunity:  Newly isolated leptospires evade the host immune system by not reacting with specific antibody which permits their multiplication. On entering the host, they also evade the host immune system by their sequestration in renal tubules, uterine lumen, eye, or brain. The humoral immune is marked by production of IgM which, together with complement and phagocytic cells, begins to clear leptospires from the host. This is followed by the production of opsonizing and neutralizing IgG. There is only low-grade cell-mediated immunity to Leptospira infection. Vaccines for use in animals consist of chemically inactivated whole Leptospira cultures which have proven to be somewhat effective. The immunity induced is serovar  specific. These antibodies do not afford protection. Virulent leptospires are better immunogens than are avirulent organisms. It is significant that antigens located on the outer envelope maintain their natural configuration. Both IgM and IgG are induced following parenteral administration of a vaccine, as the IgM is a more efficient agglutinating and neutralizing antibody for leptospires than is IgG. Moro test  is a variant of the tuberculin test in which tuberculin is incorporated into an ointment that is applied to the skin to permit the tuberculin to enter the body by inunction. Mycobacterium  is a genus of aerobic bacteria, including Mycobacterium tuberculosis, which can survive within phagocytic cells and produce disease. Cell-mediated

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immunity is the principal host defense mechanism against mycobacteria. MOTT (mycobacteria other than Mycobacterium tuberculosis)  is an acronym for mycobacteria other than those that induce tuberculosis. Their recognition is increasing. TB  is the abbreviation for tuberculosis. Mycobacteria immunity:  Immunity to tuberculosis is highly complex and involves cell-mediated mechanisms. The host immune response to tuberculosis is inappropriate, leading to tissue injury through immune mechanisms rather than elimination of the invading microorganism. In the mouse, immunity depends upon TNF-α, a TH1 cytokine pattern and MHC class II. Mouse murine macrophages activated by IFN-γ inhibit proliferation of M. tuberculosis. β2 Microglobulin is required for immunity to mycobacteria, which may point to the participation of CD8+ MHC class I restrictor cytotoxic T cells. γδ T cell receptors identify mycobacterial antigens such as heat shock proteins. These cell types secrete IFN-γ, and are cytotoxic and classified as a type I response. A type II response renders mice more susceptible to tuberculosis. Immunity in humans is also associated with a TH1 type of response associated with macrophage activation and cytotoxic removal of infected cells. Human tuberculosis patients form specific IgE and IgG4 antibodies, both of which are IL-4 dependent. IL-10 levels may also be increased. A TH2 response is associated with progressive disease in humans. M. tuberculosis

can cause release of TNF-α from primed macrophages. This cytokine is required for protection but also has a role in immunopathology. Tuberculosis patients develop necrotic lesions which are believed to help wall off established infections. TNF-α’s toxicity in a mycobacterial lesion depends on whether or not the T-cell response is TH1 or TH2. Necrosis is not produced when TNF-α is injected into a TH1 inflammatory site but marked tissue injury results when it is injected into a TH1-/TH2-mediated site. Infection by Mycobacterial leprae is weakly associated with MHC haplotypes. It appears to determine the type of disease that will develop instead of susceptibility. In tuberculoid leprosy, both in vivo and in vitro equivalents of T  cell mediated responsiveness such as skin test reactivity in lymphoproliferation in response to antigens of M. leprae are intact. TH1 cytokines are produced in these lesions. Unless treated, these will develop into lepromatous leprosy in which the TH1 response is impaired. In leprosy, immunity probably requires macrophage activation and cytotoxic T cells activated by the TH1 response. In lepromatous leprosy, there is depressed T cell mediated immunity in vivo and in vitro. Numerous macrophages packed with bacilli are present in the lesions, but there is no response. There is a continuous heavy antigen load and lack of a T cell mediated response to the microorganism. The T  cells express a TH2 cytokine pattern. Caseous necrosis  (Figure  24.21) is tissue destruction, as occurs in tuberculosis, that has the appearance of cottage cheese. It is a type of necrosis present at the center of large

IL-12

APC

Antigen

T helper (CD4 + TH1 cell)

T

Interleukin 2 TNF Granuloma forms

INFγ

Lymphocyte Macrophage Monocyte Epithelioid cell Fibroblast Giant cell

Figure 24.20  Granuloma.

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Class II MHC

M. tuberculosis

T cell receptor

Antigen presenting cell

Cytokines CD4 Bacillary antigenic peptide

CD4+ T cell Delayed hypersensitivity

Immunity

Granuloma formation

Sensitized T cell Lymphocyte

Epithelioid cell

Activated macrophage

Caseous necrosis Fibroblast Giant cell

Figure 24.21  Caseous necrosis (tuberculosis).

granulomas such as those formed in tuberculosis. The white cheesy appearance of the central necrotic area is the basis for the term. Caseation necrosis  is a type of necrosis present at the center of large granulomas such as those formed in tuberculosis. The white cheesy appearance of the central necrotic area is the basis for the term. Invasin  is a membrane protein derived from Yersina pseudotuberculosis that binds to α4β1 integrins and has the capacity to induce T cell costimulatory signals. MAC:  Mycobacterium avium complex, is a systemic infection that regularly affects subjects with AIDS, up to 66% of whom still have peripheral blood CD4+ T lymphocytes. Infection with this complex is a clear indication of immunosuppression. MAC is successfully treated with clarithromycin. MAIS complex:  Mycobacterium avium-intracellularescrofulaceum. Three species of mycobacteria that express the same antigens and lipids on their surfaces and also have the same biochemical reactions, antibiotic susceptibility, and pigment formation. They frequently occur together clinically. MAIS complex is relatively rare, but it occurs in 5 to

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8% of AIDS patients when their CD4+ T lymphocyte levels diminish to less than 100 cells per cubic millimeter of blood. Affected patients have persistent diarrhea, night sweats and fever, abdominal pain, anemia, and extrahepatic obstruction. Ciproflozacin, clofazimine, ethambutol, and rifampicin, as well as rifabutin, clarithromycin, and azithromycin have been used in treatment. Mantoux test  is a type of tuberculin reaction in which an intradermal injection of tuberculin tests for cell-mediated immunity. A positive test signifies delayed (type IV) hypersensitivity to Mycobacterium tuberculosis, which indicates previous or current infection with this microorganism. The Mitsuda reaction  is a graduated response to an intracutaneous inoculation of lepromin, a substance used in the lepromin test. A nodule representing a subcutaneous granulomatous reaction to lepromin occurs 2 to 4 weeks after inoculation and is maximal at 4  weeks. It indicates granulomatous sensitization in a leprosy patient. Although not a diagnostic test, it can distinguish tuberculoid from lepromatous leprosy in that this test is positive in tuberculoid leprosy, as well as in normal adult controls, but is negative in lepromatous leprosy patients. Ghon complex  is the combination of a pleural surfacehealed granuloma or scar on the middle lobe of the lung,

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together with hilar lymph node granulomas. The Ghon complex signifies healed primary tuberculosis. Neisseria immunity:  Even though patients with gonococcal infection develop increased levels of serum and mucosal antibody of IgG and IgA classes reactive with gonococcal surface antigens, they develop repeated episodes of urogenital infections. Thus, natural infection does not confer protective immunity. The surface antigens may be antigenically heterogeneous or this may be attributable to the brevity of the mucosal antibody response and the lack of activity of serum antibody in the mucosal infection. Attachment and colonization of the mucosa with gonococci in their encounter with polymorphonuclear neutrophils play an important role. Protective immunity against meningococcal infection is associated with complement-dependent, bactericidal serum antibodies specific for capsular polysaccharide. Cellmediated immunity does not appear to have a significant role. There is no successful gonococcal vaccine. Polysaccharide vaccines to protect against N. meningitidis of serogroup A and C induce protective bactericidal antibodies. The current vaccine contains polysaccharide from serogroups A, C, Y, and W-135. Polysaccharide A and C vaccine has been conjugated to tetanus toxoid or another protein carrier to enhance immunogenicity in young children. Polysaccharide vaccines may not induce protection in serogroup B meningococcal disease. Protection against these strains may be linked to antibody specific for outer membrane proteins which can be used to induce immunity. Pasteurella immunity:  Immunity against pasteurella multocida is mainly humoral antibody-mediated, yet cell-mediated immune responses also occur. Naturally acquired immunity to this organism can develop in unvaccinated cattle and water buffalo. Even though the organism is surrounded by a capsule that contributes to its virulence, antibody acting as opsonin can render these microorganisms readily available for phagocytosis by monocytes, macrophages, and polymorphonuclear neutrophils. Protein toxins from certain stains can induce the formation of neutralizing antitoxins, but purified proteins have not been shown to induce protective immunity. Cattle, buffalo, and poultry vaccinated with bacterins, formalinkilled organisms in a water-in-oil emulsion, form antibodies against lipopolysaccharide (LPS). Anti-LPS antibodies are also associated with naturally acquired immunity. Not only bacterins but also live-attenuated vaccines have been employed to control P. multicida infections. Proteus immunity:  Proteus mirabilis administered to mice by the transurethral route can induce antibodies that react with purified lipopolysaccharide, flagella, outer membrane protein (OMP), and mannose-resistant proteus-like MR/P fimbriae. Murine vaccination leads to partial immunity. Rheumatoid arthritis patients’ blood sera also react with P. mirabilis. A hexamer peptide within hemolysin is the target of the immunogenic response, probably due to its close similarity to susceptibility sequences of HLA/DR1 and

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DR4. Several proteins encoded by chromosomal genes serve as P. mirabilis virulence factors. These help the host evade immune defenses and lead to cell and tissue injury. These include urease and HPM homolysin. The latter leads to lysis of erythrocytes or epithelial cell membrane injury in culture. The organism also produces four types of fimbriae, surface proteins that have a role in adherence. It also secretes a protease that degrades IgA. Pseudomonas aeruginosa immunity:  Pseudomonas aeruginosa infection is followed by the development of antibodies that facilitate opsonophagocytosis in protection against subsequent infections. Thus, an adequate antibody response is required for protection. Antibody alone may be insufficient for protection since cystic fibrosis patients’ lungs continue to be chronically colonized even in the presence of potent serum antibody responses to several antigens of this microorganism. Active vaccination is made less desirable by the fact that P. aeruginosa infections cannot be reliably predicted. Thus, passively administered hyperimmune intravenous immunoglobulin from immunized volunteers has been used to confer protection. Nevertheless, contemporary investigations show that antibodies against LPS serotypes and to exotoxin A did not significantly protect recipients. Salmonella immunity:  Natural and adaptive immunity are necessary for survival following primary infection with virulent microorganisms of this genus. Salmonellae grow exponentially in the reticuloendothelial system and may reach 108 microorganisms, which is a lethal number, apparently due to endotoxin as a contributing lethal factor. The balance between strain virulence and host resistance controls the rate at which bacterial cells increase in the reticuloendothelial system. Bone marrow-derived cells TNF-α, IFN-γ, and IL-12 but not T  lymphocytes control early growth. This is termed the plateau phase after which an immune response clears the microorganisms from the tissue and provides effective immunity to reinfection. CD4+ and CD8+ T lymphocytes, together with TNF-α, IFN-γ, and IL-12 help to clear bacteria from the tissues, but antibody is also required in addition to cell-mediated immunity. The antibody may be directed against lipopolysaccharides (LPS) in animal infections with these microorganisms. In humans, antibodies to Vi antigen are believed to be significant. The diagnosis of typhoid fever is based on the detection of antibodies to O and H antigen and to Vi antigens in carriers. Immunization with killed samonellae does not induce cell-mediated immunity and confers less protection than immunization with live organisms that induce both cell-mediated and humoral immunity. Antibody alone is also not protective. The protective immunogen is probably LPS O antigen or Vi antigen in S. typhi, other protein antigens, or some combination of these. IgA provides partial immunity. Since killed microorganisms used in vaccines in the past do not induce appropriate cell-mediated immunity and are highly reactogenic, live vaccines of superior efficacy in experimental models are being developed.

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Vi antigen  is a virulence antigen linked to Salmonella microorganisms. It is found in the capsule and interferes with serological typing of the O antigen, a heat-stable lipopolysaccharide of enterobacteriaceae. Cholera toxin  is a Vibro cholerae enterotoxin comprised of five B subunits which are cell-binding 11.6-kDa structures that encircle a 27-kDa catalase which conveys ADP-ribose to G protein, leading to continual adenyl cyclase activation. Other toxins that resemble cholera toxin in function include diphtheria toxin, exotoxin A, and pertussis toxin. Exotoxin  is an extracellular product of pathogenic microorganisms. Exotoxins are 3- to 500-kDa polypeptides produced by such microorganisms as Corynebacterium diphtheriae, Clostridium tetani, and C. botulinum. Vibrio cholerae produces exotoxins that elevate cAMP levels in intestinal mucosa cells and increase the flow of water and ions into the intestinal lumen, producing diarrhea. Exotoxins are polypeptides released from bacterial cells and are diffusible, thermolabile, and able to be converted to toxoids that are immunogenic but not toxic. Bacterial exotoxins are either cytolytic, acting on cell membranes, or bipartite (A-B toxins), linking to a cell surface through the B segment of the toxin and releasing the A segment only after the molecule reaches the cytoplasm where it produces injury. Some may serve as superantigens. Coccidiodin  is a Coccidioides immitis culture extract that is used in a skin test for cell-mediated immunity against the microorganism in a manner analogous to the tuberculin skin test. Listeria:  A genus of small Gram-positive motile bacilli that have a palisading pattern of growth. The best known is L. monocytogenes, which has a special affinity for monocytes and macrophages in which it takes up residence. It may be transmitted in contaminated milk and cheese. Approximately one-third of the cases are in pregnant females, resulting in transplacental infection that may induce abortion or stillbirth. Infected infants may develop septicemia, vomiting, diarrhea, cardiorespiratory distress, and meningoencephalitis. Individuals with defective immune reactivity may develop endocarditis, meningoencephalitis, peritonitis, or other infectious processes. Treatment is with ampicillin, erythromycin, gentamycin, or chloramphenicol. Listeria immunity:  Immune responsiveness to Listeria has been investigated mostly in a mouse model. The microorganism results are only in macrophages but are also in hepatocytes of infected host. Natural immunity in mice is controlled by Lr1 locus on chromosome II. Mice resistant to Listeria respond to inoculated listeriae with large numbers of inflammatory phagocytes. Neutrophils and mononuclear phagocytes kill L. monocytogenes in vitro although resident macrophages are less effective. Oxygen-dependent and independent bactericidal mechanisms facilitate destruction of listeriae. Multiple cell types and mediators are involved

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in resistance against Listeria. T  cell-mediated immunity is significant but not the only factor in resistance to listeriosis. Neutrophils are also significant to resistance against Listeria. Other cells implicated in resistance to Listeria infections include granulocytes, NK cells, and cytotoxic T  cells. Infection with the microorganism is followed by the expression of several cytokines and include IFN-γ, IL-1β, TNF-α, and GM-CSF among others. The two cytokines most critical for resistance to Listeriosis are IFN-γ and TNF-α. IFN-γ (TH1) is necessary for resistance, but IL-4 and IL-10 (TH2) hinder resistance. Recombinant cytokines that can increase resistance to L. monocytogenes infection include IFN-γ, TNF-α, IL-1β, and IL-12 but potentiate in inflammation. Experimental animals injected with a sublethal dose of viable listeriae develop resistance to rechallenge for a few months followed by decreased resistance. Killed microorganisms failed to provide effective immunity. Listeria monocytogenes:  Specific immune responses can be mounted against intracellular bacteria and fungi. Some bacteria reproduce inside cells of the host. For example, mycobacteria and Listeria monocytogenes (Figure  24.22) are organisms of high pathogenicity that survive in phatocytic cells such as macrophages where they resist dissolution. Within the macrophage, they are not exposed to specific antibody. In addition to mycobacteria and Listeria species, a number of fungi are also intracellular pathogens. Group agglutination:  In the serologic classification of microorganisms, the identification of group-specific antigens rather than species-specific antigens by the antibody used for serotyping. A virus  is an infectious agent that ranges from 106 D for the smallest viruses to 200 × 106 D for larger viruses such as the poxviruses. Viruses contain single or double-stranded DNA or RNA that is either circular or open and linear. The nucleic acid is enclosed by a protein coat, termed a capsid, comprised of a few characteristic proteins. Most viruses are

Macrophage

Macrophage

Figure 24.22  Listeria.

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helical or icosahedral. There may be a lipid envelope which may contain viral proteins. Viruses may be incubated with cells in culture, where they produce characteristic cytopathic effects. Inclusion bodies may be produced in cells infected by viruses. Viruses infect host cells through specific receptors. Examples of this specificity include cytomegalovirus linking to β2 microglobulin, Epstein–Barr virus linking to C3d receptor (CR2), and HIV-1 binding to CD4. These sub-microscopic acellular pathogens are comprised of either a DNA or RNA nucleic acid genome enclosed in a protein coat. To replicate, the virus must invade a host cell and use its protein synthesis machinery, which the virus does not possess, that is required for independent life. It is also called a virion. A noncytopathic virus  is a virus that appropriates cellular functions and reproduction without injuring the host cell. Virion  is a complete virus particle. Viroid  is a 100-kDa 300-bp subviral infectious RNA particle comprised of a circular single-stranded RNA segment. It induces disease in certain plants. Viroids may be escaped introns. Viropathic  refers to host tissue injury resulting from infection by a pathogenic virus. A capsid  is a virus protein envelope comprised of subunits that are called capsomers. Viral interference  is resistance of cells infected with one virus to infection by a second virus. Latency  is a condition in which the viruses enter a cell but do not replicate. Once reactivated, the virus may replicate and cause disease. Lysogeny  is the condition in which a viral genome (provirus) is associated with the genome of the host in such a manner that the genes of the virus remain unexpressed. Preactivation  is a term that refers to a provirus that continues untranscribed in the genome of a host cell because of a lack of host cell stimulation. Neuraminidase  is an enzyme that cleaves the glycosidic bond between neuraminic acid and other sugars. Neuraminic acid is a critical constituent of multiple cell surface glycoproteins and confers a negative charge on the cells. Cells treated with neuraminidase agglutinate more readily than do normal cells because of the diminished coulombic forces between them. Cells treated with neuraminidase activate the alternate complement pathway. Neuraminidase is produced by myxoviruses, paramyxoviruses, and such bacteria as Clostridium perfrigens and Vibrio cholerae. Neuraminidase together with hemagglutinin is found on the spikes of the influenza virus.

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Viral immunity:  Congenital immunodeficiency patients have given insight into the relative significance of various constituents of the immune system. Subjects with isolated defects of cell-mediated immunity contract severe and often fatal viral infections that include measles and chicken pox. By contrast, those individuals with isolated immunoglobulin deficiency usually recover normally from most viral infections except enteroviruses which may lead to chronic infection of the central nervous system. Certain generalizations may be reached concerning viral immunity. These include the following.  ntibodies act mainly by neutralizing virions, rendering A them noninfectious. By contrast, cell-mediated immunity is against virus antigens present in infected cells. Antibody prevents primary infection and reinfection through neutralization of viruses on mucosal surfaces and limiting their spread in body fluids, whereas cell-mediated immunity eliminates intracellular infection and limits reactivation of persistent viruses. The immune system may be considered in three phases, i.e., immediate (less than 4 h), early (4 to 96 h), and late (greater than 96 h). It may also be divided into humoral and cell-mediated components that include both specific and nonspecific mechanisms. Neutralizing antibody:  See neutralization and neutralization test. Viral hemagglutination:  Selected viruses may combine with specific receptors on surfaces of red cells from various species to produce hemagglutination. The ability of antiviral antibodies to inhibit this reaction constitutes hemagglutination inhibition, which serves as an assay to quantify the antibodies. One must be certain that inhibition is due to the antibody and not to a nonspecific agent such as mucoproteins with myxoviruses or lipoproteins with arbor viruses. Blood sera to be assayed by this technique must have the inhibitor activity removed by treatment with neuraminidase or acetone extraction, depending upon the chemical nature of the inhibitor. The attachment of virus particles to cells is termed hemadsorption. Among viruses causing hemagglutination are those which induce influenza and parainfluenza, mumps, Newcastle disease, smallpox and vaccinia, measles, St. Louis encephalitis, Western equine encephalitis, Japanese B encephalitis, Venezuelan equine encephalitis, West Nile fever, Dengue viruses, respiratory syncytial virus, and some enteroviruses. Herpes simplex virus can be absorbed to tanned sheep red cells and hemagglutinated in the presence of specific antiserum against the virus. This method is termed indirect virus hemagglutination. Virus-associated hemophagocytic syndrome  is an aggre­ ssive hemophagocytic state that occurs in both immunocompromised and nonimmunocompromised individuals, usually those with herpetic infections including CMV, EBV, and herpes virus, and may occur in infections by adenovirus and rubella, as well as in brucellosis, candidiasis, leishmaniasis,

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tuberculosis, and salmonellosis. There is lymphadenopathy, hepatosplenomegaly, pulmonary infiltration, skin rash, and pancytopenia. The disease is sometimes confused with malignant histiocytosis, lymphoma, sinus histiocytosis, and lymphomatoid granulomatosis. Virus-neutralizing capacity  refers to serum’s ability to prevent virus infectivity. Neutralizing antibody is usually of the IgM, IgG, or IgA class. Neutralization  by antibodies is a specific immune response to viruses. Viruses are obligate intracellular parasites that multiply within host cells whose nucleic acids and protein synthesis capability are subverted and appropriated for virus propagation. Viruses may injure cells they have infected by interfering with the cell’s protein synthesis and normal functioning, leading to host cell death. This constitutes a cytopathic effect. Viruses that are not cytopathic may induce a latent infection in which they remain inside host cells and induce synthesis of proteins that provoke a specific immune response. This consists of cytolytic T lymphocytes that are specific for the virus and destroy the virus-infected cell. Viral proteins may induce delayed-type hypersensitivity that leads to cellular injury. Both antibodies from B cells and specifically sensitized T  cells confer immunity against viruses. Before host cell invasion, specific antibodies may neutralize virions through a process known as neutralization (Figure  24.23). Neutralization is the inactivation of a microbial product such as a toxin by antibody or counteraction of the infectivity of a microorganism, especially the neutralization of viruses. Antibodies bind to viral antigen, physically blocking the antigen from binding to and infecting a host cell. However, following penetration of host cells, T cell-mediated immunity is required for destruction of the virus-infected host cells. Papovaviruses  are minute tumor viruses that are icosahedral and contain double-stranded DNA. Included in the Antibody molecule Virion

group are SV40 and polyomavirus that may cause malignant and benign tumors. Permissive or nonpermissive infections occur with papovavirus. Following permissive infection of monkey cells, papovavirus replicates, leading to lysis. T antigens, which are early papovavirus proteins that occur in nonpermissive rodent cells, can lead to transformation of the cells that is not reversible if the viral genome is integrated into the host genome. It is reversible if the cell can eliminate the viral genome. HPV,  human papilloma virus, is a human virus that has the potential to be oncogenic and occurs most frequently in individuals with multiple sexual partners. There are 46 HPV genotypes. HPV can be demonstrated by in situ hybridization in proliferations of epithelial cells that are benign, such as condyloma accuminatum, or malignant, such as squamous cell carcinoma of the uterine cervix. Whereas HPV types 6 and 11 are not usually premalignant, HPV types 16, 18, 31, 33, and 35 are linked to cervical intraeptithelial neoplasia (CIN), cervical dysplasia, and anogenital cancer. HPV is predicted to induce derepression as a neoplastic mechanism. HPV encodes E6, a viral protein that combines with the tumor suppressor protein p53. HHV  is the abbreviation for human herpes virus. Herpesvirus  (Figure  24.24) is a DNA virus family that contains a central icosahedral core of double-stranded DNA. There is a lipoprotein envelope that is trilaminar and 100 nm in diameter and a nucleus that is 30 to 43 nm in diameter. Herpes viruses may persist for years in a dormant state. Six types have been described. HSV-1 (herpes simplex virus-1) can account for oral lesions such as fever blisters. HHV-2 (human herpes virus-2) produces lesions below the waistline and is sexually transmitted. It may produce venereal disease of the vagina and vulva, as well as herpetic ulcers of the penis. Both simplex-1 and simplex-2 may infect the brain (Figure 24.25). HHV-3 (herpes varicella-zoster) occurs clinically as either an acute form known as chicken pox or a chronic form termed shingles. HHV-4 (Epstein–Barr

Cellular receptor of host

Double-stranded linear DNA Envelop glycoproteins Icosahedral nucleocapsid Tegument proteins

Figure 24.23  Neutralizing antibody molecules.

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Figure 24.24  Herpesvirus.

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involved in the delayed-type hypersensitivity response to HSV, LCTL activity, and T cell help in the antibody production. CD4+ T cells are critical for clearance of the virus. The TH1 subset is protective. HSV survives for many years in the host through establishment of a latent infection without discernible protein expression. The efficacy of avirulent or inactivated virus and subunit vaccines has not been definitely established in human trials. Animal trials have revealed that protection can be induced with glycoprotein D using recombinants Salmonella introduced orally.

Figure 24.25  Herpes simplex in the brain.

virus), HHV-5 (cytomegalovirus), and HHV-6 (human B cell lymphotrophic virus) are the other types of herpes virus.

Herpesvirus-6 immunity:  Humoral immune responses during primary infection include an IgM response. Secondary infection is associated with an increase in IgG titer as well as a recrudescence of IgM reactivity. The humoral immune response is strongly cross-reactive among HHV-6 variants. The T cell response to infection remains to be elucidated. HBLV (human B lymphotropic virus):  Herpesvirus 6.

Herpes simplex virus 1 and 2 (HSV 1 and 2) polyclonal antibody  is an antibody used to identify specific and qualitative localization of herpes simplex virus (HSV) types 1 and 2 in formalin-fixed, paraffin-embedded, or frozen tissue sections. HSV-1 is most often acquired during early childhood by nonvenereal means. It causes gingivostomatis (fever blisters). HSV-2 causes Herpes genitalis. HSV-2 is usually acquired by venereal contact but can also be acquired by a neonate at the time of delivery from an infected mother’s genital tract. HSV  is the abbreviation for herpes simplex virus. Herpes simplex virus immunity:  The two related subtypes of herpes simplex virus are designated type I (HSV-1) and type II (HSV-2). They are presently designated as human herpes viruses 1 and 2, respectively. Immune response to HSV has been investigated mainly in mouse, rabbit, and guinea pig animal models. Genetic resistance of a host animal is by several mechanisms such as the effective killing action of macrophages and natural killer cells and interferon synthesis. NK cell and interferon activity can restrict an infection but cannot clear the virus, which depends on the host-specific immune response. Both antibody and T  cell immunity are induced. Antibodies interact with the virus, infected cell glycoproteins, capsid proteins, and selected infected cell polypeptides. In mice, passively transferred antibodies against HSV can protect them from lethal doses of the virus. Antibody alone is insufficient to clear virus from the nervous system or the periphery and may retard virus spread through the nervous system. Antibody activities of HSV-specific antibody in vitro include virus neutralization, complement-mediated cytotoxicity, and ADCC. Reactivation and recurrence or reinfection with HSV in humans often occurs in the presence of high titers of neutralizing antibodies, which reveals that the antibody fails to protect. T cells are vitally important in HSV infections. The cytotoxic T lymphocyte response to HSV is mediated by CD8+ T lymphocytes in both humans and mice. CD4+ (MHC) class II-restricted lymphocytes are

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Herpesvirus-8 immunity:  Antibodies specific for a latent HHV-8 antigen and to a recombinant structural antigen present in Kaposi’s sarcoma patients or at those at risk for developing this disease. There is strong evidence for a link between HHV-8 in the pathogenesis of Kaposi’s sarcoma. Antibodies to HHV-8 antigens occur more often in HIVuninfected homosexual men than in the general population including blood donors. Antibodies to undefined structural HHV-8 antigens are present in one quarter of all blood donors in North America. Yet antibodies to the recombinant capsidrelated and latent HHV-8 proteins are present in 0 to 2% of blood donors in North America and Northern Europe. Herpes zoster  is a viral infection that occurs in a bandlike pattern according to distribution in the skin involved nerves. It is usually a reactivation of the virus that causes chickenpox. Cytomegalovirus (CMV)  (Figure 24.26) is a herpes (DNA) virus group that is distributed worldwide and is not often a problem, except in individuals who are immunocompromised, such as the recipients of organ or bone marrow transplants or individuals with acquired immunodeficiency syndrome (AIDS). Histopathologically, typical inclusion bodies that resemble an owl’s eye are found in multiple tissues. CMV is transmitted in the blood. Two classes of antiviral drugs are used to treat HIV infection and AIDS. Nucleotide analogues inhibit reverse transcriptase activity. They include azidothymidine (AZT), dideoxyinosine, and dideoxycytidine. They may diminish plasma HIV RNA levels for considerable periods but often fail to stop disease progression because of development of mutated forms of reverse transcriptase that resist these drugs. Viral protease inhibitors are now used to block the processing of precursor proteins into mature viral capsid and core proteins. Currently, a triple-drug therapy consisting of protease inhibitors are used to reduce plasma viral RNA to very low levels in patients treated for more than

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Figure 24.27  Owl eye appearance.

CMV inclusion bodies Intranuclear and intracytoplasmic

Figure 24.26  Cytomegalovirus.

1 year. It remains to be determined whether or not resistance to this therapy will develop. Disadvantages include their great expense and the complexity of their administration. Antibiotics are used to treat the many infections to which AIDS patients are susceptible. Whereas viral resistance to protease inhibitors may develop after a few days, resistance to the reverse transcriptase inhibitor zidovudine may occur only after moths of administration. Three of four mutations develop in the viral resistance to zidovudine, yet only one mutation can lead to resistance to protease inhibitors. Cytomegalovirus (CMV) immunity:  Cytomegalovirus that induces injury only if the host immune response is impaired, which makes it a significant pathogen for the fetus, allograft recipients, and individuals with acquired immune deficiency syndrome (AIDS). Host immune response to CMV is both cellmediated and humoral. Cytotoxic T lymphocytes are specific for viral structural phosphoproteins. Cell-mediated immunity appears to be the major mechanism that controls CMV replication in murine CMV, and NK cells are also important. Viral proteins induce a limited humoral response to two surface glycoproteins, gB and gH, which are neutralizing domains. Both cell-mediated and humoral immunity are insufficient to block reactivation of latent virus or to protect against reinfection from an exogenous source. CMV upregulates adhesion molecules. CMV pneumonitis is an immunopathologic. CMV is linked to infection of solid organ grafts and with graft-vs.-host disease after bone marrow transplants. It causes immunosuppression by unknown mechanisms. A live attenuated vaccine strain has no effect on the incidence of CMV infection, and a recombinant gB vaccine is in phase I clinical trials. Owl eye appearance  (Figure  24.27) describes inclusions found by light microscopy in cytomegalovirus (CMV) infection. CMV-infected epithelial cells are enlarged and exhibit prominent eosinophilic intranuclear inclusions that are half the size of the nucleus and are encircled by a clear halo.

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Chicken pox (varicella)  is a human herpes virus type 3 (HHV-3) induced in acute infection that occurs usually in children less than 10 years of age. There is anorexia, malaise, low fever, and a prodromal rash following a 2-week incubation period. Erythematous papules appear in crops and intensify for 3 to 4 d. They are very pruritic. Complications include viral pneumonia, secondary bacterial infection, thrombocytopenia, glomerulonephritis, myocarditis, and other conditions. HHV-3 may become latent when chicken pox resolves. Its DNA may become integrated into the dorsal route ganglion cells. This may be associated with the development of herpes zoster or shingles later in life. A picornavirus  is a small RNA virus with a naked capsid structure. More than 230 viruses categorized as enteroviruses, rhinoviruses, cardioviruses, and aphthoviruses comprise this family. ECHO virus (enteric cytopathogenic human orphan virus)  is comprised of 30 types within the picornavirus family. It is cytopathic in cell culture and produces clinical manifestations in patients that include upper respiratory tract infections, diarrhea, exanthema, viremia, and sometimes poliomyelitis and viral meningitis. Abelson murine leukemia virus (A-MuLV)  is a B cell murine leukemia-inducing retrovirus that bears the v-abl oncogene (Figure 24.28). The virus has been used to immortalize immature B-lymphocytes to produce pre-B cell or less differentiated B cell lines in culture. These have been useful in unraveling the nature of immunoglobulin differentiation, such as H and L chain immunoglobulin gene assembly, as well as class switching of immunoglobulin. HAV  is an abbreviation for hepatitis A virus.

+ Immature B cell

Abelson murine leukemia virus (A-MuLV) Bearing the v-abl oncogene

Tumor B cell clones corresponding to earlier stages in B cell development (i.e., Pre-B cells)

Figure 24.28  Abelson murine leukemia virus.

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C107

C185

HBe/b

Arg region

N HBc/α & HBc/β

C

150

Capsid assembly domain

Nucleic acid binding domain

Figure 24.31  Schematic structure of the HBV core polypeptide. The 185 residue p21.5 polypeptide (genotype A) is shown with the amino-terminus (N) at the left and the carboxyl-terminus (C) at the right. The open region depicts the hydrophobic assembly domain (a.a.s 1–149; open): the Arg-rich nucleic acid binding region, also known as the protamine domain (a.a.s 150–185), is shown shaded. Hatched ovals indicate the approximate locations of the Hbe/a and Hbe/b antigenic determinants. The shaded rectangle portrays the capsid-specific Hbc/α and Hbc/β epitopes which supposedly overlap Hbe/a. Also indicated are the four Cys residues 48, 61, 107, and 185 (vertical bars).

Figure 24.29  Hepatitis A virus 3C proteinase.

Hepatitis A  (Figure  24.29) is a picornavirus which is also called enterovirus 72. It is spread either person to person by the fecal–oral route or by consumption of contaminated water or food. Hepatitis serology  refers to hepatitis B antigens and antibodies against them (Figure  24.30). Core antigen is designated HBc. The HBc particle is comprised of doublestranded DNA and DNA polymerase. It has an association with Hbe antigens. Core antigen signifies persistence of replicating hepatitis B virus. Anti-HBc antibody is a serologic indicator of hepatitis B (Figures  24.31 and 24.32). It is an IgM antibody that increases early and is still detectable 20 years postinfection. The IgM anti-HBc antibody assay is the one best antibody assay for acute hepatitis B. The Hbe antigen (Hbe) follows the same pattern as HbsAg antigen. When found, it signifies a carrier state. The antiHbe increases as Hbe decreases. It appears in patients who

Concentration

HBsAg Anti-HBc

HBeAg

Exposure to antigen

Anti-HBe Anti-HBs

Onset of Onset of disease recovery

Complete recovery

Figure 24.30  Hepatitis serology.

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are recovering and may last for years after the hepatitis has been resolved. The first antigen that is detectable following hepatitis B infection is surface antigen (HBs). It is detectable a few weeks before clinical disease and is highest with the first appearance of symptoms. This antigen disappears 6 months from infection. Antibody to HBs increases as the HbsAg levels diminish. Anti-HBs often is detectable for the lifetime of the individual. Hepatitis B  is a DNA virus that is relatively small and has four open reading frames. The S gene codes for HBsAg. The P gene codes for a DNA polymerase. There is an X gene and a core gene that code for HBcAg and the precore area that codes for HBeAg. HBx  is a regulatory gene of hepatitis B that codes for the production of an HBx protein which is a transcriptional transactivator of viral genes. This modifies expression of the

w.t.

TTVV

150 • RRRDRGR I

160 • SPRRRTP II

170 • SPRRRRSQ III

∆172

TTVV

RRRDRGR

SPRRRTP

∆162

TTVV

RRRDRGR

SPRRRT

∆157

TTVV

RRRDRGR

∆149

TTVV

∆149R4

TTVV

180 • SPRRRRSQ IV

SPRRRRSQ S

SRESQC

Sqc

RRRR

Figure 24.32  The protamine region of p21.5 contains four Argrich repeats that mediate interactions between the core protein and nucleic acid. Shown are the C-terminal amino acid sequences (residues 150–185) of wild-type (w.t.) p21.5 (top), as well as a series of truncated core proteins with defined endpoints.

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HBsAg particles

HBsAg

Dane particle

Figure 24.34  Hbs antigen in liver cells. Figure 24.33  DANE particle.

host gene. In transgenic mouse-induced hepatomas, it may promote development of hepatocellular carcinoma. DANE particle  (Figure  24.33) is a 42-nm structure identified by electron microscopy in hepatitis B patients in the acute infective stage. The DANE particle has a 27-nm diameter icosahedral core that contains DNA polymerase. HBV  is the abbreviation for hepatitis B virus. Australia antigen (AA)  is hepatitis B viral antigen. The name is derived from detection in an Australian aborigine. Australia antigen is demonstrable in the cytoplasm of an infected hepatocyte. In early hepatitis B, there is sublobular cell involvement, but later in the disease, only some hepatocytes are antigen positive. There is a positive correlation between the presence of hepatitis B antigen in the liver of a group of people and that group’s incidence of hepatocellular carcinoma. Hepatitis B surface antigen (HbsAg) antibody  is a murine monoclonal antibody specific for the HbsAg phenotype. HbcAg  is hepatitis B core antigen. This 27-nm core can be detected in hepatocyte nuclei. HbsAg  (Figure 24.34) is hepatitis B virus envelope or surface antigen. Hepatitis B virus protein X:  See HBx. HbeAg  is hepatitis B nucleocapsid constituent of relatively low molecular weight which signifies an infectious state when it appears in the serum. Hepatitis B virus immunity:  Hepatitis B virus (HBV) infection leads to chronic liver disease and hepatocellular carcinoma (HCC). The virus is not believed to cause direct cytopathic injury of liver cells. Liver injury is likely

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due to the host immune response. During acute hepatitis B infection, IgM anti-HBc, a thymus-independent response, appears in the early phases of the infection together with HBsAg and HBeAg. Anti-pre-S1 also appears early in the infection together with a potent MHC class I restricted cytolytic T lymphocyte (CTL) response specific for the numerous epitopes of the structural and nonstructural proteins of the virus. The CDLs are critical for viral clearance, as are cytokines, interferon, IFN-γ, and TNFα. In the early phase of acute hepatitis B, nucleocapsid antigens, HBcAg, and HBeAg induce a powerful MHC class II restricted T helper cell proliferative response. The MHC class II locus DRB1*-1302 is associated with recovery. The CD4 response has a significant role in recovery. When the virus is eliminated from the liver, HBeAg is lost from the serum, and anti-HBe is detected. A short time thereafter, HBsAg is lost and anti-HBs antibodies appear. Immune response fails to eliminate HBV in selected patients, who become chronically infected. HBsAg either isolated from infected serum or created by recombinant DNA technology represents a successful immunogen in the induction of protective immunity against HBV infection. Hepatitis, non-A, non-B (C) (NANBH)  is the principal cause of hepatitis that is transfusion related. Risk factors include intravenous drug abuse (42%), unknown risk factors (40%), sexual contact (6%),  blood transfusion (6%), household contact (3%), and health professionals (2%). There are 150,000 cases per year in the U.S. Of those cases, 30 to 50% become chronic carriers, and one-fifth develop cirrhosis. Parenteral NANBH is usually hepatitis C, and enteric NANBH is usually hepatitis E. Hepatitis C virus immunity:  HCV-infected subjects develop specific antibodies whose clinical and biological significance remains to be determined. They are not protective. Most antiHCV positive sera are also RT-PCR positive. Antibodies to the envelope glycoproteins may have neutralizing activity. Cellular immune response against recombinant viral antigens have been investigated in proliferation and cytotoxicity assays. Chronic progressive liver disease may be linked

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to TH2 cytokine profiles from liver-derived T  cells. HCVspecific cytotoxic CD8+ T  cells have been found in HCVinfected patients. HCV escapes surveillance by the immune system by altering its antigenic determinants. Problems for vaccine development include diversity of HCV genotypes and subtypes in the hypervariability of HCV quasi-species within the host. Hepatitis D virus:  See δ agent. Delta agent (hepatitis D virus [HDV])  is a viral etiologic agent of hepatitis that is a circular single-stranded incomplete RNA virus without an envelope. It is a 1.7-kb virus and consists of a small, highly conserved domain and a larger domain manifesting epitopes. HDV is a subviral satellite of the hepatitis B virus (HBV), on which it depends to fit its genome into virions. Thus, the patient must first be infected with HBV to have HDV. Individuals with the delta agent in their blood are positive for HBsAg, anti-HBC, and often HBe. This agent is frequently present in IV drug abusers and may appear in AIDS patients and hemophiliacs. Hepatitis E virus (HEV)  is enteric non-A, non-B hepatitis. A single-stranded RNA virus that has an oral–fecal route of transmission and can introduce epidemics under poor sanitary conditions where drinking water is contaminated and the population is poorly nourished. E antigen  refers to a hepatitis B virus antigen present in the blood sera of chronic active hepatitis patients. Hepatitis E virus immunity:  Immunity against hepatitis E virus (HEV), a self-limiting disease that resembles hepatitis A, consists of an immunoglobulin M (IgM) antibody response against HEV in the acute phase of the disease. Once a peak titer is reached, it declines to undetectable levels 5 months after the immune response begins. IgG titers reach their height during the early convalescent phase and decline during the following months. Up to 50% of postinfection patients reveal undetectable levels of HEV-specific IgG. Yet, some persons still have IgG antibodies 2 to 14 years following infection. HEV-specific IgG antibodies prevent reinfection and clinical hepatitis E in young adults. Hepatitis immunopathology panel  is a profile of assays that are very useful to establish the clinical and immune status of a patient believed to have hepatitis. The panel for acute hepatitis may include HBsAg, anti-HBc, anti-ABs, antihepatitis A (IgM), anti-HBe, and antihepatitis C. The panel for chronic hepatitis (carrier) includes all of these except for the antihepatitis A test. Chronic active hepatitis (autoimmune)  (Figure 24.35) is a disease that occurs in young females who may develop fever, arthralgias, and skin rashes. They may be of the HLA-B8 and -DR3 haplotype and suffer other autoimmune disorders. Most develop antibodies to smooth muscle, principally

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Figure 24.35  Chronic active hepatitis.

against actin, and autoantibodies to liver membranes. They also have other organ- and nonorgan-specific autoantibodies. A polyclonal hypergammaglobulinemia may be present. Lymphocytes infiltrating portal areas destroy hepatocytes. Injury to liver cells produced by these infiltrating lymphocytes produces piecemeal necrosis. The inflammation and necrosis are followed by fibrosis and cirrhosis. The T  cells infiltrating the liver are CD4+. Plasma cells are also present, and immunoglobulins may be deposited on hepatocytes. The autoantibodies against liver cells do not play a pathogenetic role in liver injury. There are no serologic findings that are diagnostic. Corticosteroids are useful in treatment. The immunopathogenesis of autoimmune chronic active hepatitis involves antibody, K cell cytotoxicity, and T  cell reactivity against liver membrane antigens. Antibodies and specific T suppressor cells reactive with LSP are found in chronic active hepatitis patients, all of whom develop T  cell sensitization against asialoglycoprotein (AGR) antigen. Chronic active hepatitis has a familial predisposition. Shingles (herpes zoster)  is a virus infection that occurs in a band-like pattern according to distribution in the skin of involved nerves. It is usually a reactivation of the virus that causes chickenpox. Parvovirus  is a minute icosahedral virus comprised of singlestranded DNA that may replicate in previously uninfected host cells or in those already infected with adenovirus. Parvovirus immunity:  Specific IgM followed by IgG antibodies that occur in the second week of exposure can effectively clear the infection. Parvovirus B19 targets the erythroid bone marrow cells. Antibodies that develop following first exposure to the virus are specific mainly for VP2 epitopes. This is followed by antibodies that are specific for VP1 epitopes. VP1 unique region linear epitopes are critical to induce effective neutralizing antibodies. IgG antibodies persist for life. Immunodeficient individuals who cannot develop an adequate antibody response may develop persistent parvovirus infection. VP1 specific antibodies are crucial for the control of parvovirus B19 infection. Commercial

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immunoglobulin preparation contains parvovirus B19neutralizing antibody and are useful for the treatment of persistent parvovirus infection. Antibody synthesis in immunocompetent subjects presents clinical manifestations as a consequence of direct viral cytotoxicity. Immunodeficient patients who have an impaired capacity to synthesize neutralizing antibodies develop severe anemia as a result of suppressed erythropoiesis by the virus. Empty parvovirus capsids enriched for VP1 epitopes induce protective antibody responses. A recombinant form has shown promise in clinical trials. Poliovirus  is a picornavirus of the genus enteroviridae. There are three polio serotypes. Polio and other enterviruses are spread mainly by the fecal–oral route. Poliomylelitis occurs around the world; however, in the Western Hemisphere the wild-type virus has been eliminated by successful vaccines. Rotavirus  is a double-stranded RNA virus that is encapsulated and belongs to the reovirus family. It is 70 nm in diameter and causes epidemics of gastroenteritis, which are usually relatively mild but may be severe in children less than 2 years of age. Rabies  is an infection produced by an RNA virus following a bite from an infected animal. The virus passes across the neuromuscular junction and infects the nerve from which it reaches the central nervous system. It also reaches salivary glands of lower animals. The virus infection leads to cerebral edema, congestion, round cell infiltration of the spinal cord and grey matter in the brain stem, and profound loss of Purkinje cells. Negri bodies are found prominently in the medulla oblongata, hippocampus, and cerebellum. Clinically, the fury associated with the disease is due to irritability of the central nervous system. There is fever, hyperethesia, and anoxia aggression; it may be paralytic. Human rabies is rare in the U.S. It is more common in other animals, with most of the cases appearing in skunks and raccoons. Fewer cases occur in bats and only 2% each in dogs and cats. The virus is transmitted from one person to another by inhalation or by corneal transplantation, but not by human bites. Prion  (Figure 24.36) is an infectious particle comprised of a protein with appended carbohydrate. It is the most diminutive infectious agent known. The three human diseases in which prions have been implicated include kuru, CreutzfeldtJakob disease, and Gerstmann-Straussler syndrome. They have also been implicated in the following animal diseases: sheep and goat scrapie, bovine spongiform encephalopathy, chronic wasting of elk and mule deer, and transmissible mink encephalopathy. Prions do not induce inflammation nor do they stimulate antibody synthesis. They resist formalin, heat, ultraviolet radiation, and other agents that normally inactivate viruses. They possess a 28-kDa, hydrophobic, glycoprotein particle that polymerizes, forming an amyloid-like fibrillar structure.

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Figure 24.36  Prion.

Spongiform encephalopathies  are fatal diseases caused by prions. They cause the brain to become “sponge-like.” This category of diseases includes variant Creutzfeldt-Jakob disease (vCJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE, also termed “mad cow disease”) in cattle. Slow viruses  are agents that induce infectious encephalitis following a lengthy latency. Slow viruses consist of conventional viruses and prions that are comprised of subverted cell proteins. Among the conventional group is measles, which induces subacute sclerosing panencephalitis, papovavirus, which induces progressive multifocal leukoencephalopathy, and rubella, which induces rare progressive rubella panencephalitis. The agents that cause kuru and Creutzfeldt-Jakob disease are among the nonconventional group of slow viruses. Viral capsids  (Figure 24.37) are envelope antigens, thereby inhibiting adherence and invasion of host cells. Antibodies may also act as opsonins that increase the attractiveness of viral particles to phagocytic cells. Secretory IgA antibody is important in neutralizing viruses on mucosal surfaces. Complement also facilitates phagocytosis and may be significant in viral lysis. Capsular polysaccharide  is a constituent of the protective coating around a number of bacteria such as the Nucleic acid Capsid

Capsomere

Figure 24.37  Capsid.

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NA

NA HA

HA

H1N1

NA HA

HA

H2N2

HA

HA

H3N2

Figure 24.38  Antigenic variation.

pneumococcus (Streptococcus pneumoniae) which is a polysaccharide, chemically, and stimulates the production of antibodies specific for its epitopes. In addition to the pneumococcus, other microorganisms such as Streptococci and certain Bacillus species have polysaccharide capsules. Capsule:  Refer to capsular polysaccharide. Antigenic variation  represents a mechanism whereby selected viruses, bacteria, and animal parasites may evade the host antibody or T cell immune response, thereby permitting antigenically altered etiologic agents of disease to produce a renewed infection. The variability among infectious disease agents is of critical significance in the development of effective vaccines. Antigenic variation affects the surface antigens of the viruses, bacteria, or animal parasite in which it occurs. By the time the host has developed a protective immune response against the antigens originally present, the latter have been replaced in a few surviving microorganisms by new antigens to which the host is not immune, thereby permitting survival of the microorganism or animal parasite and its evasion of the host immune response. Thus, from these few surviving viruses, bacteria, or animal parasites, a new population of infectious agents is produced. This cycle may be repeated, thereby obfuscating the protective effects of the immune response.

Epstein–Barr virus (Figure 24.42) may induce immunosuppression by mechanisms that are yet to be determined but possibly attributable to genes that encode substances which dampen antiviral immune responsiveness. Epstein–Barr virus (EBV)  is a DNA herpes virus linked to aplastic anemia, chronic fatigue syndrome, Burkitt lymphoma, histiocytic sarcoma, hairy cell leukemia, and immunocompromised patients. EBV may promote the appearance of such lymphoid proliferative disorders as Hodgkin and nonHodgkin lymphoma, infectious mononucleosis, nasopharyngeal carcinoma, and thymic carcinoma. It readily transforms B lymphocytes and is used in the laboratory for this purpose to develop long-term B lymphocyte cultures. Antibodies produced in patients with EBV infections include those that appear early and are referred to as EA, antibodies against viral capsid antigen (VCA), and antibodies against nuclear antigens (EBNA). Influenza viruses  are infectious agents that induce an acute, febrile, and respiratory illness often associated with myalgia and headache. The classification of influenza A viruses into subtypes is based on their hemagglutinin (H) and neuraminidase (N) antigens. Three hemagglutinin (H1, H2, and H3)

NA

 iruses are able to evade immune mechanisms. Besides the V sanctuary viruses enjoy once they have entered host cells, these disease agents have additional means to escape host immune mechanisms. Viruses are especially adept at antigenic variation (Figure  24.38) whereby they may alter their surface antigenic structure once antibodies are formed against their original epitopes. This process may be repeated many times leading to the production of numerous strains of a particular virus that are antigenically and, therefore, serologically distinct. The influenza (Figures 24.39 to 24.41) and AIDS viruses are especially versatile in this regard.  iruses may also induce immunosuppression in the host they V infect. The AIDS virus (HIV-1) is well known to target CD4 + helper/inducer lymphocytes that are central to mounting any type of immune response. Selected viruses such as the

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0

50 nm

PB2 PB1 PA

NP

M1 M2

NS1 and NS2 HA

Figure 24.39  Influenza virus.

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Figure 24.41  Influenza B/LEE/40 neuraminidase (sialidase).

Figure 24.40  Influenza A subtype N2 neuraminidase (sialidase). A/Tokyo/3/67.

and two neuraminidase (N1 and N2) subtypes are the principal influenza A virus antigenic subtypes that produce disease in man. Due to antigenic change (antigenic drift), infection or vaccination by one strain provides little or no protection against subsequent infection by a distantly related strain of the same subtype. Influenza B viruses undergo less frequent antigenic variation. Influenza virus immunity:  Influenza A viruses undergo changes in their surface hemagglutinin (HA) and neuraminidase (NA) glycoproteins, leading to repeated epidemics and pandemics. Mucosal IgA and serum IgG antibodies are specific for the HA molecule-neutralized virus infectivity and mainly confer resistance to reinfection. This is the reason for vaccination against epidemic strains with killed virus. The antibody response to HA is subtype specific but antigenic drift facilitates escape of infectious virus from antibody-mediated destruction. Five protective antigenic sites are present on the globular head of the HA molecule. Antibodies against neuraminidase fail to prevent infection but diminish spread of the virus. Enzyme-active centers on each of the four subunits of the NA are in a central cavity encircled by

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two antigenic sites. Influenza A viruses undergo variability which includes antigenic drift that yields variance of contemporary epidemic strains that are sufficiently different to avoid neutralization by antibody. The more extensive antigenic shift may be a consequence of dual infection with a human and an animal influenza A virus. This can lead Release into oropharynx

E-B virus particles

Epithelium with productive infection EBV-infected B cell

B Circulates

B cell pool B with latent infection B

B

B

T cell activated

Lytic cycle T

T T cell pool with EBV-specific T memory T cell activated

T B B

B cell transformed by EBV

Figure 24.42  Epstein–Barr virus–host interaction.

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to the development of a novel pandemic strain for which humans have no preexisting antibody. CD8+ virus-immune cytotoxic T lymphocytes can clear influenza A viruses from infected lungs. It is necessary for the effector lymphocyte to come into direct contact with the virus-infected target cell. Immune CD4+ T lymphocytes are involved in lung consolidation in influenza penumonia and have a significant role in clearance of the virus. CD4 + T  cells facilitate antibody production. They also facilitate the generation of “helper cytokine” such as IL-4 and IL-5 that promote clonal expansion and differentiation of B cells into antibody-secreting plasma cells. Influenza A virus infection primes the host for a secondary CD8+ T lymphocyte response to other influenza A viruses. Influenza-immune CD4 + helper T cells are specific for viral peptides presented in the context of self MHC class II glycoproteins. Influenza hemagglutinin  is an influenza virus coat glycoprotein that binds selected carbohydrates on human cells, the initial event in viral infection. Antigenic shift results from changes in the hemagglutinin. A v-myb oncogene  is a genetic component of an acute transforming retrovirus that leads to avian myeloblastosis. It represents a truncated genetic form of c-myb. Creutzfeldt-Jakob syndrome  is a slow virus infection of brain cells that reveal membrane accumulations. Street virus  is a natural or genetically unmodified virus such as rabies that can be isolated from animals. Subacute sclerosing panencephalitis  is a slow virus disease that occurs infrequently as a complication of measles and produces progressively destructive injury to the brain through slow replication of defective viruses. Acyclovir 9 (2-hydroxyethoxy-methylguanine)  is an antiviral nucleoside analog that blocks herpes simplex virus-2 (HSV-2), the causative agent of genital herpes. HSV thymidine kinase activates acyclovir, through monophosphorylation, followed by triple phosphorylation with host enzymes to yield a powerful blocking action of the DNA polymerase of HSV-2. Acyclovir is prescribed for the treatment of HSV-2 genital infection. Q fever  is an acute disease caused by the rickettsia Coxiella burnetii. Cattle, sheep, goats, and several small marsupials serve as reservoirs. Ticks are the main vector. The microorganism is highly infectious and multiplies readily to produce clinical infection. The onset is abrupt and is accompanied by headaches, high fever, myalgia, malaise, hepatic dysfunction, interstitial pneumonitis, and fibrinous exudate. Q fever may also induce atypical pneumonia, rapidly progressive pneumonia, or be a coincidental finding to a systemic illness. The disease has a relatively low mortality. It is treated successfully with tetracycline and chloramphenicol.

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Rickettsia immunity:  The immune response in rickettsial infection involves both humoral and cell-mediated immune responses that are both powerful and persistent. Antibodies that act as opsonins render microorganisms susceptible to destruction by macrophages even though complement and antibody are not bactericidal. Antibody-dependent cellular cytotoxicity has also been demonstrated. Interferon γ-activated macrophages effectively destroy rickettsia. Interferon γ and to a lesser degree tumor necrosis factor can control reproduction of rickettsiae in nonprofessional phagocytes. Protection depends upon cell-mediated immunity rather than antibody alone. Rickettsial infections activate NK cells. Both LAK and cytotoxic T cells can kill rickettsiainfected cells. Numerous attenuated rickettsial and killed subunit vaccines are available. Chlamydia immunity:  Chlamydiae infect many animal species and various anatomical sites. No single pattern of host response can be described, but there may be similarities in effector mechanisms. The strain or serovar of the infecting microorganism is also significant. In vitro studies and genital respiratory and ocular animal models have provided most of the information about both protective and pathologic host responses. Acute inflammation is the initial response with participation by polymorphonuclear leukocytes (PMNs) that may counteract the microorganisms but also cause pathologic changes. When chlamydiae infect epithelial cells, IL-1 is released as well as IL-8, a potent PMN chemoattractant. When these microorganisms infect macrophages, LPS triggers the synthesis of TNFα, IL-1, and IL-6. They may also activate the alternative pathway of complement. IL-8, TNFα, and complement may induce chemotaxis of PMNs to the local site. ICAM-1, VCAM-1, and MAdCAM-1 are all detectable early in the infection and could be addressins responsible for PMN extravasation at sites of infection. NK cells may appear early after infection of the genital tract. Chlamydial infection produces both humoral and cell-mediated immune responses. Cell-mediated immunity has been found important in both mouse and guinea pig models. The CD4+ T cells are the main subset responsible for protective cell-mediated immunity. In genital infections, the TH1 subset of CD4 + T cells is the principal cell type leading to the formation of high levels of IFN-γ which is believed to have a protective role. In some models antibody appears to be important in the resolution of infection. Antibody may neutralize chlamydial elementary bodies in vitro. Immunity to chlamydial infections is short lived. There is currently no effective vaccine for chlamydia infections in humans, but a veterinary vaccine is available. Lymphogranuloma venereum (LGV)  is a sexually transmitted disease induced by Chlamydia trachomatis that is divided into L1, L2, and L3 immunotypes. It is rare in the United States but endemic in Africa, Asia, and South America. Clinically, patients develop papulo ulcers that heal spontaneously at the inoculation site. This is followed by development of inguinal and perirectal lymphadenopathy.

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There is skin sloughing, hemorrhagic proctocolitis, purulent draining, fever, headache, myalgia, aseptic meningitis, arthralgia, conjunctivitis, hepatitis, and erythema nodosum. Various antibody assays used in the diagnosis of LGV include complement fixation, with a titer of greater than 1:32, and immunofluorescence. Also, the Frei test, which consists of the intracutaneous inoculation of a crude antigen into the forearm, is used and can be read after 72 h. It is considered positive if the area of induration is greater than 6 mm. Adenoviruses, infection, and immunity:  Species-specific icosahedral DNA viruses that belong to numerous serotypes. They produce multiple clinical syndromes in humans, including respiratory, genitourinary, gastrointestinal, and conjunctival infections. They are resistant to most antiviral chemotherapy. An oral vaccine has been very successful in preventing acute respiratory disease in military personnel. Humans develop serotype-specific neutralizing antibodies to the structural proteins, thereby preventing reinfection with the same serotype. Early (E) nonstructural proteins produce significant immunologic effects. The virus has a double-stranded linear DNA and more than 12 structural proteins. There is no virus envelope. The viral polypeptides serve as antigens for host immune responses generated as a consequence of infection as a result of immunization with a vaccine. The internal structural proteins are not believed to be involved in humoral or cell-mediated immunity. Anticytomegalovirus (CMV) antibody  is a mouse monoclonal antibody that reacts with CMV-infected cells, giving a nuclear staining pattern with early antigen and a nuclear and cytoplasmic reaction with the late viral antigen. The antibody shows no cross-reactivity with other herpesviruses or adenoviruses. Arenavirus immunity:  Even though arenaviruses may not cause ill effects in carrier rodents, when transmitted to primates they may induce severe encephalitis, hepatitis, or hemorrhagic syndrome. LCMV is a classic member of this group and has been used to investigate virus-induced cellmediated immunity. Infection of mice may be either acute, which induces strong cell-mediated immunity, or a persistent infection that is associated with little cell-mediated immunity. The immune response to the acute infection either kills the mouse by causing meningitis, or subsides and renders the mouse LCMV-immune. CD8+ T  cells not only clear the virus infection but may mediate pathological changes of choriomeningitis when injected intracerebrally. LCMV infection induces interferon γ and tumor necrosis factor α which affect the host immune response. IFN-γ induces NK cell proliferation that eliminates pathogen-infected cells and selected tumors. Arenaviruses persist in long-term carriers through antigenic variation that evades the host immune response. Mice may be protected from lethal LCMV challenge by peptide vaccination or by vaccinia vectors that express viral epitopes.

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Bunyaviridae immunity:  Bunyaviridae are very immunogenic, stimulating the formation of neutralizing antibodies that are type specific and reveal limited cross-reactivity within the genus. Solid immunity that follows infection protects against reinfection. The immune response in some individuals may be too late to prevent central nervous system or liver invasion. For example, Rift Valley fever virus may lead to liver necrosis and high mortality. Some American hantaviruses produce the greatest effect on the lungs rather than the liver, kidneys, or central nervous system, leading to hantavirus pulmonary syndrome. G1 glycoprotein antibodies neutralize viral infectivity and prevent hemagglutination. The dominant antigen is nucleocapsid protein in complement fixation assays. Little is known concerning the role of cellmediated immunity in resistance. Formalin-inactivated veterinary vaccines against Rift Valley fever and Nairobi sheep disease are available. B-type virus (Aspergillus macaques)  is an Old World monkey virus that resembles herpes simplex. Clinical features include intermittent shedding and reactivation in the presence of stress and immunosuppression. Humans who tend these monkeys may become infected with fatal consequences. B-type viruses possess an eccentric nuclear core. Calcivirus immunity:  Human calciviruses have been shown to cause gastric distress. Antibodies against members of this group such as small round-structured viruses (SRSV) that include Norwalk virus (NV) may cross-react with other viruses of this same form; thus, a rise in antibody titer is insufficient for diagnosis. IgA antibody responses appear more specific. Reinfection by NV is common as preformed antibody correlates with susceptibility to the illness. Therefore, people who recover often become susceptible again on rechallenge. High titers attained after several infections are protective in some studies although this has not been confirmed. Antibody to HuCV is protective and mainly type specific. Vaccines are available for FCV and RHDV. Canine distemper virus  induces disease in dogs and is associated with demyelination, probably induced by myelinsensitive lymphocytes. Coronavirus immunity:  Neutralizing and fusion-inhibiting antibodies are directed mainly against the protein S antigen. Antibodies against HE, N, M, and sM are also significant in neutralization together with complement factors. Among coronavirus antigens, there is high antigenic variability of the S1 subunit of IBV. This subunit induces neutralizing antibodies that bind to discontinuous epitopes. The S2 subunit contains linear epitopes and an aminodominant region. The S1 subunit of the molecule contains the most immunogenic sites of BCV and MHV. The S2 subunit of MHV also induces neutralizing and fusion-inhibiting antibodies. BCV-specific antibodies against the HE protein participate in neutralization. The M protein of MHV and TGEV stimulate complementdependent neutralizing antibodies. The TGEV sM protein is

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involved in neutralization. T  lymphocyte responses against SN protein facilitate virus elimination and may confer protection against encephalomyelitis in mice and rats. An immunodominant T  cell antigenic site in the N protein of MHV induces neutralizing S-protein-specific antibodies.

the infection. Even though little neutralizing antibody can be detected in human convalescent sera, it is believed that passive immunization with antibodies against Ebola virus afford some benefit in treatment. No vaccines for humans are presently available.

Coxsackie  is a picornavirus family from Enteroviridiae. Coxsackie A viruses have 23 virotypes, and Coxsackie B viruses have six types. Clinical conditions produced by Coxsackie viruses include herpangina, epidemic pleurodynia, aseptic meningitis, summer grippe, and acute nonspecific pericarditis and myocarditis.

Flavivirus immunity:  Yellow fever, dengue, Japanese encephalitis, and tickborne encephalitis are the most important viruses of the flavivirus group. The E protein plays a critical role in infection and immunity since it possesses cellular receptor-binding determinants, a membrane fusion activity, and epitopes for neutralizing antibodies. Macrophages clear the viremia, yet antiviral function may be affected by their state of activation and levels of virus-specific antibodies. West Nile virus infection is associated with impaired NK cell function, which may be related to MHC class I antigen expression on virus-infected cells, which could represent an immune escape mechanism. Virus-specific antibodies provide protection against flavivirus disease. Anti-E protein antibodies are protective in various species and are believed to play a major role in immunity and natural infections. Previous infections are thought to ameliorate or protect from subsequent infections with heterologous viruses by inducing formation of group-specific antibodies. Flavivirus NS 1 protein stimulates protective antibodies. Antibody-dependent cellular cytotoxicity (ADCC) occurs in dengue fever. Cellular immunity is believed to be required for control of infection since T cells adoptively transferred into unimmunized mice can protect them against lethal encephalitis. In dengue fever, there is antibody-enhanced infection of mononuclear phagocytes. Primary dengue infection sensitizes serotype cross-reactive memory T lymphocytes for activation during the secondary infection, leading to inflammatory cytokine release that facilitates the development of capillary leak syndrome. Both CD4 and CD8 cytotoxic lysis of virus-infected cells has been absorbed in dengue fever. Dengue antigen stimulates CD4+ T cells to synthesize interferon γ. Memory responses are primed for major activation during secondary infections. The YF17D strain of yellow fever virus was the first live-attenuated vaccine for this virus family. It has proven safe and highly effective in inducing long-lasting immunity. Other members of this virus group are also candidates for vaccine development. Tick-borne encephalitis virus vaccine is a formalin-inactivated preparation which is highly effective in producing few side effects. Vaccination against Japanese encephalitis has included both inactivated and live attenuated viruses.

Cytopathic effect (of viruses)  refers to injurious effects of viruses on host cells produced by various biochemical and molecular mechanisms that are independent of host immunity against the virus. Selected viruses produce disease even though they have little cytopathic effect because the immune system recognizes and destroys the virus-infected cells. Defective endogenous retroviruses:  Partial retroviral genomes that are integrated into host-cell DNA and carried as host genes. Dengue  is an infection produced by the group B arbovirus, flavivirus, which the mosquito (Stegomyia Aedes aegypti) transmits. Dengue fever that occurs in the tropical regions of Africa and America may either be benign or produce malignant dengue hemorrhagic shock syndrome, in which patients experience severe bone pain (break-bone fever). They have myalgia, biphasic fever, headache, lymphadenopathy, and a morbilliform maculopapular rash on the trunk. They also manifest thrombocytopenia and lymphocytopenia. EBNA (Epstein–Barr virus Epstein–Barr nuclear antigen.

nuclear

antigen):  See

Filovirus immunity:  No effective immune responses are associated with fatal filovirus infections that cause fulminating hemorrhagic fever with severe shock syndrome and high mortality in humans and nonhuman primates. Those antibodies that do develop in monkeys against Ebola Reston virus are nonprotective. No significant role for neutralizing antibodies has been found for viral clearance. Extensive alterations of the parafollicular regions in the spleen and lymph nodes lead to destruction of antigen-presenting dendritic cells, pointing to disruption of cell-mediated immunity during filoviral hemorrhagic fever. Besides these cytolytic effects, the high carbohydrate content of these viruses may suppress immune reactivity. A nonstructural glycoprotein secreted from cells infected with the Ebola virus may interfere with an immune response against the virus. A fragment of spike GP released by infected cells may have a similar action. Filovirus GP possesses a sequence motif homologous to an immunosuppressive domain of retroviral glycoproteins. Filoviruses are now known to induce immunosuppression in the infected host, which contributes to the rapid course and severity of

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Infectious mononucleosis  is a disease of teenagers and young adults who have a sore throat, fever, and enlarged lymph nodes. Atypical large lymphocytes with increased cytoplasm, which is also vacuolated, are found in the peripheral blood and have been shown by immunophenotyping to be T cells. They are apparently responding to Epstein–Barr virus-infected B lymphocytes. There is also lymphocytosis, neutropenia, and thrombocytopenia. Patients also develop heterophile antibodies which agglutinate horse, ox, and sheep red blood cells as revealed by the Paul-Bunnell test.

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Infectious mononucleosis is the most common condition that EBV causes. There may be splenomegaly and chemical hepatitis. Infectious mononucleosis syndrome(s)  include conditions induced by viruses that produce an acute and striking peripheral blood monocytosis and lead to symptoms resembling those of infectious mononucleosis induced by Epstein–Barr virus. Examples include herpes virus, cytomegalovirus, HIV-1, HHV-6, and Toxoplasma gondii infections. Kuru  is a slow virus disease of some native tribes of Guinea that practice cannibalism. Transmission is through skin lesions of individuals preparing infected brains for consumption. The virus accumulates in brain cell membranes. Newcastle disease  is follicular conjunctivitis induced by an avian paramyxovirus that blocks the oxidative burst in phagocytes. Cytokines that produce fever are formed. There is recovery in approximately 1 to 2 weeks. In birds the agent induces pneumoencephalitis, which is fatal. P1 kinase  is a serine/threonine kinase activated by interferons α and β. It prevents translation by phosphorylating eIF2, the eukaryotic protein synthesis initiation factor. This facilitates inhibition of viral replicaton. Papillomavirus immunity:  Papillomaviruses induce skin and mucosa neoplasia. Humans who have been infected with this virus develop antibodies that react with papillomavirus capsid proteins. Cervical cancer patients frequently form antibodies against the E7 protein of HPV 16 and HPV 18. Patients’ sera have also demonstrated antibodies against E2, E6, and E7 proteins. Chronic infection in immunologically competent hosts point to the possibility that the viral antigens may not be recognized by the immune system. HPV disease occurs in immunosuppressed transplant patients and in acquired immune deficiency syndrome (AIDS). Thus, cell-mediated immunity is significant for the control HPV infection. NK cells are significant in the cellular response to HPV infection. NK cell activity is decreased in patients with HPV-induced neoplasia. Decreased nomers of the potent antigen-presenting cells known as Langerhans cells occur in HPV precancerous lesions. Viral antigens are presented to T lymphocytes via class I MHC molecules which are downregulated in HPV-induced cervical and laryngeal lesions. The papillomavirus evades the immune system through downregulation of MHC class I molecules and Langerhans cells and diminished susceptibility to NK cells. Vaccines have been developed in animal modes, especially cattle. Therapeutic vaccines for subjects already infected with these viruses hold promise. Parainfluenza virus (PIV) immunity:  Immunity against parainfluenza virus infection (presented clinically as croup, upper respiratory infections, and pharyngitis) is manifested as an increase in IgG antibodies to PIV in 93%, 81%, and

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80% of PIV type 1, 2, and 3 infections, respectively. IgM antibodies occur in 40 to 90%. They are common cross-reactions of IgG antibodies to PIV 1 and 3 but not of antibodies to PIV 2 and PIV 1 and 3. Cross-reactions are less frequent with IgM antibodies. EIA is more sensitive than complement fixation but is of lower specificity because of cross-reactions of PIV with mumps virus. Type-specific PIV antigens are found in 94 to 100% of culture-positive nasopharyngeal aspirates. Paramyxovirus immunity:  Both serum antibody and cellmediated immunity are induced by infection with human paramyxoviruses that cause such common childhood diseases as measles, mumps, and respiratory tract infections. Both limbs of the immune response are important for recovery from disease although the relative significance of these varies with the particular virus of this group. Secretory antibody is important in some of them such as respiratory infections, but it is only partially protected. Most all of the virus-encoded proteins induce serum antibody detectable after infection. Antibodies specific for M protein and F protein are usually at low titers. Even though antinucleocapsid antibody is often present in high titer, the only neutralizing antibodies are those specific for the attachment protein and the fusion protein and are thus protected. Antibodies against either F or HN proteins are protective but the greatest protection is induced when both antigens are used for immunization. The cell-mediated immune response to paramyxoviruses remains to be defined. These viruses may evade the host immune response and nonspecifically suppress cell mediated immunity through infection of monocytes and macrophages as observed in measles infection. They also evade the host immunity by establishing a persistent infection. Measles, mumps, Newcastle disease, canine distemper, and rhinderpest virus vaccines are presently available. These are all live attenuated virus vaccines. Poxvirus immunity:  Multiple antibodies are produced in response to poxvirus infection. These antibodies can be detected by numerous assays including complement fixation, virus neutralization, and ELISA among others. Not all of these antibodies are protective. Significant are the antibodies that neutralize enveloped or nonenveloped virus infectivity, those that combine with circulating antigens to facilitate immune clearance by phagocytes, and those that together with effector cells and complement lyse infected cells. Neutralizing antibody may diminish thyremia by acting on extracellular virus. The passive transfer of antibodies has been shown to protect mice and sheep from infection by viruses of this group, but the passive protection is brief and of limited effectiveness compared with active immunization with live virus that induces long-lasting immunity. Crossreactive neutralizing antibodies may be detected within a week following infection and last for a generation. But the level of neutralizing antibody and the host immune status are not directly correlated. The cell-mediated response is the principal mechanism of protection and recovery from poxvirus diseases. Immune T lymphocytes and monocytes and macrophages are necessary for regression of ectromelia

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infection of mice. They develop delayed-type hypersensitivity that is a T cell-mediated response. Vaccinia virus infection of Rhesus monkeys and hamsters is mediated by NK cells, but by cytotoxic T cells in sheep. Vaccinia virus shows interferon sensitivity. Poxvirus synthesis remains intracellular and is transmitted from one cell to another without exposure to neutralizing antibody, which is effective only against virus budding from infected cells. The virus may also survive in the skin, where epidermal Langerhans’ cells process and present antigen to lymphocytes to generate a protective immune response. Poxviruses encode functional homologues of host cell factors that regulate the immune system. Edward Jenner in 1798 showed that cowpox cross-protects against smallpox virus infection. Cowpox vaccinia was invaluable in ridding the world of smallpox. Thereafter, it was judged that the effects of vaccination were a liability as the disease smallpox had been eliminated. Vaccinia has also been used in the control of pox in various animal species. Vaccinia is currently used as a vector for genes of other viral pathogens in the development of vaccines. Provirus  is the DNA version of a retrovirus that has been integrated into the host cell genome where it may remain inactive transcriptionally for prolonged periods. Picornavirus  is a small RNA virus with a naked capsid structure. More than 230 viruses categorized as enteroviruses, rhinoviruses, cardioviruses, and aphthoviruses comprise this family. Picornavirus immunity:  Neutralizing antibodies have an important role in protection against picornaviruses as shown by the ability of passively transferred antibodies to block virus replication and disease progression. The early IgM response is less specific than the subsequent IgG and IgA responses. There is considerable cross-reactivity among the different serotypes. Virus neutralization by antibody involves Fc receptor-mediated endocytosis (opsonization) and interactions that prevent virus penetration and uncoating or induce lethal RNA unpackaging. CD4+ T  cells have also been shown to be significant in picornavirus infections that induce cell-mediated responses with the production of cytokines. CD4+ and CD8+ T lymphocytes recognize specific epitopes. Picornaviruses evade the immune system through antigenic variation of neutralizing antibody epitopes and may also involve variation at T cell sites/MHC-binding structures, which interferes with help for humoral immune responses and cell-mediated killing of infected cells. Vaccines against picornaviruses depend on their ability to induce neutralizing antibodies in the host following administration of either a live attenuated virus or a chemically inactivated intact virus. Reovirus immunity:  Most humans acquire a reovirus-specific antibody response in infancy or early childhood. Reovirus induces strong humoral and cell-mediated immune responses. Reovirus-specific antibodies are directed mainly against the outer capsid protein σ1. Yet serum antibodies to

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other viral proteins are also induced. Reovirus-specific antibodies to the σ1 hemagglutinin protein are mainly serotype specific. By contrast, reovirus-specific monoclonal antibodies reactive against other external capsid proteins reveal serotype nonspecific neutralizing or hemagglutinin inhibiting properties. Reovirus-specific monoclonal antibodies can block attachment of the virus to host cells as well as inhibit internalization and intracellular proteolytic uncoating. Virusspecific cytotoxic T lymphocytes are also elicited in addition to T  cell-mediated delayed-type hypersensitivity reactions. The DTH response is serotype specific, whereas the CDLs are mainly serotype nonspecific. These viruses may induce antigen-specific as well as antigen-nonspecific immunosuppression. Reoviruses can induce specific T  cell-responses in systemic tissues. A virus-specific B cell response occurs in Peyer’s patches, and a cytotoxic T lymphocyte response in Peyer’s patches. The ability of reovirus to enter Peyer’s patches via M cells reveals that these viruses might be used in a mucosal vaccine. Rhabdovirus immunity:  Resistance to rabies is in part genetically controlled, as has been shown in mice where it is controlled by one or two genes. Resistance is a dominant trait. There is a difference among species in susceptibility to rabies virus infection. Immunity may be either nonspecific or specific. Interferon plays a critical role since rhabdoviruses are quite susceptible to interferon action. In rabies there is no serological evidence of infection prior to onset of the disease which is usually fatal. Vaccination studies have yielded the most information concerning specific immunity. Vaccination during the incubation period, if not repeated, can cause the “early death phenomenon.” Passively transferred specific antibodies can protect against rabies. The relative significance of cell-mediated immunity as a protective mechanism remains to be demonstrated. Yet antibody titers and protection are closely correlated. Protective mechanisms following postexposure treatment of humans with rabies vaccine involve T lymphocytes. Rabies virus infection progresses silently in the nervous system without inducing any detectable humoral immune response. Anti-rabies vaccination must distinguish between preventive vaccination and postexposure treatment. Several vaccine injections together with specific immunoglobulin inoculation are warranted in postexposure treatment for humans when an individual has been badly exposed. Preventive vaccination is usually carried out in veterinary medicine. Contemporary vaccines confer partial or no protection against selected rabies-related virus infections. Only inactivated vaccines are licensed for use in humans. Those previously used that contain nervous tissue are dangerous because of their myelin content which may induce hypersensitivity reactions that lead to paralysis. Most current vaccines are prepared from virus grown in cell culture. While attenuated virus vaccines have been used in domestic animals in the past, they have been replaced by newer potent inactivated vaccines. Recombinant vaccines make use of a vaccinia recombinant virus containing the rabies virus glycoprotein gene which is able to induce production of virus

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glycoprotein in infected cells, to induce rabies virus neutralizing antibodies, and to protect susceptible hosts. It is active by oral administration. Rhinovirus immunity:  Although most rhinovirus infections resolve spontaneously, respiratory tract infections may occur in immunocompromised hosts. There are nearly 100 serotypes of rhinovirus. Studies with human rhinovirus 2 (HRV-2) reveal that HRV-2 specific immunoglobulins in blood sera and nasal secretions increase 1 to 2 weeks following inoculation. HRV-2 antibodies peak at 35 d following inoculation. Serum-neutralizing antibodies remain elevated for many years following infection. Local specific antibodies cannot be detected after 2 years. Preinoculation IgA levels in nasal washings are diminished in those who become infected compared with those who do not. Aspirin and acetaminophen suppress serum antibody responses. Smallpox:  See variola. Theiler’s virus myelitis:  Murine spinal cord demyelination that is considered to be an immune-based consequence of a viral infection. Togavirus immunity:  Lifelong immunity is induced by infection with a number of the togaviruses. Attenuated vaccines have been used to successfully control Venezuelan equine encephalitis virus in horses. Induction of vaccinal immunity in livestock can protect humans through vaccination of the intermediate host. Antibodies against E1 protein and the E2 protein can neutralize and passively protect against alpha virus infection in mice and monkeys. Nonstructural protein antibodies can recognize surface components of infected cells. Anti-NS-1 antibodies are highly efficient in activating complement on cell surfaces, leading to lysis of infected cells. Maturation of these viruses from infected cells is by budding through the cytoplasmic membrane. The recognition of nonstructural proteins on infected cell surfaces by T lymphocytes is a significant immunity mechanism. Vaccinia virus live vaccine expressing nonstructural proteins have been used as experimental vaccines. E1 proteins of alphaviruses participate in cell surface adsorption and fusion. E2 proteins contain significant virulence determinants. TORCH panel  is a general serologic screen to identify antenatal infection. Elevated levels of IgM in a neonate reflect in utero infection. The panel may be further refined by determining IgM antibody specific for certain microorganisms. TORCH is an acronym for toxoplasma, other, rubella, cytomegalic inclusion virus, herpes (and syphilis). There are both false-positive and false-negative reactions in the quantitative TORCH screen. If the TORCH panel is positive, it is indicative of in utero infection which may have major consequences. Toxoplasmosis may result in microglial nodules, thrombosis, necrosis, and blocking of the foramina, leading to hydrocephalus. Rubella may cause hepatosplenomegaly, congenital heart disease, petechiae and purpura, decreased weight

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at birth, microcephaly, cataracts, and central nervous system manifestations including seizures and bulging fontanelles. Cytomegalovirus is characterized by hepatosplenomegaly, hyperbilirubinemia, microcephaly, and thrombocytopenia at birth, followed later by deafness, mental retardation, learning disabilities, and other manifestations. Herpes simplex can lead to premature birth. The central nervous system manifestations include seizures, chorioretinitis, paralysis that is either flaccid or spastic, and coma. Syphilis is an addendum to the TORCH designation, but congenital syphilis has increased in recent years and is not associated with specific clinical findings. Varicella  is a human herpes virus type 3 (HHV-3) induced in acute infection that occurs usually in those less than 10 years of age. There is anorexia, malaise, low fever, and a prodromal rash following a 2-week incubation period. Erythematous papules appear in crops and intensify for 3 to 4 d. They are pruritic. Complications include viral pneumonia, secondary bacterial infection, thrombocytopenia, glomerulonephritis, mycocarditis, and other conditions. HHV-3 may become latent when chicken pox resolves. Its DNA may become intergrated into the dorsal route ganglion cells. This may be associated with the development of herpes zoster or shingles later in life. Varicella-zoster virus immunity:  Varicella-zoster virus (VZV) causes two separate illnesses, i.e., chicken pox or varicella and shingles or herpes zoster. Chicken pox is the primary infection, and reactivation of the virus in adulthood causes shingles, a dermatomal exanthem. The immune response to chicken pox includes IgM response at the end of the incubation period when a vesicular rash appears. Immunofluorescence can be used to detect VZV-specific antibodies reacting with outer membrane of live VCV-infected cells. The initial antibodies are specific for VCV gB, followed soon thereafter by antibodies to gH and gE. Chicken pox patients also develop a cellular immune response which reacts with the same viral glycoproteins recognized by the antibody as well as the regulatory protein termed IE62. Chickenpox patients develop lymphocyte proliferative responses to VZV gE, gI, gB, gH, and IE62 antigens. CD8+ class I-restricted T cells and CD4+ class II-restricted T  cells mediate VZV-specific cytotoxicity. The VZV cellular immune responses control the severity of the chicken pox exanthem in normal individuals. A latent VZV infection develops in most children in whom the virus remains dormant in the dorsal root ganglia for many years. In senior adult years, the virus may become reactivated and induce herpes zoster (shingles). This is associated with decreased immunity that accompanies increasing age. In the period prior to the development of zoster, anti-VZV glycoprotein antibody is greatly diminished in the serum, and cellular immunity is likewise decreased. Cells synthesizing IFN-γ (TH1 cells) diminishes more than those producing interleukin-4 (IL-4) (TH2 cells). Several weeks after the appearance of herpes zoster, high titers of VZV-specific antibody appear and there is an increased lymhoproliferative

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response to VZV antigens. Varicella-zoster immune globulin (VZIG) of high titer can prevent chicken pox if injected intramuscularly. It is important to give the globulin within 3 to 4 d after exposure to chicken pox. A live attenuated varicella vaccine is available for use in the United States, Europe, and Japan. Vaccination is followed by the development of both humoral and cellular immune responses. Variola (smallpox):  Variola major is a Poxvirus variolaeinduced disease that has now been eliminated from the worldwide human population. This virus-induced disease caused vesicular and pustular skin lesions, leading to disfigurement. It produced viremia and toxemia. Approximately one-third of the people who were unvaccinated succumbed to the disease. V. minor (alastrim) is a mild form of smallpox. It was produced by a different strain that was so weak it was unable to induce the formation of pocks on the chick chorioallantoic membrane. The term “variola” describes both the smallpox virus and the disease it causes. Fungi  are single-celled and multicellular eukaryotic microorganisms such as yeast and molds. They readily invade and colonize a host with compromised immunity, producing a variety of diseases. Immunity to fungi involves both cellmediated and humoral immune responses. Mycoses  are diseases produced by a fungus infection. Fungal immunity:  Nonspecific immune mechanisms of the host that form a first line of defense against fungal infections include the mechanical barrier provided by the skin and mucous membranes, competition by the normal bacterial flora for nutrients, and the respiratory tract’s mucociliary clearance mechanism. Fungi are not lysed by the terminal components of the complement system and specific antibody. Yet complement components serving as opsonins facilitate phagocytosis of fungi by phagocytic cells. Fungi are powerful activators of the alternative complement system. Neutrophils are very significant in protection against various mycoses including disseminated candidiasis and invasive aspergillosis. Monocytes and resident macrophages vary in their ability to kill fungi. Few specific anti-fungal activities of activated human macrophages have been demonstrated. Bronchoalveolar macrophages play an important role in the immune response to inhaled fungi. Natural killer cells inhibit the growth of C. neoformans and P. brasiliensis in vitro. NK cells have also been shown to clear cryptococcus from mice. Specific antibodies are of little use in host defense against most mycoses. But specific cell-mediated immunity is paramount for a protective immune response to C. neoformans disease and the dimorphic fungi. It is also important for protection against dermatophyte infections. Cell-mediated immunity plays an important role in protection against mucocutaneous candidiasis. AIDS patients have a high incidence of fungal infections. Cytokines formed in a specific cell-mediated immune response facilitate the antifungal action of natural killer cells, nonspecific T lymphocytes, and neutrophils. Cytokines

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such as tumor necrosis factor, granulocyte macrophage colony stimulating factor (GM-CSF), and interleukin-12 (IL-12) released during a cell-mediated immune response may activate effector cells to kill fungi. Immunosuppressive cytokines such as IL-10 and transforming growth factor β (TGF-β) are also formed in response to fungal infection. Severe fungal infections usually occur in profoundly immunosuppressed patients who have diminished responses to immunization. Defects in host immunity associated with fungal infections are being identified in order that cytokines such as interferon γ (IFN-γ) may be administered to chronic granulomatous disease patients, and granulocyte colony-stimulating factor and GM-CSF may be administered to neutropenic patients. Monoclonal antibodies against capsular glucuronoxylomannan have been given to patients with cryptococcosis. Candida immunity:  Resistance against Candida begins with the nonspecific barriers such as intact skin and mucosal epithelium in addition to the indigenous bacterial flora that competes for binding sites. Once these protective barriers have been breached, neutrophils are the major cellular defense by phagocytosing the Candida microorganisms with intracellular killing through oxidative mechanisms. Monocytes and eosinophils also participate in this process. Microabscesses may form in infected tissues. Mononuclear cells constitute the main inflammatory response in more chronic infections. IgG, IgM, and IgA immunoglobulin classes of Candidaspecific antibodies have been found in infected patients. Local mucosal immunity such as in the vagina is associated with the development of IgA antibodies in secretions. Even though antibody titers were elevated in infected patients, the humoral immune response does not have a principal role in host defense against Candida. Patients with defects in cellmediated immunity, such as AIDS patients, and those with chronic mucocutaneous candidiasis have increased susceptibility to Candida infections. Vaccination has been determined to be ineffective in preventing Candida infections. Coccidioides immunity:  Immunity against Coccidioides immitis depends upon T lymphocytes. IFN-γ plays an important role in protection which can be conferred by the recombinant form of this cytokine in experimental mice. Monocytes have a precise role in limiting infection before a specific immune response develops. Spherules and arthroconidia induce the synthesis of TNF-α by human monocytes in vitro. TNF-α alone or combined with IFN-γ promotes killing of spherules by human monocytes in vitro. TNF-α and IL-6 levels have been shown to be elevated in patients with overwhelming infection by this organism. Antigen overload and specific suppressor T lymphocyte activity from a circulating humoral suppressor substance may sometimes suppress the T lymphocyte. A Coccidioides-specific response occurs in some patients. A positive skin test indicates that the patient has been previously infected. This usually remains positive for the patient’s lifetime. Up to 90% of all infected individuals develop an antibody response to C. immitis. Mycelial phase antigens are most often used in serodiagnosis. IgM

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forms early but disappears after 6 months, whereas IgG elevated titers may indicate dissemination. Immunity induced by infection is species- and, in some instances, strain-specific, yet immunization with purified antigens may induce heterologous protection. Immunity is mediated by T  cells and is far more significant to resistance than is the humoral immune response which is also stimulated. Antibodies act mainly against extracellular parasites to reduce invasion. CD4+ T cells control primary infections whereas CD8+ lymphocytes are more significant in later stages of infection. There is a vaccine for chickens, but it is expensive. Cryptosporidium immunity:  Mucosal immunization might prove useful to prevent cryptosporidiosis in AIDS patients. Little is known of the host immune response to C. parvum. The infection is increased in severity and duration in immunosuppressed individuals, indicating that a specific mucosal immune response must be induced in the host. IFN-γ limits the infection whereas CD4+ T cells limit the duration of the infection. Thus both IFN-γ and CD4+ T cells are critical to inducing resistance to and resolution of the infection. IL-12 activates both natural killer cells and cytotoxic T lymphocytes and induces IFN-γ synthesis. The administration of IL-12 prevents C. parvum murine infection. This proves that exogenous IL-12 therapy can prevent the infection through an IFN-γ-dependent specific immune mechanism and that endogenous IL-12 synthesis helps to limit C. parvum infection. Cytochalasins  are metabolites of various species of fungi that affect microfilaments. They bind to one end of actin filaments and block their polymerization. Thus, they paralyze locomotion, phagocytosis, capping, cytokinesis, etc. Cryptococcus neoformans immunity:  The polysaccharide capsule of C. neoformans serves as an anti-phagocytic mechanism. It blocks binding sites recognized by phagocytic receptors for β-glucan and mannan that could mediate phagocytosis and secretion of TNF-α. The capsule also covers IgG bound to the cell wall but it is the site of complement activation in the alternative pathway in which IC3b fragments might facilitate opsonization. Neutrophils, monocytes, and NK cells all show anticryptococcal activity in vitro. Nonencapsulated C. neoformans generate elevated levels of IL-2 and IFN-γ in vivo. The polysaccharide capsule may also induce suppressor T cells that synthesize a factor which inhibits binding of the organism by macrophages. Critical to immunity to this fungus is the recognition of encapsulated C. neoformans by antigen-specific mechanisms. A specific immune response is essential to control encapsulated C. neoformans. NK and T cells exert their antifungal action against C. neoformans independent of oxygen or nitrogen radicals. T cell-mediated immunity is critical for acquired immunity against C. neoformans. NK cells have also been shown to play an important role. Histoplasma immunity:  Cell-mediated immunity is the main host defense against infection with Histoplasma

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capsulatum. The specific cell-mediated response in humans occurs in lymphoid organs and other tissues 7 to 18 d following exposure to conidia. This leads to the initiation of healing of lesions and organs with the formation of granulomas that have central necrosis. Lymph nodes that drain sites of infection are enlarged, encapsulated, and may calcify. Delayed-type hypersensitivity responses to histoplasmin are detectable a month after infection. Macrophages and neutrophils may harbor H. capsulatum. Yeasts and conidia that are not coated with opsonins bind to epitopes of the lymphocyte function-associated antigen 1 (CD11a/CD18), complement receptor type III (CD11b/CD18), and the P150-95 complex (CD11c/CD18) of adhesion-promoting receptors on human macrophages. Yeast bonding requires diovalent cations. The unopsonized use induces formation of hydrogen peroxide by human macrophages. Yet Histoplasma yeasts fail to induce a respiratory burst when phagocytosed. Antigen-specific CD4+ T lymphocytes mediate both protective immunity and delayed-type hypersensitivity. Cytokines that influence the outcome of infection in naïve animals include IL-12 and TNF-α. IL-3, GMCSF, and macrophage CSF activate human microphages to block yeast cell growth. There is no licensed vaccine for H. capsulatum but its heat shock protein system may be a promising candidate for a future vaccine. Ketokonazole  is an antifungal drug used to treat chronic mucocutaneous candidias. Mucocutaneous candidiasis:  Cellular immunodeficiency is associated with this chronic Candida infection of the skin, mucous membranes, nails, and hair, with about 50% of patients manifesting endocrine abnormalities. Cell-mediated immunity to Candida antigens alone is absent or suppressed. The individual manifests anergy following the injection of Candida antigen into the skin. Immunity to other infectious agents, including other fungi, bacteria, and viruses, is not impaired. The B cell limb of the immune response, even to Candida antigens, does not appear to be affected. The antibody response to Candida and other antigens is within normal limits. The relative numbers of both T and B lymphocytes are normal, and immunoglobulins are at normal or elevated levels. Four clinical patterns have been described. The most severe is known as early chronic mucocutaneous candidiasis with granuloma and hyperkeratotic scales on the nails or face. These have an associated endocrinopathy in about 50% of the cases. The second type is late-onset chronic mucocutaneous candidiasis, which involves the oral cavity or occasionally the nails. The third form is transmitted as an autosomal recessive trait and is usually not associated with endocrine abnormalities. It is a mild to moderately severe disorder. The fourth form is known as juvenile familial polyendocrinopathy with candidiasis, which may be associated with hypoparathyroidism with or without Addison’s disease. Those individuals in whom endocrinopathy is associated with mucocutaneous candidiasis may demonstrate autoantibodies against the endocrine tissue involved. In addition to the immunologic abnormalities described above, there

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Immunity against Microorganisms

is diminished formation of lymphokines, e.g., macrophage migration inhibitory factor (MIF), directed against Candida antigens. Recommended treatment includes antimycotic agents and immunologic intervention designed to improve resistance of the host. Nocardia immunity:  Infection by microorganisms of this genus usually begin as a pulmonary infection that may be either localized or disseminated. Nocardia species have a complex antigenic structure. Immunocompromised patients have an increased likelihood of developing infections by Nocardia. Defects in the mononuclear phagocyte system increase host susceptibility. The microorganisms grow in monocytes and macrophages with the addition of recombinant IFN-γ and TNF-α enhance nocardial growth. T cell of mice immunized against whole cells of Nocardia species become immunologically reactive against the microorganism and kill it directly. Whereas T  cell-deficient athymic mice show increased susceptibility to nocardial infection, B cell-deficient mice do not. Thus, T  cells rather than B cells are critical for host immunity against nocardial infection. No effective vaccine against Nocardia is presently available. Spherulin  is an antigen derived from spherules of Coccidioides immitis that has been used for the delayedtype hypersensitivity skin test for coccidioidomycosis. Parasites  are organisms that derive sustenance from a living host such as worms and protozoa. They are eukaryotic organisms with several chromosomes in a nucleus surrounded by a membrane. These include single-celled protozoa and multicellular helminth worms that range from a few micrometers to several meters. Protozoans  are single-celled parasites. Parasite immunity:  The cytokine network is critical in parasitic infection. Contemporary research is attempting to untangle this complex network in order to develop appropriate mechanisms to combat infections. Partial success has been achieved in attempting to control the direction of an immune response by incorporating cytokines into a vaccine against leishmaniasis. IL-12 has been used to prevent granuloma formation in schistosomiasis. A fine balance must be maintained between ensuring protection while reducing the possibilities of counter protection. The primary concern in parasitic infections is not to determine whether an immune response occurs, but whether the interaction between parasite and host will lead to protection or pathological changes or a combination of the two. It is necessary to reveal which antigens induce protection, how this may be induced artificially, and to ascertain what causes the pathological changes and how they may be countered. No commercially available vaccines against any human parasitic diseases exist. There are only a few against parasites of veterinary importance.

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Ascaris immunity:  The roundworm Ascaris lumbricoides infects 1.3 billion people. Infected subjects mount strong IgG antibody responses specific for the parasite, but most individuals respond to only a subset of parasite constituents. Only 20% of individuals respond to the ABA-1 antigen/allergen. Laboratory studies have shown that immune responses to Ascaris antigens in laboratory rodents is restricted by the class II MHC region. People may vary in the specificity of their hypersensitivity reactions to Ascaris lumbricoides. The IgE response appears to be protective in ascariasis and is believed to be a protective mechanism in other helminth infections. Mouse experiments show that the immune response to Ascaris infection is dominated by TH2 cells, which helps to explain the elevated IgE levels, eosinophilia, and mastocytosis observed in these infections. The TH2 response is critical for immune elimination of the parasites. Although the parasite is able to alter its surface and secreted antigens, it remains to be proven that this serves as an effective mechanism to evade the host immune system. No vaccine is available. Babesiosis immunity:  The host immune response to babesiosis, a malaria-like disease transmitted by parasitized Ixodes ticks, depends in part on the spleen, which has a central role in immune defense. Patients in whom the spleen has been removed are more susceptible to infection by Babesia and manifest elevated parasitemia. Complement activation by Babesia might lead to formation of TNF-α and IL-1, promoting local defense. Complement levels decrease in babesiosis. Patients develop increased circulating C1q binding activity and decreased C4, C3, and CH50 levels. The formation of TNG-α and IL-1 could account for many of the clinical features of the disease. Besides macrophages, other cellular immune functions are a critical part of the response to Babesia. T  cell-deficient mice manifest significantly increased parasitemia. Cellular immunity is diminished by the disease itself, which is also associated with increased CD8+ T lymphocytes, diminished monocyte mitogen responsiveness, and polyclonal hypergammaglobulinemia. Chagas’ disease:  The immune response effectively controls the high number of parasites in the acute phase, leading to essentially undetectable parasitemia in the chronic phase, yet sterile immunity and complete parasite clearance and cure have not been achieved in humans or in experimental animals infected with Trypanosoma cruzi. The immune response does not achieve a cure but maintains a host–parasite balance that lasts for the lifetime of the infected person. Various antigens have been used in vaccine trials but most only reduce the parasitemia during the acute phase of the disease and transform lethal to nonlethal infections. No vaccination has produced complete protection, and the vaccinated animals still become infected. Decreased parasitemia may diminish the incidence and severity of the chronic phase. Echinococcus immunity:  The genus Echinococcus includes four species of tapeworm parasites, among which is

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E. granulosus. Its cycle of transmission involves interaction between a definitive host, such as carnivores, and an intermediate host (herbivores/omnivores). Each host may reveal two morphologically distinct parasite stages. Definitive hosts are infected with the tapeworm stage of Echinococcus. Immunological methods have been used to diagnose infection in definitive hosts and to develop a recombinant vaccine that is highly effective in protecting sheep from hydatid infection. Ovine hydatid cyst fluid is rich in antigen. The principal parasite antigens are designated antigen 5 and antigen B. Antibodies against these are useful in immunodiagnosis of hydatid infection in humans. Protein antigens of 27 kDa and 94 kDa from protoscoleces  are recognized by sera of dogs infected with E. granulosus. Oncosphera antigens of 22, 30, and 37 kDa are specific for E. granulosus and are stage specific for oncospheres. Little is known regarding cellular responses against E. granulosus infection in dogs, but T cells and activated macrophages are believed to play a significant role in cellular immunity against Echinococcus in intermediate hosts. With respect to the humoral immune response to infection, dogs form IgA, IgG, and IgM antibodies against E.  granulosus. IgA antibody against E. granulosus is produced in the intestinal mucosa, but some dogs manifest elevated levels of IgE. IgA, IgE, IgG, and IgM antibodies are synthesized in intermediate hosts infected with this microorganism. Immunodiagnosis can be carried out by the ELISA in the definitive host by detecting circulating antibodies specific for the microorganism. Coproantigens of Echinococcus may be detected in the feces of infected dogs. The mucosal IgA produced in dogs has little effect on the worms, which have the capacity to suppress cytotoxic and effector activity in the region of the scolex. E. granulosus is believed to modulate the immune system of the intermediate host through production of cytotoxic substances, immunosuppressive and immunostimulatory cytokines. Concomitant immunity is critical in ensuring the survival of E. granulosus in intermediate hosts. This refers to the capacity of established hydatid cysts to avoid the immune system of the intermediate host while inducing an effective immune response against subsequent infection by this microorganism. Concomitant immunity is mediated by antibody and is directed against oncospheres. Killing is induced through an antibody-dependent, complement-mediated lysis of the parasite. Elephantiasis  is enlargement of extremities by lymphedema caused by lymphatic obstruction during granulomatous reactivity in filariasis. Entamoeba histolytica antibody  is a specific serum antibody that develops in essentially all individuals infected with E. histolytica. The antibody responses comprise mainly of IgG and to a lesser degree IgA. IgM declines quickly whereas specific IgG remains increased for months or years. Coproantibodies are found in the feces of amebic dysentery patients, whereas amebic liver abscess patients have secretory IgA in their saliva and colostrum. There is little evidence of cellular immunity and granuloma formation in amebic

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dysentery ulcers and in amebic liver abscesses. E. histolytica trophozoites not only resist normal human leukocytes but in fact kill them. IFN-γ induced by antigenic stimulation activates macrophages to kill trophozoites. The organism itself is potently chemotactic for neutrophils, which are killed on contact with the parasite. Lytic enzymes released from dead cells induce tissue injury. IFN-γ and TNF-α-activated neutrophils are able to kill trophozoites. There have been conflicting reports of the relative complement sensitivity of E. histolytica. Filarial immunity:  Increased levels of parasite-specific antibodies are synthesized following filarial infection. Subjects with asymptomatic microfilaremic infections develop high titers of filarial-specific IgG4 antibodies, yet patients with chronic lymphatic obstruction develop mainly IgGl, IgG2, and IgG3. Most infected subjects develop antifilarial IgE antibodies. IgE and IgG4 antibodies are usually directed against the same epitopes and are regulated by IL-4 and IL-13. Little or no proliferative response to parasite antigens occurs in lymphocytes from asymptomatic microfilaremia individuals. This lack of T cell reactivity is parasite antigen-specific since responsiveness to nonparasite antigens and mitogens is unaffected. Asymptomatic microfilaremic subjects are unable to produce IFN or IFN-γ but retain the ability to synthesize IL-4 and IL-5. Subjects with chronic lymphatic pathology synthesize IFN-γ, IL-4, and IL-5 following exposure to the parasite antigen. IL-10 modulates the synthesis of IFN-γ in microfilaremia. Prenatal exposure to microfilarial stage antigens can lead to long-term anergy to filarial antigens once naturally infected. Protective immunity can be induced by attenuated larvae or by repeated infections. Individuals who develop resistance to new infection while maintaining adult parasites acquire concomitant immunity. The few individuals who remain free of infection in spite of long-term residence in high endemic areas are said to have putative immunity. A helminth  is a parasitic worm. Infections by helminths induce a TH2-regulated immune response that is associated with inflammatory infiltrates rich in eosinophils and IgE synthesis. Hookworm immunity:  Hookworms in humans induce various antibody responses that may be assayed by either the ELISA or the radioimmunoassay technique. There is a prominent immune response to excretory–secretory (ES) products as well as to surface cuticular antigens. There is also a sharp rise in specific immunoglobulin isotypes during infection. There is a marked elevation of total serum IgE in human hookworm disease. The remaining additional immunoglobulin in the circulation is not specific for parasite antigens. Serum IgA may be diminished during hookworm disease because hookworm proteases are able to digest host IgA. The greater the burden of worms, the more intense the antibody response to adult antigens. Those subjects who have fewer worms develop higher titers of antilarval antibodies, which increases resistance to larval challenge.

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 ookworms induce TH2 type responses together with specific H IgE antibodies against ES products and eosinophilia. Evidence is lacking to support the concept that TH2-dependent immunity plays a major part in host protective immunity against hookworms. Little is known concerning cellular responses to hookworms. Eosinophilia is a common finding in hookworm infection. There is increased production of superoxide and enhanced chemotaxins of eosinophils from infected donors, yet the eosinophilia has not been linked to host-protective immunity to hookworms. No vaccines are available for immunization against hookworm disease in humans. Leishmania  is an obligate intracellular protozoan parasite with an affinity for infecting macrophages. It produces chronic inflammatory disease of numerous tissues. In mice, TH1 responses to Leishmania major that include IFN-γsynthesis, control infection. By contrast, TH2 responses to IL-4 synthesis result in disseminated lethal disease. Leishmaniasis  is a parasitic human infection that can lead to the development of different disease conditions ranging from cutaneous lesions to fatal visceral infection. Mouse models of infection have yielded much information on mechanisms of susceptibility and resistance to this infection, rendering experimental leishmaniasis a fine model system to evaluate T cell subset polarization and its relationship to pathogenesis. These intracellular protozoan parasites affect 12 million people worldwide. Leishmania major or L. tropica may cause cutaneous leishmaniasis; L. braziliensas cause mucocutaneous leishmaniasis; and L. donovini or L. infantum that induce viseral leishmaniasis produce the clinical disease known as Kala-azar (black disease), dum dum fever, or ponos. This clinical disease follows spread of the parasite from the skin lesion to tissue macrophages in the liver, spleen, and bone marrow. Patients develop fever, malaise, weight loss, coughing, and diarrhea, as well as anemia, darkening skin, and hepatosplenomegaly. Immunity depends on polarization of CD4+ TH cell subsets. In a TH2 response to infection, IL-4 and IL-10 correlate with disease susceptibility, whereas a murine TH1 response is associated with production of interferon γ (IFN-γ) and IL-2, which lead to resolution of lesions in animals that remain refractory to further challenge. Malaria  is a disease induced by protozoan parasites (Plasmodium species) with a complex life cycle in a mosquito and a vertebrate host. Four species of the parasite are responsible for human malaria. Numerous immunogenic proteins are formed at each morphologically distinct stage in the life cycle. The asexual stage in the blood stream causes the disease. The parasite employs various mechanisms to evade a protective immune response. However, immunity against the parasite and the disease eventually develops from repeated exposure. Malaria vaccine development is in progress. Metronidazole  is the drug of choice to treat intestinal and extraintestinal amebiasis and neurogenital trichomoniasis,

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and an alternative drug in Giardia lamblia, Balantidium coli, and Blastocystis hominis and infections. A MOTT  cell  is a type of plasma cell in which refractile eosinophilic inclusion bodies that resemble Russell bodies are found. It is associated with African sleeping sickness. It is demonstrable in periarteriolar cuffs in the brain of patients in late stages of African trypanosomiasis. Opisthorchiasis–clonorchiasis immunity:  Antibodies synthesized in patients infected with opisthorchiasis or chlonorchiasis react with the various developmental stages of the parasites. Stage-specific and cross-reactive common antigens have been detected and identified. IgG is the predominant class of antibodies in the serum but IgE have also been detected to a lesser degree. However, the bile contains secretory IgA as the principal immunoglobulin. The parasite antigens manifest an IgE-potentiating activity. Even though high-antibody titers are achieved in infected patients, the protective ability of these antibodies is doubtful. Some investigations have suggested that complement-fixing antibodies might have a role but Opisthorchis may be able to activate complement by way of the alternative pathway leading to lymphocyte killing. Cell-mediated immunity also occurs following natural infection or immunization with the parasite antigen. The role of T cells remains to be determined. Primary infection does not appear to protect against reinfection by the same parasite. Thus there appears to be a lack of protective acquired immunity. These liver flukes survive within the biliary system that may serve as a type of immunologically privileged site. The parasites can shed their surface tegument following injury by immune mechanism, representing yet another mechanism to evade host defenses. Onchocerciasis volvulus immunity:  The filarial parasite that can induce dermal and ocular complications contains antigens that induce antigen-specific IgG, IgM, IgE, and IgA antibodies, in addition to polyclonal bead stimulation. Humoral immunity develops early in chimpanzees, but the exact role of antibodies in protection remains to be determined. Antibodies against microfilariae facilitate adherence of granulocytes in vitro, and eosinophils and neutrophils mediate antibody-dependent killing. Massive eosinophil degranulation may lead to tissue injury. Infected subjects develop elevated IgE which may worsen ocular lesions by contributing to acute inflammation. Much of the IgE antibody is O. volvulus antigen specific. Immune complexes may also contribute to acute inflammation. Cell-mediated immunity is downregulated to antigens that are specific and nonspecific for the infectious agent. Onchocerciasis is marked by a predominant TH2 cytokine response in subjects with ocular pathology, whereas a TH1 response is associated with immunity. Thus the host immune response is significant in both pathology and protection in onchocerciasis. The HLA-D allele influences the pathogenesis of O. volvulus infection. Ocular pathology is the most serious effect of this disease affecting both anterior and posterior segments of the eye.

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Protection against onchocerciasis has been directed to vectal control but development of a protective vaccine would be a better solution. Larval antigens are the best targets for a prophylactic vaccine. Sabin-Feldman dye test  is an in vitro diagnostic test for toxoplasmosis. Serial dilutions of patient’s serum are combined with Toxoplasma gondii microorganisms, and complement is added. If specific antibodies against Toxoplasma organisms are present in the  serum, complement interrupts the integrity of the toxoplasma membrane admitting methylene blue which has been added to the system and stains the interior of the organism. That dilution of patient’s serum in which one half of the Toxoplasma organisms have been fatally injured is the titer. Schistosoma immunity:  The immune response to the blood flukes classified as schistosomes is complex. Repeated exposure to schistosome larval antigens may lead to hypersensitivity and cercarial dermatitis (swimmers itch). Exposure to large numbers of S. mansoni or S. japonicum can lead to a serum sickness or immune complex-like disease, whereas immune reactions to later stages of the infection may be associated with resistance against infection. Many of the pathological changes in schistosome infections are linked to deposition of eggs which induce granulomatous reactions in the tissues resulting in fibrosis. The granuloma is a delayed-type hypersensitivity reaction that is T  cell dependent. In addition to the cells expected in a granuloma, eosinophils, lymphocytes, and macrophages are also present. The fibrosis is also egg antigen induced. Multiple immune parameters are activated by these eggs and their antigens leading to a modulation in chronically infected individuals. Egg antigens may induce a protective as well as an immunopathological response. Whereas the eggs induce mostly immunopathologic effects, reactions to the schistosomulum are mostly protective of the host. Adult worms from a primary infection can continue to survive in individuals resistant to reinfection with fresh cercariae through “concomitant immunity.” Irradiated cercariae can be used to induce immunization of mice and other experimental animals against cercarial challenge. The main target of destructive immunological attack is the migrating schistosomulum. Humans develop concomitant immunity slowly. Resistance is correlated with peripheral blood eosinophilia in S. haematobium infections. Infection-protective immunity may be associated in elderly persons with IgE antibodies against adult worm antigens. More than 90% of the surface antigens of the young schistosomulum are carbohydrates. Anti-egg antibodies may cross-react with these antigens. Adult worms activate TH1 responses, whereas eggs induce TH2 responses. Protection in mice is mediated mainly by TH1 cells and can be potentiated with IL-12. TNF-α is associated with granuloma formation. Adult worms are usually not susceptible to immune attack either by coating themselves with host-derived macromolecules that mask parasite surface antigens or the worms may shed antigenic macromolecules from the outer tegument, rendering their outer

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surfaces immunologically inert. Even though egg identification in human excreta has long been the method of diagnosis, indirect diagnosis using antibody detected by the ELISA technique is used with increasing frequency. Schistosomederived carbohydrate antigens in the blood are also helpful in immunodiagnosis. There is no vaccine for protection against schistosomiasis in humans, yet irradiated larvae have been used to immunize cattle. Antigen masking  is the ability of some parasites (e.g., S. mansoni) to become coated with host proteins, theoretically rendering them “invisible” to the host’s immune system. Strongyloides immunity:  Immunoglobulin G (IgG), IgA, and IgE classes of immunoglobulin form in response to antigens of Strongyloides stercoralis filariform larvae. The principal humoral responses by the IgG4 subclass may be directed to more than 50 different 15- to 100-kDa antigens. The remaining IgG subclasses recognize fewer than 20 antigens. Zinc endopeptidase together with 31- and 28-kDa proteins are antigens that induce specific immune responses; however, none of these antibodies is protective against dissemination in the host. Patients without detectable humoral responses remain asymptomatic. Immunocompromised patients with disseminated infection may manifest high titers of parasitespecific antibodies. Impaired cell-mediated immunity has been claimed by some to facilitate parasite dissemination, but this has not been proven, especially since AIDS patients have not developed this as an opportunistic infection. Strongyloides hyperinfection:  Strongyloides stercoralis larvae may invade the tissues of immunosuppressed patients with enteric strongyloides infection to produce this condition. Suramin (Antrypol, 8,8'-(carbonyl-bis-(imino-3,1-phe­ nylenecarbonylimino))-bis-1,3,5-naphthalene trisulfonic acid)  is a therapeutic agent for African sleeping sickness produced by trypanosomes. Of immunologic interest is its ability to combine with C3b, thereby blocking factor H and factor I binding. The drug also blocks lysis mediated by complement by preventing attachment of the membrane attack complex of complement to the membranes of cells. Taenia solium immunity:  Preencystment immunity (early immunity) refers to the immune response at the oncosphere penetration site. Late postoncospheral or postencystment immunity refers to the immune response at the final establishment site. Secretory IgA in gut secretions likely attacks invading oncospheres since it is resistant to intestinal enzymes. Mast cells surround invading oncospheres and developing larvae, which suggests that IgE might react with antigen leading to degranulation of these cells whose products cause increased vascular permeability. This would permit IgG antibodies to reach the invading site. Eosinophils also surround invading oncospheres but there is no evidence that they induce injury. When oncospheres that are newly hatched reach their establishment site, they transform

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from a stage in which they are highly vulnerable to attack to one in which they are completely resistant. Both humoral and cellular responses to T. solium is heterogeneous in both pigs and humans. 90% of the serum antibody is IgG. The T. solium cysticerci induce a chronic granulomatous reaction in pig muscle with extensive eosinophil infiltration and degranulation. T  cell immunity to larval cestodes remains to be demonstrated. Larval cestodes may contain blocking antibodies on the surface. Molecular mimicry is also believed to represent an evasive strategy whereby this parasite avoids the host immune response. Some larvae produce inhibitors of proteolytic enzymes such as trypsin and chymotrypsin. Some cestode larvae may induce immunosuppression of the host. Vaccination with living eggs can induce complete protection against challenge infection, but there is only a limited supply of the eggs. This has been remedied by the development of a recombinant taeniid vaccine antigens which have been able to induce protection in animals.

host during acute infection. Activation of macrophages by IFN-γ is a principal effector mechanism in toxoplasma infection. Macrophages kill by both the oxidative and nonoxidative pathways. Both NK and T cells are essential components of the efferent limb of the protective cell-mediated immune response. CD8+ T cells produce gamma interferon to activate macrophages whereas CD4+ T cells synthesize interleukin 2 which facilitates IFN-γ synthesis by CD8+ T cells. NK cells also produce IFN-γ which helps to induce a TH1-type response (IL-2 and IFN-γ synthesis) of CD4+ T cells. TGF-β and IL-10 downregulate IFN-γ production by NK cells during infection. AIDS patients may develop toxoplasmic encephalitis, pointing to the significance of cell-mediated immunity which they have lost. Tachyzoites in the acute stage of infection are the principal targets for the protective immune response. Tachyzoites induce both antigen-specific and nonspecific suppressor cells to inhibit induction of the immune response to the parasite. No vaccine for T. gondii is available.

Theileria immunity:  Immunity against these tick-transmitted intracellular protozoan parasites of domestic animals depend upon cell-mediated immune responses for protection. Humoral immune responses directed against schizonts and piroplasms are insignificant in the development of natural protective immunity. Animals that recover may be resistant to homologous challenge and developing immunity that persists for 3 to 5 years. Infection with sporozoites and the development of schizonts are critical for the development of natural immunity. Live attenuated, subunit and recombinant vaccines have been used. T. annulata live vaccine is prepared from attenuated cell lines that produce infection in cattle without causing disease. These induce protective immunity. T. parva subunit vaccine contains p67 antigen, the recombinant form of which protects 70% of immunized cattle against experimental challenge.

Toxoplasmosis  is the disease induced by the protozoan parasite Toxoplasma gondii.

Toxocara canis immunity:  The immune response in humans to the domestic dog roundworm or Toxocara canis includes the development of antibodies that are useful for immunodiagnosis to detect infection. The seroprevalence rate in the U.S. general population is 2.8% for adults but 23.1% for children. The immune response is also characterized by development of eosinophilic granulomata which may appear throughout the body except for the brain. Larvae within liver granulomata may be killed. T.  canis infection induces a powerful TH2 response in experimental animals. There is no available vaccine. Toxoplasma gondii immunity:  Both humoral and cell-mediated responses follow infection with T. gondii. The cellular immune response is the principal mediator of resistance to infection, although both cellular and humoral confer resistance. Antibodies activate complement by the classical pathway to lyse extracellular parasites. Tachyzoites coated with immunoglobulin are killed within macrophages. Both monocytes and neutrophils also mediate effective killing. Whereas antibodies mediate only a protective effect that is partial, cell-mediated immunity is critical for survival of the

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Trichuris trichiura immunity:  Immune responses against these worm infections of humans include specific IgG, IgA, and IgE antibody responses. The IgG level increases with the level of the infection, but elevated IgA levels may be associated with diminished worm burdens. The cellular response remains to be defined. Macrophages release TNF-α and there is IgE-mediated release of mast cell mediators that contribute to inflammation and enteropathy associated with infection. In mice the immune response to T. muris consists of a powerful T cell response that leads to premature expulsion of worms from the intestine. Primary infections are followed by the development of resistance to reinfection. Immunity can be passively transferred with either CD4+ T cells or immune sera. Inflammatory responses are not believed to contribute to immunity. Murine stains that effectively eliminate the infection develop responses mediated by TH2 cells. There is no vaccine for Trichuris. Tropical eosinophilia  is a hypersensitivity to filarial worms manifested in the lungs. It has been reported in the Near East and in the Far East. Patients develop wheezing, productive cough, and a cellular infiltrate comprised of eosinophils, lymphocytes, and fibroblasts. Fibrosis may result. Trypanosome immunity:  Vaccination against African sleeping sickness known as trypanosomiasis has thus far been unattainable. Even though the antigen that induces a protective humoral response is well known as a surface glycoprotein that covers the entire trypanosome, the organism repeatedly changes the antigenic structure of this glycoprotein (the variant surface glycoprotein VSG), thereby evading destruction by the host immune response. Only a single VSG is expressed by a trypanosome at a time. Trypanosomes that express the same VSG are classified as belonging to the same variable antigenic type (VAT). Only short-term immunity

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development of fibrosis interrupts the venous blood supply to the liver, leading to hypertension and cirrhosis. Trypanosome Trypanosome proliferation

+ Ablastin

No trypanosome proliferation

Figure 24.43  Ablastin.

can be induced by allowing cattle to become infected by fly bites and then treating the infection as soon as the parasitemia becomes patent. The powerful humoral response in trypanosomal infections is characterized by the appearance of IgM and IgG antibodies that are associated with the elimination of each VAT. These antibodies kill parasites and clear them from the blood by complement activation by way of the classic pathway and opsonization that results in uptake by the liver’s Kupffer cells. The immune response in experimental mice consists of both T-dependent and T-independent components. Lymphocyte responsiveness is profoundly depressed in trypanosome infections. Variable surface glycoproteins  are surface coat constituents of African trypanosomes. By a process similar to gene conversion, the trypanosome may alter its surface glycoprotein coat by expressing different glycoprotein genes. Released antigen  is antigen derived from trypanosomes during an infection that may appear in the patient’s serum. It corresponds to the antigenic type of the trypanosome infecting the individual. Ablastin  is an antibody with the exclusive property of preventing reproduction of such agents at the rat parasite Trypanosoma lewisi (Figure 24.43). It does not demonstrate other antibody functions.  arasites such as Schistosoma mansoni produce eggs that P induce granuloma formation and isolation of the eggs. The

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I ntracellular protozoa often activate specific cytotoxic T cells. They represent a critical mechanism to prevent dissemination of intracellular malarial parasites. Parasite antigen–antibody (immune) complexes may be trapped in the renal microvasculature, leading to immune complex glomerulonephritis. Parasites are capable of evading immune mechanisms. Animal parasites have developed remarkable mechanisms to establish chronic infections in the vertebrate host. Natural immunity to them is weak, and the parasites have devised novel and ingenious mechanisms to circumvent specific immunity. Parasites either camouflage their own antigens or interfere with host immunity. Some parasites mask their antigens by coating themselves with host proteins, which prevents their detection by host immune mechanisms. Some develop resistance by biochemical alterations of their surface coat. They are also adept at changing their surface antigens by antigenic variation, which may frustrate attempts to prophylactic immunization. Other parasites, such as Entamoeba histolytica, may shed their antigenic coats. Schistosomiasis  is a schistosome infection that is characteristically followed by a granulomatous tissue reaction. Fasciola immunity:  An inflammatory response in the liver is associated with primary infection of a host with the liver fluke Fasciola hepatica. The cellular infiltrate includes neutrophils, eosinophils, lymphocytes, and macrophages. A significant eosinophilia occurs in the peritoneal cavity and the blood. There is no cellular response encircling the juvenile flukes themselves. Numerous macrophages and fibroblasts occur in injured areas in the chronic phase of a primary infection. This is followed by liver fibrosis which is accompanied by numerous CD8+ and y8_ TCR+T  cells. Numerous lymphocytes and eosinophils infiltrate larger bile ducts, and in cattle a granulomatous reaction is followed by bile duct calcification. Resistance to reinfection follows primary infection of cattle, rats, and possibly humans, yet this fails to occur in sheep and mice. The failure of sheep to develop resistance against reinfection may be associated with cellular immune deficiencies. Products released from F. hepatica may adversely affect the host immune response such as cathepsin proteases which flukes secrete and which may cleave immunoglobulins, thereby inhibiting attachment to host effector cells. Other immunosuppressive molecules may be toxic to host cells.

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Vaccines and Immunization

A vaccine  may contain live attenuated or killed microorganisms or parts or products from them capable of stimulating a specific immune response comprised of protective antibodies and T cell immunity. A vaccine should stimulate a sufficient number of memory T and B lymphocytes to yield effector T cells and antibody-producing B cells from memory cells. Viral vaccine should also be able to stimulate high titers of neutralizing antibodies. Injection of a vaccine into a nonimmune subject induces active immunity against the modified pathogens.  ther than macromolecular components, a vaccine may conO sist of a plasmid that contains a cDNA encoding an antigen of a microorangism. Other vaccines include antiinsect vector vaccines, fertility-control vaccines, peptide-based preparations, antiidiotype preparations and DNA vaccines, among others. There is no antiparasite vaccine manufactured by conventional technology in use at present. Vaccines can be prepared from weakened or killed microorganisms, inactivated toxins, toxoids derived from microorganisms, or immunologically active surface markers extracted from microorganisms. They can be administered intramuscularly, subcutaneously, intradermally, orally, or intranasally; as single agents or in combination. An ideal vaccine should be effective, well tolerated, easy and inexpensive to produce, easy to administer, and convenient to store. Vaccine side effects include fever, muscle aches and injection site pain, but these are usually mild. Reportable adverse reactions to vaccines include anaphylaxis, shock, seizures, active infection, and death. Vaccination  is immunization against infectious disease through the administration of vaccines for the production of active (protective) immunity in humans or other animals. It may be induced with killed, attenuated, or nonpathogenic forms of the pathogenic agent or its antigens to generate protective adaptive immune responses characterized by antigenspecific memory T cells and memory B cells specific for the pathogen. Subsequent exposure to the pathogen will then induce a secondary or anamnestic response. To vaccinate  is to inoculate with a vaccine to induce immunity against a disease. Vaccinable  is the capability of being vaccinated successfully. Attenuation  is the decrease of a particular effect, such as exposing a pathogenic microorganism to conditions that destroy its virulence, but leave its antigenicity or immunogenicity intact.

An attenuated pathogen  is one that has been altered to the point that it will grow in the host and induce immunity without causing clinical illness. To attenuate  is the process of diminishing the virulence of a pathogenic microorganism, rendering it incapable of causing disease. Attenuated bacteria or viruses may be used in vaccines to induce better protective immunity than would have been induced with a killed vaccine. Attenuated  is an adjective that denotes diminished virulence of a microorganism. Mass vaccination  refers to immunization with vaccines during an outbreak of a communicable disease in an effort to prevent an epidemic. For example, mass vaccinations may be carried out in schools and hospitals during meningitis or hepatitis epidemics. Therapeutic vaccination  is a vaccine administered to alleviate a pre-existing allergic or autoimmune condition, cancer, or other disease. Coverage (vaccine):  Refer to efficacy. International Unit of Immunological Activity  refers to the use of an international reference standard of a biological preparation of antiserum or antigen of a precise weight and strength. The potency or strength of biological preparations such as antitoxins, vaccines, and test antigens derived from microbial products and antibody preparations may be compared against such standards to reflect their strength or potency. In vaccine standardization, the immunizing dose (ImD50)  is that amount of the immunogen required to immunize 50% of the test animal population, as determined by appropriate immunoassay. The LD50  is the dose of a substance such as a bacterial toxin or microbial suspension that leads to the death of 50% of a group of test animals within a certain period following administration. This has been employed to evaluate toxicity or virulence and to evaluate the protective qualities of vaccines administered to experimental animals. A challenge  is the deliberate administration of an antigen to induce an immune reaction in an individual previously exposed to that antigen to determine the state of immunity. 769

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Challenge stock  is an antigen dose that has been precisely measured and administered to an individual following earlier exposure to an infectious microorganism.

polio, and rubella. Live attenuated virus vaccines contain live viruses in which accumulated mutations impede their growth in human cells and their disease-causing capacity.

A polyvalent vaccine  is comprised of multiple antigens from more than one strain of a pathogenic microorganism or from a mixture of immunogens such as the diphtheria, pertussis, and tetanus toxoid preparation.

Dead vaccine:  See inactivated vaccine.

A mixed vaccine  is a preparation intended for protective immunization that contains antigens of more than one pathogenic microorganism. Thus, it induces immunity against those disease agents whose antigens are represented in the vaccine. It may also be called a polyvalent vaccine. Multivalent vaccine:  See polyvalent vaccine. Combination vaccines  are immunizing preparations that contain immunogens (antigens) from more than one pathogenic microorganism. They induce protection against more than one disease. Combined prophylactic:  See mixed vaccine. A live vaccine  is an immunogen for protective immunization that contains an attenuated strain of the causative agent, an attenuated strain of a related microorganism that crossprotects against the pathogen of interest, or the introduction of a disease agent through an avenue other than its normal portal of entry or in combination with an antiserum. Live attenuated vaccine  is an immunizing preparation consisting of microorganisms whose disease-producing capacity has been weakened deliberately in order that they may be used as immunizing agents. Response to a live attenuated vaccine more closely resembles a natural infection than does the immune response stimulated by killed vaccines. The microorganisms in the live vaccine are actually dividing to increase the dose of immunogen, whereas the microorganisms in the killed vaccines are not reproducing, and the amount of injected immunogen remains unchanged. Thus, in general, the protective immunity conferred by the response to live attenuated vaccines is superior to that conferred by the response to killed vaccines. Examples of live attenuated vaccines include those used to protect against measles, mumps,

Cold chain (vaccination)  refers to the continuous refrigeration of a labile vaccine from its preparation and transport to the geographic site where is it to be used. A killed vaccine  is an immunizing preparation comprised of microorganisms, either bacterial or viral, that are dead but retain their antigenicity, making them capable of inducing a protective immune response with the formation of antibodies and/or stimulation of cell-mediated immunity. Killed vaccines do not induce even a mild case of the disease which is sometimes observed with attenuated (greatly weakened but still living) vaccines. Although the first killed vaccines contained intact dead microorganisms, some modern preparations contain subunits or parts of microorganisms to be used for immunization. Killed microorganisms may be combined with toxoids, as in the case of the DPT (diphtheria–pertussis– tetanus) preparations administered to children. Killed virus vaccines  are immunogen preparations containing virons deliberately killed by heat, chemicals, or radiation. An inactivated vaccine  is an immunizing preparation that contains microorganisms such as bacteria or viruses that have been killed to stop their replication while preserving their protection-inducing antigens. Formaldehyde, phenol, and β-propiolactone have been used to inactivate viruses, whereas formaldehyde, acetone, phenol, or heating have been methods used to kill bacteria to be used in vaccines. Inactivated virus vaccine:  Refer to killed virus vaccine. β propiolactone  is a substance employed to inactivate the nucleic acid core of pathogenic viruses without injuring the capsids. This permits the development of an inactivated vaccine, as the immunizing antigens that induce protective immunity are left intact. An autogenous vaccine  (Figure  25.1) is prepared by isolating and culturing of microorganisms from an infected

A

Infected subject

A

Microorganisms from infected subject grown in culture and vaccine prepared

Killed vaccine used to immunize infected host

Figure 25.1  Autogenous vaccine.

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subject. The microorganisms in culture are killed and used as an immunogen, i.e., a vaccine, to induce protective immunity in the same subject from which they were derived. In earlier years, this was a popular method to treat Staphylococcus aureus-induced skin infections.

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A bacterin  is a vaccine comprised of killed bacterial cells in suspension. Inactivation is by either chemical or physical treatment. Sensitized vaccine  is an immunizing preparation that contains bacteria treated with their homologous immune serum.

A homologous vaccine  is an autogenous vaccine. A heterologous vaccine  induces protective immunity against pathogenic microorganisms which the vaccine does not contain. Thus, the microorganisms that are present in the heterologous vaccine possess antigens that cross-react with those of the pathogenic agent absent from the vaccine. Measles vaccine can stimulate protection against canine distemper. Vaccinia virus was used in the past to induce immunity against smallpox because the agents of vaccinia and variola share antigens in common. Heterotypic vaccine:  See heterologous vaccine. A lapinized vaccine  is a preparation used for immunization that has been attenuated by passage through rabbits until its original virulence has been lost. A caprinized vaccine  is a preparation used for therapeutic immunization, which contains microorganisms attenuated by passage through goats. An edible vaccine  is a genetically altered food containing microorganisms or related antigens that may induce active immunity against infection. It is a plant constituent that has been altered genetically to express an antigen of a pathogen and can be ingested as food. SMAA  is a solid matrix antibody-antigen complex. It is a vaccine comprised of monoclonal antibodies against a vaccine epitope that are bound chemically to a microbead after which the vaccine antigenic determinant interacts with the monoclonal antibody. A microbead coated with monoclonal antibodies against different epitopes can exhibit epitopes to both B cells and T cells. A reassortant vaccine  is an immunizing preparation in which antigens from several viruses or from several strains of the same virus are combined. A virosome  is a vaccine comprised of a spherical artifical virus employed to direct vaccine antigens directly into a cell of the host. It consists of a liposome comprised of the vaccine antigen and the hemagglutinin and neuraminidase proteins of influenza. It has the membrane fusion properties, conformational stability, and ability to invade host cells of the native virus. The antigen is either present in the virosome lumen or cross-linked to its surface chemically. A bacterial vaccine  is a suspension of killed or attenuated bacteria prepared for injection to generate active immunity to the same microorganism.

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The prime-boost strategy  is a vaccination protocol requiring primary vaccination with a recombinant vector or naked DNA vaccine with a subsequent booster injection of a recombinant protein subunit vaccine. Catch-up vaccine  (or vaccination) refers to the immunization of unvaccinated children at a convenient time, such as the first day of school, rather than at the optimal time for antibody synthesis. This procedure provides many children who have missed vaccines at regularly scheduled intervals a second opportunity for disease prevention and control. A subunit vaccine  is an immunizing preparation comprised of a specific component of a pathogen, such as a viral protein or bacterial polysaccharide. The vaccine employs whole micromolecules or large macromolecular fragments that contain the protective epitopes. A peptide vaccine  is an immunizing preparation comprised of a small antigenic peptide capable of generating an immune response. Reverse vaccinology  is the development of a vaccine antigen based on sequences in a pathogenic organism’s genome or proteome that are most likely to represent epitopes that T cells recognize. This strategy is also termed epitope-driven vaccine design. A DNA vaccine  is an immunizing preparation comprised of a bacterial plasmid containing a cDNA encoding a protein antigen. The mechanism apparently consists of professional antigen-presenting cells transfected in vivo by the plasmid, which then express immunogenic peptides that induce specific immune responses. The CpG nucleotides present in the plasmid DNA serve as powerful adjuvants. DNA vaccines may induce powerful cytotoxic T lymphocyte responses. This is an immunizing preparation made by genetic engineering in which the gene that encodes an antigen is inserted into a bacterial plasmid which is injected into the host. Once inside, it employs the nuclear machinery of the host cell to manufacture and express the antigen. In contrast to other vaccines, DNA vaccines may induce cellular as well as humoral immune responses. A naked DNA vaccine  is an immunizing preparation that is comprised of an isolated DNA plasmid that encodes the vaccine antigen. Following introduction into the host body, the plasmid becomes incorporated into the host cells that form pathogen protein.

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CpG nucleotides:  Bacterial DNA unmethylated cytidine-guanine sequences that facilitate immune responses acting as an adjuvant. They are believed to enhance DNA vaccine efficacy.

constituents. It was considered to slow the release of immunogen from the site of injection and to induce B cells capable of forming antibody at the site of antigen deposition.

DNA vaccination  consists of imunization with plasmid DNA to induce an adaptive immune response against the encoded protein. Bacterial DNA which is rich in unmethylated CpG dinuleotides serves as an adjuvant for this kind of vaccination.

Serum virus vaccination  is a method no longer used that consisted of administering an immunizing preparation of an infectious agent such as a live vaccine virus together with an antiserum specific for the virus. It was intended to ameliorate the effects of the live infectious agent. This method was abandoned because it was considered a dangerous practice.

Synthetic vaccines  are substances used for prophylactic immunization against infectious disease prepared by artificial techniques such as from cloned DNA or through peptide synthesis. A recombinant vaccine  is an immunogen preparation for prophylactic immunization, comprised of products of recombinant DNA methodology, prepared by synthesizing proteins employing cloned complementary DNA. A recombinant vector vaccine  is an immunizing preparation in which a vector containing the DNA that encodes the vaccine antigen enters host cells and facilitates vaccine antigen translation directly inside them. An idiotype vaccine  is an antibody preparation that mimics antigens at the molecular level. Such vaccines induce immunity specific for the antigens they mimic. They are not infectious to the recipient, are physiologic, and can be used in place of many antigens, e.g., idiotype vaccine related to Plasmodium falciparum circumsporozote (CS) protein. An antiidiotypic vaccine  is an immunizing preparation of antiidiotypic antibodies that are internal images of certain exogenous antigens. To develop an effective antiidiotypic vaccine, epitopes of an infectious agent that induce protective immunity must be identified. Antibodies must be identified which confer passive immunity to this agent. An antiidiotypic antibody prepared using these protective antibodies as the immunogen, in some instances, can be used as an effective vaccine.  ntiidiotypic vaccines have effectively induced protecA tive immunity against such viruses as rabies, coronavirus, cytomegalovirus, and hepatitis B; such bacteria as Listeria monocytogenes, Escherichia coli, and Streptococcus pneumoniae; and such parasites as Schistosoma mansoni infections. Antiidiotypic vaccination is especially desirable when a recombinant vaccine is not feasible. Monoclonal antiidiotypic vaccines represent a uniform and reproducible source for an immunizing preparation. Anavenom  is a toxoid consisting of formalin-treated snake venom which destroys the toxicity but preserves immunogenicity of the preparation. Saponin  is a glucoside used in the past for its adjuvant properties to enhance immune reactivity to certain vaccine

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Although several types of rinderpest vaccines  have been used in the past, the most satisfactory contemporary vaccine contains a virus adapted to tissue culture. T  cell vaccination (TCV)  is a technique to modulate an immune response in which T lymphocytes are administered as immunogens. The vaccine is comprised of T  cells specific for the target autoantigen in an autoimmune response to be modulated. For example, antimyelin basic protein (MBP) CD4+8– T  cells, serving as a vaccine, were irradiated (1500 R) and injected into Lewis rats. Vaccination with the attenuated anti-MBP T  cells induced resistance against subsequent efforts to induce experimental allergic encephalomyelitis (EAE) by active immunization with myelin basic protein in adjuvant. This technique could even induce resistance against EAE adoptively transferred by active anti-MBP T cells. This method was later successfully applied to other disease models of adjuvant arthritis, collagen-induced arthritis, experimental autoimmune neuritis, experimental autoimmune thyroiditis, and insulin-dependent diabetes mellitus (IDDM). TCV was demonstrated to successfully abort established autoimmune disease and spontaneous autoimmune disease in the case of IDDM. Immunological contraception  is a method to prevent an undesired pregnancy. Vaccines that induce antibodies and cell-mediated immune responses against either a hormone or gamete antigen significant to reproduction have been developed. Such vaccines control fertility in experimental animals. They have undergone exhaustive safety and toxicological investigations which have shown the safety and reversibility of some of the vaccines, and with approval of regulatory agencies and ethics commissions have undergone clinical trials in humans. Six vaccines, three in women and three in men, have completed phase I clinical trials showing their safety and reversibility. One vaccine has successfully completed phase II trials in females, proving efficacy. The trials have determined the titers of antibodies and other immunological features. The fertilized egg makes the hormone hCG. Antibodies that inactivate one or more hormones involved in the production of gametes and sex steroids could be expected to impair fertility. Blocking the action of LHRH would also inhibit the synthesis of sex steroids. This might prove useful in controlling fertility of domestic animals but would not be acceptable for contraception in humans. Two

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vaccines against LHRH are in clinical trials in prostate carcinoma patients. An FSH vaccine would act at the level of male fertility since FSH is required for spermatogenesis in primates.

Immunization Passive immunization  describes the transfer of a specific antibody or of sensitized lymphoid cells from an immune to a previously nonimmune recipient host. Unlike active immunity, which may be of a relatively long duration, passive immunity is relatively brief, lasting only until the injected immunoglobulin or lymphoid cells have disappeared. Examples of passive immunization include (1) the administration of γ globulin to immunodeficient individuals and (2) the transfer of immunity from mother to young, i.e., antibodies across the placenta or the ingestion of colostrumcontaining antibodies. The source of passively administered antibodies may be human blood donors, immunized humans or other animals, or hybridoma cell lines. No immunological memory is established. Prophylactic immunization  is a procedure to prevent disease through either active immunization or passive immunization. Active immunization usually induces longer lasting protection than does passive immunization. Immunoprophylaxis  describes disease prevention through the use of vaccines to induce active immunization or antisera to induce passive immunization. Diathelic immunization  describes protective immunity induced by injecting antigen into the nipple or teat of a mammary gland. Genetic immunization  consists of the inoculation of plasmid DNA encoding a protein into muscle for the purpose of inducing an adaptive immune response. For reasons yet to be explained, the plasmid DNA is expressed and induces T cell responses and antibody formation to the protein which the DNA encoded.

Vaccines against Viruses Smallpox:  Historically, the intracutaneous inoculation of pus from lesions of smallpox victims into healthy, nonimmune subjects to render them immune to smallpox was known as variolation. I n China, lesional crusts were ground into a powder and inserted into the recipient’s nostrils. These procedures protected some individuals, but often led to life-threatening smallpox infection in others. Edward Jenner’s introduction of vaccination with cowpox to protect against smallpox rendered variolation obsolete. Cowpox  (Figure 25.2) is a bovine virus disease that induces vesicular lesions on the teats. It is of great historical significance in immunology because Edward Jenner observed that milkmaids who had cowpox lesions on their hands failed to develop smallpox. He used this principle in vaccinating humans with the cowpox preparation to produce harmless vesicular lesions at the site of inoculation (vaccination). This stimulated protective immunity against smallpox (variola) because of shared antigens between the vaccinia virus and the variola virus. Vaccinia  refers to a virus termed Poxvirus officinale derived from cowpox and used to induce active immunity against smallpox through vaccination (Figure  25.3). It differs from both cowpox and smallpox viruses in minor antigens. Vaccinia virus:  Refer to vaccinia. Vaccinia gangrenosa:  Chronic progressive vaccinia. Eczema vaccinatum:  On occasion, in subjects receiving smallpox vaccination in the past, the virus in the vaccine superinfected areas of skin affected by atopic dermatitis. This led to generalized vaccinia, which was severe and frequently fatal. It was also referred to as Kaposi’s varicelliform eruption. Progressive vaccinia  describes an adverse reaction to smallpox vaccination in children with primary cell-mediated

Vaccine extraimmunization  refers to the administration of excessive or repeated doses of a vaccine to children or adults, usually as a consequence of poor record-keeping. Hyperimmune  is a descriptor for an animal with a high level of immunity that is induced by repeated immunization of the animal to generate large amounts of functionally effective antibodies, in comparison to animals subjected to routine immunization protocols, perhaps with fewer boosters. Hyperimmunization  is the successive administration of an immunogen to an animal to induce the synthesis of antibody in relatively large amounts. This procedure is followed in the preparation of therapeutic antisera by repeatedly immunizing animals to render them “hyperimmune.”

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Figure 25.2  James Gillray’s cartoon of the cowpox.

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Vaccination

“Take”

Antigens

Delayed skin reaction

Lymphatic system

Scar

Venule Lymph node Release of sensitized lymphocytes into circulation

Figure 25.3  Vaccination against smallpox.

immunodeficiency, such as severe combined immunodeficiency. The vaccination lesion would begin to spread from the site of inoculation and cover extensive areas of the body surface, leading to death. Postvaccinal encephalomyelitis  is a demyelinating encephalomyelitis that occurs approximately 2 weeks following vaccination of infants less than 1 year of age, as well as of adults, with vaccinia virus to protect against smallpox. This rare complication of the smallpox vaccination frequently leads to death. Vaccinia immune globulin  is hyperimmune gammaglobulin used to treat dermal complications of vaccination for smallpox, such as eczema vaccinatum and progressive vaccinia. This is no longer used as smallpox is believed to have been eradicated throughout the world. Chronic progressive vaccinia (vaccinia gangrenosa) (historical):  An unusual sequela of smallpox vaccination in which the lesions produced by vaccinia on the skin became gangrenous and spread from the vaccination site to other areas of the skin. This occurred in children with cell-mediated immunodeficiency. Generalized vaccinia  is a condition observed in some children being vaccinated against smallpox with vaccinia virus. There were numerous vaccinia skin lesions that occurred in these children who had a primary immunodeficiency in antibody synthesis. Although usually self-limited, children who also had atopic dermatitis in addition to the generalized vaccinia often died. Smallpox vaccine  an immunizing preparation prepared from the lymph of cowpox vesicles obtained from healthy vaccinated bovine animals. This vaccine is no longer used as smallpox has been eradicated throughout the world.

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Smallpox vaccination  involves the induction of active immunity against smallpox (variola) by immunization with a related agent, vaccinia virus, obtained from vaccinia vesicles on calf skin. Shared, and therefore cross-reactive, epitopes in the vaccinia virus provide protective immunity against smallpox. This ancient disease has now been eliminated worldwide with the only laboratory stocks maintained in Atlanta (United States) and Moscow (Russia). These stocks are supposed to have been destroyed by both agencies once the virus was sequenced. Smallpox vaccination was first developed by the English physician Edward Jenner, whose method diminished the mortality from 20% to less than 1%. Following application of vaccinia virus by a multiple pressure method, vesicles occur at the site of application within 6 to 9 d. Maximum reactivity is observed by day 12. Initial vaccinations were given to 1- to 2-year-old infants with revaccination after 3 years. Children with cell-mediated immunodeficiency syndromes sometimes developed complications such as generalized vaccinia spreading from the site of inoculation. Postvaccination encephalomyelitis also occurred occasionally in adults and in babies less than 1 year old. The procedure was contraindicated in subjects experiencing immunosuppression due to any cause. Rabies vaccine:  In humans, significant levels of neutralizing antibody can be generated by immunization with a virus grown in tissue culture in diploid human embryo lung cells. A rabies vaccine adapted to chick embryos, especially egg passage material, is used for prophylaxis in animals prior to exposure. The “historical” vaccine originally prepared by Pasteur made use of rabbit spinal cord preparations to which the virus had become adapted. However, they were discontinued because of the risk of inducing postrabies vaccination encephalomyelitis. HEP  is the abbreviation for high egg passage, which signifies multiple passages of rabies virus through eggs to achieve

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attenuation for preparation of a vaccine appropriate for use in immunizing cattle. LEP (low egg passage)  is a type of vaccine for rabies that has been employed for the immunization of dogs and cats. Human diploid cell rabies vaccine (HDCV)  is an inactivated virus vaccine prepared from fixed rabies virus grown in human diploid cell tissue culture. Rabies vaccination:  See postrabies vaccination encephalo­ myelitis. Postrabies vaccination encephalomyelitis  is a demyelinating disease produced in humans actively immunized with rabies vaccine containing nervous system tissue to protect against the development of rabies. Serial injections of rabbit brain tissue containing rabies virus killed by phenol could induce demyelinating encephalomyelitis in the recipient. This method of vaccination was later replaced with a vaccine developed in tissue culture that did not contain any nervous tissue. Yellow fever vaccine  is a lyophilized attenuated vaccine prepared from the 17D strain of liver-attenuated yellow-fever virus grown in chick embryos. A single injection may confer immunity that persists for a decade. Influenza virus vaccine  is a purified and inactivated immunizing preparation made from viruses grown in eggs. It cannot lead to infection. It contains (H1N1) and (H3N2) type A strains and one type B strain. These are the strains considered most likely to cause influenza in the United States. Whole virus and split virus preparations are available. Children tolerate the split virus preparation better than the whole virus vaccine. The influenza virus vaccine is a polyvalent immunizing preparation that contains inactivated antigenic variants of the influenza virus (types A and B) either individually or combined for annual use. It protects against epidemic disease and the morbidity and mortality induced by influenza virus, especially in the aged and chronically ill. The vaccine is reconstituted each year to protect against the strains of influenza virus present in the population.  he influenza virus vaccine is for active immunization against T specific strains of influenza virus. It is highly recommended for anyone six months of age or older who, because of age or underlying medical conditions, is at increased risk of complications from influenza. Healthcare workers and others in close contact with high risk people should be vaccinated. Fluvirin is not indicated in children younger than 4  years of age. FluMist® is indicated in healthy children and adolescents (5–17 years of age) and healthy adults 18–49 years of age. Influenza A viruses are classified into subtypes based on their surface antigens designated hemagglutinin (H) and neuraminidase (N). Immunity against these surface antigens, especially the hemagglutinin, diminishes the likelihood of

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infection and severity of disease if infection occurs. Influenza vaccines are standardized to contain hemagglutinins of strains, typically two type A and one type B, representing the influenza viruses postulated to circulate in the United States in the upcoming influenza season. The vaccine is prepared from highly purified egg-grown viruses made from noninfectious (inactivated) preparations. Subvirion and purified surface-antigen preparations are also available. Since the vaccine viruses are initially in embryonated hens’ eggs, the vaccine could contain minute quantities of residual egg protein. The trivalent influenza vaccine prepared for the 2006– 2007 season contained A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004 antigens. These viruses were used because they were postulated to be representative of the influenza viruses likely to circulate in the United States. during the 2006–2007 influenza season. Influenza vaccine is available in a split-virus preparation, which causes diminished febrile reaction. Thus, the split-virus vaccines are recommended for use in children younger than 13 years of age. Split-virus vaccines may also be referred to as “subvirion” or “purified-surface-antigen” vaccines. FluMist®  contains live attenuated influenza viruses that reproduce in the nasopharynx of the recipient and are present in respiratory secretions. FLU/v  is a newly developed vaccine against influenza that focuses on parts of the virus that do not mutate from year to year. Murine studies show that 57% of mice given the new vaccine survived a lethal dose of the flu compared with none that were given a controlled flu vaccine. This new vaccine purportedly protects against all strains of influenza, including bird flu, permanently. Current vaccines only protect against specific strains for a limited amount of time. Investigators attempt to predict which version will be prominent in the forthcoming flu season. Because of the propensity for the influenza virus to mutate, the vaccine for a particular year may not be as effective as hoped, and new vaccines must be developed each flu season. FLU/v can be produced quicker and more easily than traditional vaccines. Salk vaccine  is an injectable poliomyelitis virus vaccine, killed by formalin that was used for prophylactic immunization against poliomyelitis prior to development of the Sabin oral polio vaccine. Inactivated poliovirus vaccine  is an immunizing preparation prepared from three types of inactivated polioviruses. Also called Salk vaccine. The inactivated poliovirus vaccine (IPV—injection)  is used to actively immunize infants, children and adults for prevention of poliomyelitis induced by poliovirus types 1, 2, and 3. The disease produced by these three types of the virus is spread by the fecal–oral route of transmission, but may also be disseminated by the pharyngeal route. Poliovirus vaccine,

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inactivated, leads to the synthesis of neutralizing antibodies against each of the three types of virus, resulting in protective efficacy and antibody responses in most children. It induces secretory antibody (IgA) in the pharynx and gastrointestinal tract and diminishes pharyngeal excretion of poliovirus type 1 from 75% of children with neutralizing antibodies at levels 1:64. The vaccine may also induce herd immunity in the vaccinated population. Sabin vaccine  is an attenuated live poliomyelitis virus vaccine that is administered orally to induce local immunity in the gut, which is the virus’s natural route of entry, thereby stimulating local as well as systemic immunity against the causative agent of the disease. Provocation poliomyelitis  is an uncommon consequence of attempted immunization against poliomyelitis in which paralysis follows soon after injection of vaccines such as those that contained Bordetella pertussis or alum. Poliomyelitis vaccines:  The three strains of poliomyelitis virus combined into a live attenuated oral poliomyelitis vaccine was first introduced by Sabin. Replication in the gastrointestinal tract stimulates effective local immunity associated with IgA antibody synthesis. Individuals to be immunized receive three oral doses of the vaccine. This largely replaces the Salk vaccine which was introduced in the early 1950s as a vaccine comprised of the three strains of poliovirus that had been killed with formalin. This preparation must be administered subcutaneously. Live oral poliovirus vaccine  is an immunizing preparation prepared from three types of live attenuated polioviruses. An advisory panel to the Centers for Disease Control and Prevention recommended in 1999 that its routine use be discontinued. It contains a live yet weakened virus that has led to eight to ten cases of polio each year. Now that the polio epidemic has been eliminated in the United States, this risk is no longer acceptable. Also called Sabin vaccine. Measles vaccine  is an attenuated virus vaccine administered as a single injection to children at 2 years of age or between 1 and 10 years old. Contraindications include a history of allergy or convulsions. Puppies may be protected against canine distemper in the neonatal period by the administration of attenuated measles virus which represents a heterologous vaccine. Passive immunity from the mother precludes early immunization of puppies with live canine distemper vaccine. Live attenuated measles (rubeola) virus vaccine  is an immunizing preparation that contains live measles virus strains. It is the preferred form except in patients with lymphoma, leukemia, or other generalized malignancies; radiation therapy; pregnancy; active tuberculosis; egg sensitivity; prolonged drug treatment that suppresses the immune response, such as corticosteroids or antimetabolites; or

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administration of gammaglobulin, blood, or plasma. Persons in these groups should be administered immune globulin immediately following exposure. Live measles virus vaccine  is a standardized attenuated virus immunizing preparation used to protect against measles. The measles virus vaccine (live—injection)  is used to immunize against measles, a common childhood disease that is induced by measles virus, which is a paramyxovirus. It can be associated with serious complications, including pneumonia and encephalitis, or death. Subjects first vaccinated with measles virus vaccine at 12 months of age or older require revaccination with measles, mumps, and rubella virus vaccine live before entering elementary school. Revaccination may induce seroconversion. The Advisory Committee on Immunization Practices (ACIP) advises that the first dose of measles–mumps–rubella vaccine live be administered at 12–15 months of age and that the second dose be given at 4–6 years of age. Rubella vaccine  is an attenuated virus vaccine used to immunize girls 10 to 14 years of age. It is used in the MMR combination, or used alone to immunize seronegative women of childbearing age, but it is not to be used during pregnancy. Live rubella virus vaccine  is an attenuated virus immunizing preparation employed to protect against rubella (German measles). All nonpregnant susceptible women of childbearing age should be provided with this vaccine to prevent fetal infection and the congenital rubella syndrome, i.e., possible fetal death, prematurity, impaired hearing, cataract, mental retardation, and other serious consequences. Mumps vaccine  is an attenuated virus vaccine prepared from virus generated in chick embryo cell cultures. It is a live attenuated immunizing preparation employed to prevent mumps. It should be administered under the same guidelines and restrictions that apply to live attenuated measles virus vaccine. Mumps virus vaccine (live—injection)  is for immunization of individuals 12 months of age or older. Not recommended for infants younger than 12 months because of the possible presence of maternal mumps-neutralizing antibodies, which might interfere with the immune response. Mumps is a common childhood disease induced by mumps virus, a paramyxovirus that may lead to such serious complications as aseptic meningitis, deafness, orchitis, and/or even death. As proven in clinical trials, it is highly immunogenic and well tolerated. A single injection can induce mumps-neutralizing antibodies in 95% of susceptible children and 93% of susceptible adults. Even though the antibody level is relatively lower than that following natural infection, it is protective and of long duration. A few (1–5%) individuals receiving the vaccine may fail

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to seroconvert following primary immunization. Protective efficacy of mumps vaccine has been established in controlled field trials. Seroconversion has been shown to parallel protection from the disease. Antibodies appearing following vaccination may be assayed by neutralization, hemagglutination-inhibition, or ELISA techniques. Antibodies are often detectable 11–13 days after primary vaccination. Those vaccinated at 12 months of age or older should be revaccinated with measles–mumps–rubella virus vaccine before admission to elementary school. Live measles and rubella virus vaccine  is a standardized immunizing preparation that contains attenuated measles and rubella viruses. Live measles and mumps virus vaccine  is a standardized immunizing preparation that contains attenuated measles and mumps viruses. Live measles, mumps, and rubella virus vaccine (live—injection)  is used for simultaneous vaccination in individuals at least 12 months of age. These subjects should be revaccinated prior to admission to elementary school. Vaccination of persons exposed to natural measles may afford protection if the vaccine is administered within 72 hours of exposure. If given a few days prior to exposure, greater protection is possible. MMR vaccine  is measles–mumps–rubella vaccine, a live attenuated virus vaccine given at 15 months of age or earlier. A booster injection is given later. The vaccine is effective in stimulating protective immunity in most cases. It might prove ineffective in children younger than 15 months of age if they still have massively transferred antibodies from the mother. This vaccine should not be given to pregnant women, immunodeficient individuals undergoing immunosuppressive therapy, or individuals with acute febrile disease. Varicella (chickenpox) vaccine  is an immunizing preparation comprised of attenuated varicella virus. Hepatitis vaccine  (Figure 25.4) is a vaccine used to actively immunize subjects against hepatitis B virus and contains purified hepatitis B surface antigen. Current practice uses an immunogen prepared by recombinant DNA technology referred to as Recombivax®. The antigen preparation is administered in three sequential intramuscular injections to individuals such as physicians, nurses, and other medical personnel who are at risk. Temporary protection against hepatitis A is induced by the passive administration of pooled normal human serum immunoglobulin which protects against hepatitis A virus for a brief time. Antibody for passive protection against hepatitis must be derived from the blood sera of specifically immune individuals. Hepatitis B vaccine:  Human plasma-derived hepatitis B vaccine (Heptavax-B), which was developed in the 1980s, was

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DNA polymerase

HBsAg

HBcAg Double-stranded circular DNA

Single-stranded DNA HBcAg

Figure 25.4  Hepatitis B virus and its antigens.

unpopular because of the fear of AIDS related to any product for injection derived from human plasma. It was replaced by a recombinant DNA vaccine (Recombivax®) prepared in yeast (Saccharomyces cervesiae). It is very effective in inducing protective antibodies in most recipients. Nonresponders are often successfully immunized by intracutaneous vaccination. An immunizing preparation containing hepatitis B protein antigen produced by genetically engineered yeast. Hepatitis B vaccine (recombinant—injection)  is for immunization against infection caused by all known subtypes of hepatitis B virus, which is one of several hepatitis viruses that produce systemic infection with major pathology in the liver. These include hepatitis A virus, hepatitis D virus, and hepatitis C and E viruses, previously referred to as “non-A, non-B” hepatitis viruses. Hepatocellular carcinoma is a serious complication of hepatitis B virus infection. Reliable studies have linked chronic hepatitis B infection with hepatocellular carcinoma. Eighty percent of primary liver neoplasms are induced by hepatitis B virus infection. The CDC has recognized hepatitis B vaccine as the first anti-cancer vaccine because it can prevent primary liver cancer. Hepatitis A vaccine (inactivated—injection)  is used for active immunization of individuals 12 months of age and older against disease caused by hepatitis A virus (HAV). Primary immunization should be administered at least 2 weeks before expected exposure to HAV. Primary immunization of children and adolescents (12 mos–18 yrs) consists of a single dose of 720 enzyme-linked immunosorbent assay (ELISA) units in 7.5mL and a booster dose (720 EL.U. in 0.5mL) should be administered anytime between 6 and 12 months later. For adults, primary immunization consists of a single dose of 1,440 EL.U. in 1mL, and a booster dose (1,440 EL.U. in 1mL) should be administered anytime between 6 and 12 months later. Hepatitis A inactivated and hepatitis B recombinant vaccine (injection)  is for active immunization of individuals 18 years of age or older against disease caused by hepatitis A

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virus (HAV) and infection by all known subtypes of hepatitis B virus (HBV). Vaccination may not protect 100% of recipients. Hepatitis D (Delta virus) fails to occur in the absence of HVB infection. Thus, vaccination with this vaccine also protects against hepatitis D. Canine parvovirus vaccine:  Initially, a feline enteritis vaccine that was live and attenuated was used based on its crossreactivity with canine parvovirus. Canine parvovirus may have originated from the feline enteritis organism by mutation. This vaccine was later replaced with attenuated canine parvovirus vaccine. The distemper vaccine  is an attenuated canine distemper virus vaccine prepared from virus grown in tissue culture or chick embryos. Newcastle disease vaccines  include: (1) an inactivated virus raised in chick embryos that is incorporated into aluminum hydroxide gel adjuvant, and (2) live virus grown in chick embryos and attenuated in a graded manner. Strains with medium virulence are administered parenterally, and those that are less virulent are given to birds either in drinking water or as an aerosol. Several experimental AIDS vaccines  are under investigation. HIV-2 inoculation into cyomologus monkeys apparently prevented them from developing simian AIDS following injection of the SIV virus. Various problems relate to the successful development of an HIV vaccine to protect against AIDS. There is no precise animal model that can be employed to evaluate a vaccine against HIV. Studies of vaccine efficacy in humans are also problematic. The simian immunodeficiency virus (SIV), a close relative of HIV that infects macaques, produces a disease that closely resembles AIDS in Asian macaques but not in the African variety. This variability of SIV points to the problem in attempting to extrapolate results of vaccine trials in macaques to trials in humans. The requirements to establish immunity against HIV infection are unknown. Unlike infections that induce long-lasting protective immunity, HIV coexists with an intensive immune response. HIV infection leads to a progressive immunodeficiency even though there is variability among individuals in resistance to HIV which could be attributable either to their infection by a mutant HIV or to resistance to HIV infection mediated by their CD8+ T cells. The type of immunity induced is also significant. A CD8+ T cell or TH1 cell immune response would probably be most desirable. AIDS patients usually produce TH2 cell cytokines and their CD4+ T cell response to HIV components is affected by TH1 cytokines. Subunit vaccines have also been attempted but these induce immunity to only some proteins of the virus and, when tested in chimpanzees, have induced immunity specific only for the exact virus strain used to make the vaccine. Such subunit vaccines fail to protect against natural infection. In addition, there are ethical issues involved in AIDS vaccine development.

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HGP-30  is an experimental vaccine for AIDS that employs a synthetic HIV core protein, p17. Human papillomavirus recombinant vaccine (quadrivalent—injection):  Females 9–26 years of age should be vaccinated to prevent disease caused by human papillomavirus (HPV) types 6, 11, 16, and 18. Diseases caused by these virus types include cervical cancer, genital warts (condyloma acuminata), and the following precancerous or dysplastic lesions: cervical adenocarcinoma in situ (AIS), cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia (VAIN), CIN grade 1. Quadrivalent HPV (types 6, 11, 16, and 18) recombinant vaccine should be given intramuscularly as three separate 0.5 mL doses according to the following schedule: first dose at the elected date; second dose two months after first dose; third dose six months after first dose. Refer also to Gardasil®.

Vaccines against Bacteria TAB vaccine  is an immunizing preparation used to protect against enteric fever. It is comprised of Salmonella typhi and S. paratyphi A and B microorganisms that have been killed by heat and preserved with phenol. The bacteria used in the vaccine are in the smooth specific phase. They also contain both O and Vi antigens. The vaccine is administered subcutaneously. Lipopolysaccharide from the Gram-negative bacteria may induce fever in vaccine recipients. If Salmonella paratyphi C is added, the vaccine is referred to as TABC. If tetanus toxoid is added, it is referred to as TABT. Typhoid vaccination:  See TAB vaccine. Typhoid vaccine:  Following ingestion, virulent strains of Salmonella typhi can penetrate the stomach acid barrier, colonize the intestinal tract, pass through the lumen, and gain access to the lymphatic system and bloodstream to produce disease. S. typhi’s ability to induce disease, as well as a protective immune response, depends on the presence of a complete lipopolysaccharide in the microorganisms. Two forms of immunizing preparation are currently in use. One is a live, attenuated S. typhi strain Ty2la that is used as an oral vaccine administered in four doses by adults and children over 6 years of age. It affords protection for 5 years. This vaccine is contraindicated in patients taking antimicrobial drugs and in AIDS patients. A second type of the vaccine, which is for parenteral use, is prepared from the capsular polysaccharide of Salmonella typhi. It is administered to 6-month-old children, divided into two doses spaced 4 weeks apart. It is effective 55 to 75% of the time, and lasts for 3 years. Cholera toxin  is a Vibro cholerae enterotoxin comprised of five B subunits which are cell-binding 11.6-kDa structures encircling a 27-kDa catalase that conveys ADP-ribose to G protein, leading to continual adenyl cyclase activation.

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Vaccines and Immunization

Other toxins that resemble cholera toxin in function include diptheria toxin, exotoxin A, and pertussis toxin. Cholera vaccine  is an immunizing preparation comprised of Vibrio cholerae smooth strains Inaba and Ogawa in addition to El Tor vibrio, which have been killed by heat or formalin treatment. It is designed to induce protective active immunity against cholera in regions where it is endemic, as well as in travelers to those locations. The immunity induced is effective for only about 12 weeks. Plague vaccine:  Yersinia pestis microorganisms killed by heat or formalin are injected intramuscularly to induce immunity against plague. It is administered in three doses 4 weeks or more apart. The duration of the immunity is approximately 6 months. A live attenuated vaccine, used mainly in Java, has also been found to induce protective immunity. Plague vaccine  is an immunizing preparation prepared either from a crude fraction of killed plague microorganisms, Yersinia pestis, or synthetically from recombinant proteins. It is rarely used except in a laboratory or for field workers in regions where plague is endemic. Diphtheria toxin  is a 62-kDa protein exotoxin synthesized and secreted by Corynebacterium diphtheriae. The exotoxin, which is distributed in the blood, induces neuropathy and myocarditis in humans. Tryptic enzymes nick the singlechain diphtheria toxin. Thiols reduce the toxin to produce two fragments. The 40-kDa B fragment gains access to cells through their membranes, permitting the 21-kDa A fragment to enter. Whereas the B fragment is not toxic, the A fragment is toxic and it inactivates elongation factor-2, thereby blocking eukaryocytic protein synthesis. Guinea pigs are especially sensitive to diphtheria toxin, which causes necrosis at injection sites, hemorrhage of the adrenals, and other pathologic consequences. Animal tests developed earlier in the century consisted of intradermal inoculation of C.  diphtheriae suspensions into the skin of guinea pigs that were unprotected, compared to a control guinea pig that had been pretreated with passive administration of diphtheria antitoxin for protection. In later years, toxin generation was demonstrated in vitro by placing filter paper impregnated with antitoxin at right angles to streaks of C. diphtheriae microorganisms growing on media in Petri plates. Formalin treatment or storage converts the labile diphtheria toxin into toxoid. Diphtheria immunization  results from the repeated administration of diphtheria toxoids [as alum precipitated toxoids (APT)]. Toxoid–antitoxin floccules (TAF) are an alternate form for adults who show adverse reaction to APT. Besides this active immunization procedure, diphtheria antitoxin can also be given for passive immunization in the treatment of diphtheria. Diphtheria toxoid  is an immunizing preparation generated by formalin inactivation of Corynebacterium diphtheriae

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exotoxins. This toxoid, which is used in the active immunization of children against diphtheria, is usually administered as a triple vaccine, together with pertussis microorganisms and tetanus toxoid; as purified toxoid which has been absorbed to hydrated aluminum phosphate (PTAP); or as alum-precipitated toxoid (APT). Infants immunized with one of these preparations develop active immunity against diphtheria. Toxoid–antitoxin floccules (TAF) may be administered to adults who demonstrate adverse hypersensitivity reactions to toxoids. Formol toxoid  is a toxoid generated by the treatment of an exotoxin such as diphtheria toxin with formalin. Although first used nearly a century ago, it was subsequently modified to contain an adjuvant such as an aluminum compound to boost immune responsiveness to the toxoid. It was later replaced with the so-called triple vaccine of diphtheria, pertussis, and toxoid vaccine. An immunizing preparation containing toxoid–antitoxin floccules  has been used to induce active immunity against diphtheria in subjects who show adverse reactions to alumprecipitated toxoid. The preparation consists of diphtheria toxoid combined with diphtheria antitoxin in the presence of minimal excess antigen. It has been used in individuals who are hypersensitive to alum-precipitated toxoid alone. Horse serum in the preparation may induce hypersensitivity to horse protein in some subjects. Diphtheria vaccine  is an immunizing preparation to protect against Corynebacterium diphtheriae. See DTaP vaccine. PTAP  is a purified diphtheria toxoid which has been ad­sorbed on hydrated aluminum phosphate. It has been em­ployed to induce active immunity against diphtheria. CRM 197  is a carrier protein used in vaccines. It is a nontoxic mutant protein related to diphtheria toxin. Tetanus antitoxin  is an antibody raised by immunizing horses against Clostridium tetani exotoxin. It is a therapeutic agent to treat or prevent tetanus in individuals with contaminated lesions. Anaphylaxis or serum sickness (type III hypersensitivity) may occur in individuals receiving second injections because of sensitization to horse serum proteins following initial exposure to horse antitoxin. One solution to this has been the use of human antitetanus toxin of high titer. Treatment of the IgG fraction yields (F(ab′)2 fragments which retain all of the toxin-neutralizing capacity but with diminished antigenicity of the antitoxin preparation. Tetanus vaccine  is an immunizing preparation to protect against Clostridium tetani. See DTaP vaccine. A toxoid  is formed by treating a microbial toxin with formaldehyde to inactivate toxicity but leave the immunogenicity (antigenicity) of the preparation intact. Toxoids are prepared

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Problem Seizures Encephalitis Severe brain damage Death

Risk of Occurrence After Vaccination Disease 1:1750 1:25–1:50 1:100,000 1:1000–1:4000 1:310,000 1:2000–1:8000

DPT vaccine  is a discontinued immunizing preparation that consisted of a combination of diphtheria and tetanus toxoids and killed pertussis microorganisms but is no longer used in pediatric immunizations because of the superiority of DTaP vaccine that contains only acellular pertussis microorganisms.

1:1,000,000

DTaP vaccine  (Figure 25.6) is an acellular preparation used for protective immunization that is comprised of diptheria and tetanus toxoids and acellular pertussis proteins that is used to induce protective immunity in children against diptheria, tetanus, and pertussis. Children should receive DTaP vaccine at the ages of 2, 4, 6, and 15 months, with a booster given at 4 to 6 years of age. The tetanus and diptheria toxoids should be repeated at 14 to 16 years of age. The vaccine is contraindicated in individuals who have shown prior allergic reactions to DTaP or in subjects with acute or developing neurologic disease. DTaP vaccine is effective in preventing most cases of the disease it addresses.

1:200–1:1000

Figure 25.5  Pertussis vaccine.

from exotoxins produced in diphtheria and tetanus. These are used to induce protective immunization against adverse effects of the exotoxins in question. Tetanus toxoid  is prepared from formaldehyde-treated toxins of Clostridium tetani. It is an immunizing preparation to protect against tetanus. Individuals with increased likelihood of developing tetanus as a result of a deep penetrating wound with a rusty nail or other contaminated instrument are immunized by subcutaneous inoculation. The preparation is available in both fluid and adsorbed forms. It is included in a mixture with diphtheria toxoid and pertussis vaccine and is known as DTP or triple vaccine. It is employed to routinely to immunize children less than 6 years old. Pertussis vaccine  (Figure 25.5) is a preparation used for prophylactic immunization against whooping cough in children. It consists of virulent Bordetella pertussis microorganisms that have been killed by treatment with formalin. It is administered in conjunction with diphtheria toxoid and tetanus toxoid as a so-called triple vaccine. In addition to stimulating protective immunity against pertussis, the killed Bordetella pertussis microorganisms act as an adjuvant and facilitate antibody production against the diphtheria and tetanus toxoid components in vaccine. Rarely does a hypersensitivity reaction occur. To reduce the toxic effects of the vaccine, an acellular product is now in use. Whooping cough vaccine:  See pertussis vaccine. Diptheria and tetanus toxoids (adsorbed—injection)  is the preparation intended for pediatric use (DT) and is indicated for active immunization against diphtheria and tetanus in infants and children from 2 months of age up to 7 years of age, for whom the use of combined vaccine containing pertussis antigen is contraindicated. The vaccine should be administered intramuscularly. The potency of tetanus and diphtheria toxoids has been determined based on immunogenicity studies, with comparison to a serological correlate of protection (0.01 antitoxin units/mL) established by the Panel on Review of Bacterial Vaccines and Toxoids. Results of the study indicated protective levels of antibody were raised in more than 90% of the study population after primary immunization with both components. Booster injections were effective in 100% of individuals with pre-existing antibody responses.

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Diptheria and tetanus toxoids and acellular pertussis vaccine (adsorbed DTaP—injection)  is intended for active immunization against diphtheria, tetanus, and pertussis simultaneously in infants and children 6 weeks to 7 years of age (prior to 7th birthday). These preparations combine diphtheria and tetanus toxoids with acellular pertussis bacterial vaccine. The acellular pertussis antigens include pertussis toxin (PT), FHA, and pertactin. Immunization with diphtheria and tetanus toxoid is believed to confer protection lasting 10 years. Nevertheless, diphtheria toxoid does not prevent carriage of Corynebacterium diphtheriae in the pharynx or nose, nor on the skin. Protection against pertussis lasts for 4 to 6 years. Serum diphtheria and tetanus antitoxin levels of 0.l IU/mL and higher are considered as protective. Efficacy of the pertussis component does not have a well-established correlate of protection. Triple vaccine  is an immunizing preparation comprised of three components and used to protect infants against diphtheria, pertussis (whooping cough), and tetanus. It is made up of diphtheria toxoid, pertussis vaccine, and tetanus toxoid. The first of four doses is administered between 3 and 6 months of age. The second dose is administered 1 month later, and the third dose is given 6 months after the second. The child receives a booster injection when beginning school (Figure 25.7). BCG (Bacille Calmette-Guérin)  is a Mycobacterium bovis strain maintained for more than 75 years on potato, bile glycerine agar, which preserves the immunogenicity but dissipates the virulence of the microorganism. It has long been used in Europe as a vaccine against tuberculosis, although it never gained popularity in the United States. It has also been used in tumor immunotherapy to nonspecifically activate the immune response in selected tumor-bearing patients, such as those with melanoma or bladder cancer. BCG has been suggested as a possible vector for genes that determine

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Diphtheria, tetanus toxoids, and whole cell pertussis vaccine

Diphtheria, tetanus toxoids and acellular pertussis vaccine

Diphtheria and tetanus toxoid (pediatric)

Tetanus and diphtheria toxoids (adult)

Diphtheria and tetanus toxoids and Hib conjugate and whole cell pertussis vaccines

Synonyms Manufacturers

DTP, DTwP several

DTP, DTaP Connaught, Lederie

DT several

Td several

DTwP-Hib Lederie

Concentration (per 0.5 ml) Diphtheria Tetanus Pertussis

6.5–12.5 Lf u 5–5.5 Lf u 4u

6.6–12.5 Lf u 5–7.6 Lf u none

2 Lf u 2–5 Lf u none

6.7 or 12.5 Lf u 5 Lf u 4u

Hib Packaging

none 5 or 7.5 ml vials

6.7–7.5 Lf u 5 Lf u either 46.8 mcg or 300 HA u none 5 or 7.5 vials

2 months to 1015 M–1) for biotin. Streptavidin is similar in properties to avidin but has a lower affinity for biotin. Many biotin molecules can be coupled to a protein, enabling the biotinylated protein to bind more than one molecule of avidin. If biotinylation is performed under gentle conditions, the biological activity of the protein can be preserved. By covalently linking avidin to different ligands, such as fluorochromes, enzymes, or EM markers, the biotin–avidin system can be employed to study a wide variety of biological structures and processes. This system has proven particularly useful in the detection and localization of antigens, glycoconjugates, and nucleic acids by employing biotinylated antibodies, lectins, or nucleic acid probes. Pancreatic islet cell hormones:  Immunoperoxidase staining of islet cell adenomas with antibodies to insulin, glucagon, somatostatin, and gastrin facilitates definition of their clinical phenotype. Autoradiography  is a method employed to localize radioisotopes in tissues or cells from experimental animals injected with radiolabeled substances. The radioisotopes serve as probes bound to specific DNA or RNA segments. Radioactivity is detected by placing the x-ray or photographic emulsion into contact with the tissue sections or nylon/nitrocellulose membranes in which they are localized to record sites of radioactivity. The technique permits the detection of radioactive substances by analytical methods involving electrophoresis, Southern blotting, and Northern blot hybridization. Intracellular cytokine staining  refers to the use of fluorescent labeled anticytokine antibodies to “stain” permeabilized cells that synthesize the cytokine in question.

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Protein

Anticytokine antibody-coated well

* * *

* * *

Antibody

Unoccupied sites blocked with protein

Addition of cytokine

Figure 28.48  Neuron-specific enolase.

PAS  (1) Abbreviation for periodic acid Schiff stain for polysaccharides. This technique identifies mucopolysaccharide, glycogen, and sialic acid among other chemicals containing 1,2-diol groups. (2) Abbreviation for para-aminosalicylic acid, which is used in the treatment of tuberculosis. Neuron-specific enolase (NSE)  is an enzyme of neurons and neuroendocrine cells, as well as their derived tumors (e.g., oat cell carcinoma of lung), demonstrable by immunoperoxidase staining. NSE also occurs in some neoplasms not derived from neurons or endocrine cells. (Figure 28.48) Enzyme immunoassay (EIA)  is a technique employed to measure immunochemical reactions based on enzyme catalytic properties. The three widely used techniques include a heterogeneous EIA technique, ELISA, and two homogeneous techniques, enzyme-multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA). The enzyme-linked immunosorbent assay (ELISA)  (Figure 28.49) is a binder-ligand immunoassay that employs an enzyme linked to either antiimmunoglobulin or antibody specific for antigen and detects either antibody or antigen. This method is based on the sandwich or double-layer technique, in which an enzyme rather than a fluorochrome is used as the label. In this method, antibody is attached to the surface of plastic tubes, wells, or beads to which the antigen-containing test sample is added. If antibody is being sought in the test sample, then antigen should be attached to the plastic surface. Following antigen–antibody interaction, the enzyme–antiimmunoglobulin conjugate is added. The ELISA test is read by incubating the reactants with an appropriate substrate to yield a colored product that is measured in a spectrophotometer. Alkaline phosphatase and horseradish peroxidase are enzymes that are often employed. ELISA methods have replaced many radioimmunoassays because of their lower cost, safety, speed, and simplicity in performing. EIA  is an abbreviation for enzyme immunoassay.

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Addition of enzyme-conjugated anticytokine antibody development with substrate

Figure 28.49  ELISA.

Enzyme labeling  is a method such as the immunoperoxidase technique that permits detection of antigens or antibodies in tissue sections by chemically conjugating them to an enzyme. Then, by staining the preparation for the enzyme, antigen or antibody molecules can be located. See immunoperoxidase method. Spot ELISA  is an assay that is a variation on standard ELISA. It is used primarily for the detection of immunoglobulin-secreting cells (ISC) or cytokine-secreting cells (CSC), although future applications may include detection of specific hormone-secreting cells. As in standard ELISA, the starting point is a plastic or nitrocellulose vessel coated with antigen or capture antibody. The ISC or CSC of interest is added and then removed, following sufficient incubation time for the cell to secrete its immunoglobulin or cytokine. The secreted product binds locally to the capture protein and is subsequently detected by enzyme-linked antibody. Finally, a substrate that yields an insoluble product is added and the resulting colored precipitate is quantified. EMIT  is an abbreviation for enzyme-multiplied immuno­ assay technique. The enzyme-multiplied immunoassay technique  is an immunoassay used to monitor therapeutic drugs such as antitumor, antiepileptic, antiasthmatic, and metabolites of cocaine and of other agents subject to abuse. It is a one-phase, competitive enzyme-labeled immunoassay. ELISA (enzyme-linked immunosorbent enzyme-linked immunosorbent assay.

assay):  See

Antihistone antibodies  are associated with several autoimmune diseases that include SLE, drug-induced lupus, juvenile RA, and RA. H-1 antibodies are the most common in

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SLE followed by anti-H2B, anti-H2A, anti-H3, and anti-H4, respectively. Antihistone antibodies are usually assayed by the ELISA technique.

or autoradiography can be used if a radioisotope was used to determine where the labeled antibodies were bound to homologous antigen. Also called Western blotting.

Collagen disease/lupus erythematosus diagnostic panel  refers to a battery of serum tests for the diagnosis of collagen vascular disease that yields the most information for the least cost.

Protein blotting:  See immunoblotting.

The ELISPOT assay  is a modification of the enzyme-linked immunosorbent assay (ELISA) which involves the capture of products secreted from cells placed in contact with antigen or antibody fixed to a plastic surface. An enzyme-linked antibody is then used to identify the captured products by cleaving a colorless substrate to yield a colored spot. Western blot (immunoblot)  (Figure  28.50) is a method to identify antibodies against proteins of precise molecular weights. It is widely used as a confirmatory test for HIV-1 antibody following the HIV-1 antibody screen test performed by the ELISA assay. Following separation of proteins by one- or two-dimensional electrophoresis, they are blotted or transferred to a nitrocellulose or nylon membrane followed by exposure to biotinylated or radioisotope-labeled antibody. The antigen under investigation is revealed by either a color reaction or autoradiography, respectively. Immunoblot (Western blot)  refers to the interaction between labeled antibodies and proteins that have been absorbed on nitrocellulose paper. See Western blot. Immunoblotting  is a method to identify antigen(s) by the polyacrylamide gel electrophoresis (PAGE) of a protein mixture containing the antigen. PAGE separates the components according to their electrophoretic mobility. After transfer to a nitrocellulose filter by electroblotting, antibodies labeled with enzyme or radioisotope and which are specific for the antigen in question are incubated with the cellulose membrane. After washing to remove excess antibody that does not bind, substrate can be added if an enzyme was used, Filter paper Nitrocellulose

The Cleveland procedure  is a form of peptide map in which protease-digested protein products, with sodium dodecyl sulfate (SDS) present, are subjected to SDS-PAGE. This produces a characteristic peptide fragment pattern that is typical of the protein substrate and enzyme used. Blot  refers to the transfer of DNA, RNA, or protein molecules from an electrophoretic gel to a nitrocellulose or nylon membrane by osmosis or vacuum, followed by immersing the membrane in a solution containing a complementary, i.e., mirror-image molecule corresponding to the one on the membrane. This is known as a hybridization blot. Southern blotting  (Figure 28.51) is a procedure to identify DNA sequences. Following extraction of DNA from cells, it is digested with restriction endonucleases to cut DNA at precise sites into fragments. This is followed by separation of the DNA segments according to size by electrophoresis in agarose gel, denaturation with sodium hydroxide, and transfer of the single-stranded DNA to a nitrocellulose membrane by blotting. This is followed by hybridization with a 35S- or 32Pradiolabeled probe of complementary DNA. Alternatively, a biotinylated probe may be used. Autoradiography or substrate digestion identifies the location of the DNA fragments that have hybridized with the complementary DNA probe. Specific sequences in cloned and in genomic DNA can be

Filter paper wick Buffer Peptides transferred to nitrocellulose (blot) Add Ab and radiolabelled conjugate

Nitrocellulose

Autoradiograph develop and fix autoradiograph

Figure 28.50  Western blot (immunoblot).

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Southwestern blot  is a method that combines Southern blotting that identifies DNA segments, with Western immunoblotting that characterizes proteins. A protein may be hybridized to a molecule of single-stranded DNA bound to the membrane. Southwestern blotting is helpful in delineating nuclear transcription-related proteins.

Ag band

Gel with separated DNA fragments Capillary action

Capillary action

Gel with separated Ag fragments

Protein separation techniques:  Proteins may be purified using both electrophoresis and chromatography. Individual techniques are discussed separately. See affinity chromato­ graphy; isoelectric focussing; SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Filter paper

Nitrocellulose Filter paper wick Buffer Side view of setup

Figure 28.51  Southern blotting.

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Cells

Rupture in detergents

Isolate RNA

Electrophoretically separate on the basis of size on a denaturing agarose gel Location of gene-specific gene transcript (identify by size)

Size Markers Blot to nitrocellulose filter

Large

Hybridize to singlestranded 32 p labeled probe

Small Gel

RNA

Filter

Autoradiogram

Figure 28.52  Northern blotting.

identified by Southern blotting. Whereas DNA analysis is referred to as Southern blot, RNA analysis is referred to as a Northern blot, and protein analysis is referred to as a Western blot. A Northwestern blot is one in which RNA-protein hybridizations are formed. Northern blotting  (Figure  28.52) is a method to identify specific mRNA molecules. Following denaturation of RNA in a particular preparation with formaldehyde to cause the molecule to unfold and become linear, the material is separated by size through gel electrophoresis and blotted onto a natural cellulose or nylon membrane. This is then exposed to a solution of labeled DNA “probe” of complementary sequence for hybridization. This step is followed by autoradiography. Northern blotting corresponds to a similar method used for DNA fragments which is known as Southern blotting. In-situ hybridization  (Figure 28.53) is a technique to identify specific DNA or RNA segments in cells or tissues or in viral plaques or colonies of microorganisms. DNA in cells or tissue fixed on glass slides must be denatured with formamide before hybridization with a radiolabeled or biotinylated DNA or RNA probe that is complementary to the tissue mRNA

being sought. Proof that the probe has hybridized to its complementary strand in the tissue or cell under study must be by autoradiography or enzyme-labeled probes, depending on the technique being used. Molecular hybridization probe  is a molecule of nucleic acid, which is labeled with a radionuclide or fluorochrome that can reveal the presence of complementary nucleic acid through molecular hybridization such as in situ. FISH (fluorescence in situ hybridization)  is a technique in which whole cells or a chromosomal spread on a microscope slide are exposed to a DNA probe that is fluorescently labeled; subsequent microscopic examination of the whole chromosome or a specific part of it may reveal tumorigenic chromosomal translocations, which are clearly visible by this technique. Multiple probes tagged with separate fluorochromes may be used simultaneously. The polymerase chain reaction (PCR)  (Figure 28.54) is a technique to amplify a small DNA segment beginning with as little as 1 μg. The segment of double-stranded DNA is placed between two oligonucleotide primers through many cycles of amplification. Amplification takes place in a thermal cycler, with one step occurring at a high temperature in the presence Target strands separated and attached to primers 1st cycle

Primers extended to make copies of targets

2nd cycle ad infinitum

Figure 28.53  In-situ hybridization.

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Figure 28.54  Polymerase chain reaction (PCR).

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DNA Sequence DR1

Protein Sequence

CAG CTT AAG TTT GAA TGT CAT TTC TTC AAT 1001

DR2(15 and 16)

c

AGG

G

Glu Leu Lys Phe Glu Cys His Phe Phe Asn

Pro

Trp Vai

1002

Figure 28.55  DNA fingerprinting.

of DNA polymerase that is able to withstand the high temperature. Within a few hours, the original DNA segment is transformed into millions of copies. PCR methodology has been used for multiple purposes, including detection of human immunodeficiency virus 1 (HIV-1), the prenatal diagnosis of sickle cell anemia, and gene rearrangements in lymphoproliferative disorders, among numerous other applications. The technique is used principally to prepare enough DNA for analysis by available DNA methods and is used widely in DNA diagnostic work. PCR has a 99.99% sensitivity. PCR  is an abbreviation for polymerase chain reaction. Reverse transcriptase polymerase chain reaction (RT-PCR)  is a technique employed to amplify RNA sequences. Reverse transcriptase is used to convert an RNA sequence into a cDNA sequence that is amplified by PCR using gene-specific primers. This technique is a variation of the polymerase chain reaction (PCR) employed to amplify a complementary cDNA of a gene of interest. Taq polymerase  or Thermus aquaticus polymerase. A heatresistant DNA polymerase that greatly facilitates use of the polymerase chain reaction to amplify minute quantities of DNA from various sources into a sufficiently large quantity that can be analyzed. DNA fingerprinting  (Figure  28.55) is a method to demonstrate short, tandem-repeated highly specific genomic sequences known as minisatellites. There is only a 1 in 30 billion probability that two persons would have the identical DNA fingerprint. It has greater specificity than restriction fragment length polymorphism (RFLP) analysis. Each individual has a different number of repeats. The insert-free wild-type M13 bacteriophage identifies the hypervariable minisatellites. The sequence of DNA that identifies the differences is confined to two clusters of 15-bp repeats in the protein III gene of the bacteriophage. The specificity of this probe, known as the Jeffries probe, renders it applicable to parentage testing, human genome mapping, and forensic science. RNA may also be split into fragments by an enzymatic digestion followed by electrophoresis. A characteristic pattern for that molecule is produced and aids in identifying it. DNA microarray  is a technique in which a different DNA is placed on a small section of a microchip. The microarray

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is then used to evaluate expression of RNA in normal or neoplastic cells. Dot blot  (Figure 28.56) is a rapid hybridization method to partially quantify a specific RNA or DNA fragment found in a specimen without the need for a Northern or Southern blot. After serially diluting DNA, it is “spotted” on a nylon or nitrocellulose membrane and then denatured with NaOH. It is then exposed to a heat-denatured DNA fragment probe that is believed to be complementary to the nucleic acid fragment whose identity is being sought. The probe is labeled with 32P or 35S. When the two strands are complementary, hybridization takes place. This is detected by autoradiography of the radiolabeled probe. Enzymatic, nonradioactive labels may also be employed. Multilocus probes (MLPs)  (Figure 28.57) are probes used to identify multiple related sequences distributed throughout each person’s genome. Multilocus probes may reveal as many as 20 separate alleles. Because of this multiplicity of alleles, there is only a remote possibility that two unrelated persons would share the same pattern, i.e., about 1 in 30 billion. There is, however, a problem in deciphering the multibanded arrangement of minisatellite RFLPs, as it is difficult to ascertain which bands are allelic. Mutation rates of minisatellite HVRs remain to be demonstrated but are recognized occasionally. Used in resolving cases of disputed parentage. Restriction fragment length polymorphism (RFLP)  refers to genome diversity in DNA from different subjects revealed by restriction map comparisons. It is based on differences in restriction fragment lengths which are determined by

Coat with BSA

Wash add substrate

Determine best concentrations of Ag and Ab Dot varying amounts of Ag on membrane

Incubate with several dilutions of labeled Ab

Figure 28.56  Dot blot.

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M C C AF1 AF2

M C C AF1 AF2

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each chromosome, one from the mother and the other from the father. When the lengths of related alleles on homologous chromosomes are the same, there will be only a single band in the DNA typing pattern. Therefore, the use of an SLP may yield either a single- or double-band result from each individual. Single locus markers such as the pYNH24 probe developed by White may detect loci that are highly polymorphic, exceeding 30 alleles and 95% heterozygosity. SLPs are used in resolving cases of disputed parentage. A λ cloning vector  is a genetically engineered λ phage that can accept foreign DNA and be used as a vector in recombinant DNA studies. Phage DNA is cleaved with restriction endonucleases, and foreign DNA is inserted. Insertion vectors are those with a single site where phage DNA is cleaved and foreign DNA inserted. Substitution or replacement vectors are those with two sites which span a DNA segment that can be excised and replaced with foreign DNA.

Multilocus probes

Single locus probes

Figure 28.57  Multilocus probes (MLPs).

sites of restriction endonuclease cleavage of the DNA molecules. This is revealed by preparing Southern blots using appropriate molecular hybridization probes. Polymorphisms may be demonstrated in exons, introns, flanking sequences, or any DNA sequence. Variations in DNA sequence show Mendelian inheritance. Results are useful in linkage studies and can help to identify defective genes associated with inherited disease. RFLP (restriction fragment length polymorphism)  is a method to identify local DNA sequence variations of humans or other animals that may be revealed by the use of restriction endonucleases. These enzymes cut double-stranded DNA at points where they recognize a very specific oligonucleotide sequence, resulting in DNA fragments of different lengths that are unique to each individual animal or person. The fragments of different sizes are separated by electrophoresis. The technique is useful for a variety of purposes, such as identifying genes associated with neurologic diseases (e.g., myotonic dystrophy) which are inherited as autosomal dominant genes or in documenting chimerism. The fragments may also be used as genetic markers to help identify the inheritance patterns of particular genes. Single locus probes (SLPs)  are probes that hybridize at only one locus. These probes identify a single locus of variable number of tandem repeats (VNTRs) and permit detection of a region of DNA repeats found in the genome only once and located at a unique site on a certain chromosome. Therefore, an individual can have only two alleles that SLPs will identify, as each cell of the body will have two copies of

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Sequence-specific priming (SSP)  is a method that employs a primer with a single mismatch in the 3′-end that cannot be employed efficiently to extend a DNA strand because the enzyme Taq polymerase, during the PCR reaction, and especially in the first PCR cycles which are very critical, does not manifest 3′ -5′ proofreading endonuclease activity to remove the mismatched nucleotide. If primer pairs are designed to have perfectly matched 3′-ends with only a single allele, or a single group of alleles and the PCR reaction is initiated under stringent conditions, a perfectly matched primer pair results in an amplification product, whereas a mismatch at the 3′-end primer pair will not provide any amplification product. A positive result, i.e., amplification, defines the specificity of the DNA sample. In this method, the PCR amplification step provides the basis for identifying polymorphism. The postamplification processing of the sample consists only of a simple agarose gel electrophoresis to detect the presence or absence of amplified product. DNA amplified fragments are visualized by ethidium bromide staining and exposure to UV light. A separate technique detects amplified product by color fluorescence. The primer pairs are selected in such a manner that each allele should have a unique reactivity pattern with the panel of primer pairs employed. Appropriate controls must be maintained. The plaque-forming cell (PFC) assay  (Figure  28.58) is a technique for demonstrating and enumerating cells forming antibodies against a specific antigen. Mice are immunized with sheep red blood cells (SRBC). After a specified period of time, a suspension of splenic cells from the immunized mouse is mixed with antigen (SRBC) and spread on a suitable semisolid gel medium. After or during incubation at 37°C, complement is added. The erythrocytes that have antiSRBC antibody on their surface will be lysed. Circular areas of hemolysis appear in the gel medium. If viewed under a microscope, a single antibody-forming cell can be identified in the center of the lytic area. There are several modifications of this assay, as some antibodies other than IgM may fix

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Plaqueforming cell

PFU  is an abbreviation for plaque-forming unit. An assay of plaques that develop in the hemolytic plaque assay and related techniques.

Hemolytic plaque Top view of Petri dish

Figure 28.58  Plaque-forming cell (PFC) assay.

complement less efficiently. In order to enhance the effects, an antiglobulin antibody called developing antiserum is added to the mixture. The latter technique is called indirect PFC assay. The PFC (plaque-forming cell)  is an in vitro technique in which antibody-synthesizing cells derived from the spleen of an animal immunized with a specific antigen produce antibodies that lyse red blood cells coated with the corresponding antigen in the presence of complement in a gel medium. The reaction bears some resemblance to β hemolysis produced by streptococci on a blood agar plate. When examined microscopically, a single antibody-producing cell can be detected in the center of the plaque-forming unit. Jerne plaque assay  (Figure  28.59) is a technique to identify and enumerate cells synthesizing antibodies. Typically, spleen cells from a mouse immunized against sheep red blood cells are combined with melted agar or agarose in which sheep erythrocytes are suspended. After gentle mixing, the suspension is distributed into Petri plates where it gels. This is followed by incubation at 37°C, after which complement is added to the dish from a pipette. Thus, the sheep erythrocytes (SRBC) surrounding cells secreting IgM antibody against SRBC are lysed by the added complement, producing a clear zone of hemolysis resembling the effect produced by β hemolytic streptococci on blood agar. IgG antibody against sheep erythrocytes can be identified by adding anti-IgG antibody to aid lysis by complement. Whereas modifications of Immunized animal

Plaqueforming cell

Spleen

Agar with spleen cells and sheep RBC Coating agar

Sheep RBC’s

Glass slide

Figure 28.59  Jerne plaque assay.

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Plaques form on glass slide

this method have been used to identify cells producing antibodies against a variety of antigens or haptens conjugated to the sheep red cells, it can also be used to ascertain the immunoglobulin class being secreted. This method is also known as the hemolytic plaque assay.

The reverse plaque method  (Figure 28.60) is a method to identify antibody-secreting cells regardless of their antibody specificity. The antibody-forming cells are suspended in agarose and incubated at 37°C in Petri plates with sheep red cells coated with protein A. Anti-Ig and complement are also present. Cells synthesizing and secreting immunoglobulin become encircled by Ig–anti-Ig complexes and then link to protein A on the erythrocyte surfaces. This leads to hemolytic plaques (zones of lysis). Thus, any class of immunoglobulin can be identified by this technique through the choice of the appropriate antibody. The Cunningham plaque technique  is a modification of the hemolytic plaque assay in which an erythrocyte monolayer between a glass slide and cover slip is used without agar for the procedure. Nylon wool  (Figure 28.61) is a material that has been used to fractionate T and B cells from a mixture of the two based upon the tendency for B cells to adhere to the nylon wool, whereas the T cells pass through. B cells are then eluted from the column. Previously, tissue typing laboratories used this technique to isolate B lymphocytes for MHC class II (B cell) typing. Magnetic beads have replaced nylon wool for lymphocyte T and B cell separation. Microlymphocytotoxicity  (Figure  28.62) is a widely used technique for HLA tissue typing. Lymphocytes are separated from heparinized blood samples by either layering over Ficoll-Hypaque, centrifuging and removing lymphocytes from the interface, or with beads. After appropriate washing, these purified lymphocyte preparations are counted and aliquots dispensed into microtiter plate wells containing predispensed quantities of antibody. When used for human histocompatibility (HLA) testing, antisera in the wells are specific for known HLA antigenic specificities. After incubation of the cells and antisera, rabbit complement is added and the plates are again incubated. The extent of cytotoxicity induced is determined by incubating the cells with trypan blue, which enters dead cells staining them blue but leaves live cells unstained. The plates are read by using an inverted phase contrast microscope. A scoring system from 0 to 8 (where 8 implies >80% of target cells killed) is employed to indicate cytotoxicity. Most of the sera used to date are multispecific, as they are obtained from multiparous females who have been sensitized during pregnancy by HLA antigens determined by their spouse. Monoclonal antibodies are

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Ig from B cell Anti-Ig

Antibody release

+

Complement Complement mediated red cell lysis, plaque formation

B Lymphocyte (Plasma cell) Antibody-coated erythrocyte

Figure 28.60  Reverse plaque method.

being used with increasing frequency in tissue typing. This technique is useful to identify HLA-A, HLA-B, and HLA-C antigens. When purified, B cell preparations and specific antibodies against B cell antigens are employed, HLA-DR and HLA-DQ antigens can be identified. In the cell-mediated lympholysis (CML) test  responder (effector) lymphocytes are cytotoxic for donor (target) lymphocytes after the two are combined in culture (Figure 28.63). Target cells are labeled by incubation with 51Cr at 37°C for 60 min. Following combination of effector and target cells in tissue culture, the release of 51Cr from target cells injured by cytotoxicity represents a measure of cell-mediated lympholysis (CML). The CML assay gives uniform results, is relatively simple to perform, and is rather easily controlled. The effector

Syringe

Nylon wool

B lymphocytes

Separation of T and B lymphocytes (T cells pass through)

cells can result from either in vivo sensitization following organ grafting or can be induced in vitro. Variations in effector to target cell ratios can be employed for quantification. In the mixed lymphocyte reaction (MLR)  lymphocytes from potential donor and recipient are combined in tissue culture (Figure 28.64). Each of these lymphoid cells has the ability to respond by proliferating following stimulation by antigens of the other cell. In the one-way reaction, the donor cells are treated with mitomycin or irradiation to render them incapable of proliferation. Thus, the donor antigens stimulate the untreated responder cells. Antigenic specificities of the stimulator cells that are not present in the responder cells lead to blastogenesis of the responder lymphocytes. This leads to an increase in the synthesis of DNA and cell division. This process is followed by introduction of a measured amount of tritiated thymidine, which is incorporated into the newly synthesized DNA. The mixed-lymphocyte reaction usually measures a proliferative response and not an effector-cellkilling response. The test is important in bone marrow and organ transplantation to evaluate the degree of histoincompatibility between donor and recipient. Both CD4+ and Cd8+ T lymphocytes proliferate and secrete cytokines in the MLR. Also called mixed-lymphocyte culture. Lymphocyte transformation  (Figure 28.65) is an alteration in the morphology of a lymphocyte induced by an antigen, mitogen, or virus interacting with a small, resting lymphocyte. The transformed cell increases in size and amount of cytoplasm. Nucleoli develop in the nucleus, which becomes lighter staining as the cell becomes a blast. Epstein–Barr

Test tube

T lymphocytes

Live cells

Figure 28.61  Nylon wool.

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Live cells

Dead cells

All cells dead

Figure 28.62  Microlymphocytotoxicity.

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added

4–5 days

3 hours

Culture medium containing 51Cr Target cells (chromium-labeled) Stimulating (sensitizing) cells Responding (effector) cells

Figure 28.63  Cell-mediated lympholysis (CML) test.

virus transforms B cells, and the human T cell leukemia virus transforms T cells. The lymphocyte transformation test involves activation of lymphocytes with mitigens, antigen, superantigens, and antibodies to components of cell

Strain A

Strain B

Stimulator lymphocytes from the spleen treat with X-rays or mitomycin C

Responder lymphocytes from the spleen

4-Day incubation in microwell culture

membranes. This leads to their synthesis of proteins that include immunoglobulins, cytokines, and growth factors. The activated lymphocyte enters the cell cycle, synthesizes DNA, and replicates and undergoes metabolic and morphologic changes. The mitogens, phytohemagglutinin and Concanvalin-A, superantigens, anti-CD3, and antigens that are presented by antigen-presenting cells activate T lymphocytes. Antiimmunoglobulin, bacterial lypopolysaccharides, and staphycoccal protein A activate B lymphocytes. The lymphocyte transformation assay is a broadly used in vitro test to evaluate lymphocyte function in patients. The lymphocyte antigen stimulation test  is an assay for the in vitro assessment of impaired cell-mediated immunity. This test is useful to evaluate patients with genetic or acquired immunodeficiencies, bacterial and viral infections, cancer, autoimmune disorders, transplantation-related disorders, antisperm antbodies, or previous exposure to a variety of antigens, allergens, pathogens, and metals/chemicals. Lymphocyte antigen stimulation is assayed by (3H)-thymidine uptake or a flow cytometric assay (based on expression of the activation antigen CD69) with (3H)-thymidine incorporation.

Addition of [3H thymidine] 18–24 hour incubation period Harvest cells from microwell culture into a filter-paper strip

Count [3H] incorporated into the DNA

Figure 28.64  Mixed lymphocyte reaction (MLR).

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Figure 28.65  Lymphocyte transformation.

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Antigen-stimulated culture supernatants can be assessed for cytokine production by EIA. The lymphocyte mitogen stimulation test  is an assay used for the in vitro assessment of cell-mediated immunity in patients with immunodeficiency, autoimmunity, infectious diseases, cancer, and chemical-induced hypersensitivity reactions. Healthy human lymphocytes have receptors for mitogens such as the plant lectin concanavalin-A (Con A), pokeweed mitogen (PWM), Staphylococcus protein A, and chemicals. Lymphocytes respond to these mitogens that stimulate large numbers of lymphocytes, without prior sensitization. In contrast to antigens, mitogens do not require a sensitized host. Mitogens may stimulate both B and T cells, and the inability of lymphocytes to respond to mitogens suggests impaired cell-mediated or humoral immunity. The lymphocyte toxicity assay  is a test to evaluate adverse reactions to drugs, especially anticonvulsants. Incubation with liver microsomes is believed to metabolize the drug to the in vivo metabolite that kills lymphocytes from sensitized patients but not from controls. Lymphocytes derived from nonreactive individuals do not show significant lymphocyte toxicity. Macrophage–monocyte inhibitory factor (MIF)  (Figure  28.66) is a substance synthesized by T lymphocytes in response to immunogenic challenge that inhibits the migration of macrophages. MIF is a 25-kDa lymphokine. Its mechanism of action is by elevating intracellular cAMP, polymerizing microtubules, and stopping macrophage migration. MIF may increase the adhesive properties of macrophages, thereby inhibiting their migration. The two types of the protein MIF include one that is 65-kDa with a pIof 3 to 4 and another that is 25-kDa with a pI of approximately 5. The macrophage migration test  is an in vitro assay of cell-mediated immunity. Macrophages and lymphocytes from the individual to be tested are placed into segments of capillary tubes about the size of microhematocrit tubes No Antigen

Ovalbumin

Toxoid

A

B

C

D

E

F

G

H

I

Figure 28.66  Macrophage–monocyte inhibitory factor (MIF).

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and incubated in tissue culture medium containing the soluble antigen of interest, with maintenance of appropriate controls incubated in the same medium not containing the antigen. Lymphocytes from an animal or human sensitized to the antigen release a lymphokine called migration inhibitory factor that will block migration of macrophages from the end of the tube where the cells form an aggregated mass. The macrophages in the control preparation (that does not contain antigen) will migrate out of the tube into a fan-like pattern. Macrophage functional assays  are tests of macrophage function. (1) Chemotaxis: macrophages are placed in one end of a Boyden chamber and a chemoattractant is added to the other end. Macrophage migration toward the chemoattractant is assayed. (2) Lysis: macrophages acting against radiolabeled tumor cells or bacterial cells in suspension can be measured after suitable incubation by measuring the radioactivity of the supernatant. (3) Phagocytosis: radioactivity of macrophages that have ingested a radiolabeled target can be assayed. Panning  (Figure 28.67) is a technique to isolate lymphocyte subsets through the use of petri plates coated with monoclonal antibodies specific for lymphocyte surface markers. Thus, only lymphocytes bearing the marker being sought bind to the petri plate surface. Immunobeads  (Figure  28.68) are minute plastic spheres with a coating of antigen (or antibody) that may be aggregated or agglutinated in the presence of the homologous antibody. Immunobeads are used also for the isolation of specific cell subpopulations such as the separation of B cells from T cells that is useful in class II MHC typing for tissue transplantation. The nitroblue tetrazolium (NBT) test  (Figure  28.69) is an assay that evaluates the hexose monophosphate shunt in phagocytic cells. The soluble yellow dye, nitroblue tetrazolium, is taken up by neutrophils and monocytes during phagocytosis. In normal neutrophils, the NBT is reduced by enzymes to insoluble, dark blue formazan crystals within the cell. Neutrophils from patients with chronic granulomatous disease are unable to reduce the nitroblue tetrazolium. The ability to reduce NBT to the insoluble deep blue formazan crystals depends on the generation of superoxide in the neutrophil being tested. Flow cytometry  (Figure  28.70) is an analytical technique to phenotype cell populations that requires a special apparatus, termed a flow cytometer, that can detect fluorescence on individual cell in suspension and thereby ascertain the number of cells that express the molecule binding a fluorescent probe. Cell suspensions are incubated with fluorescent-labeled monoclonal antibodies or other probes and the quantity of probe bound by each cell in the population is assayed by passing the cells one at a time through a spectrofluorometer

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Add a mixture of CD4+ and CD8+ T cells CD8+

mAb against CD4

Incubation

CD4+

Discard top layer containing CD8+ CD8+ CD4+

CD8+ CD4+

CD4+ T cells remain in petri plate

Figure 28.67  Panning.

with a laser-generated incident beam. Sample cells flow single file past a narrowly focused excitation light beam that is used to probe the cell properties of interest. As the cells pass the focused excitation light beam, each cell scatters light and may emit fluorescent light, depending on whether or not it is labeled with a fluorochrome or is autofluorescent. Scattered light is measured in both the forward and perpendicular directions relative to the incident beam. The fluorescent emissions of the cell are measured in the perpendicular directions by a photosensitive detector. Measurements of light scatter and fluorescent emission intensities are used to characterize each cell as it is processed. Flow cytometry is a fast, accurate way to measure multiple characteristics of a single cell simultaneously. These objective measurements are made one cell at a time, at routine rates of 500 to 4000 particles per second in a moving fluid stream. A flow cytometry measures relative size (FSC), relative granularity or internal complexity (SSC), and relative fluorescence. Three-color flow cytometry is used to analyze blood cells by size, cytoplasmic granularity, and surface markers labeled with different fluorochromes. Flow cytometry serves as the basis for numerous very different, highly specialized assays. It is a multifactorial analysis

technique and provides the capability for performing many of these assays simultaneously. The neutrophil microbicidal assay  is a test that assesses the capacity of polymorphonuclear neutrophil leukocytes to kill intracellular bacteria. Bright  is an adjective used in flow cytometry to indicate the relative fluorescence intensity of cells being analyzed, with bright designating the greatest intensity and dim representing the lowest intensity of fluorescence. FACS®  is an abbreviation for fluorescence-activated cell sorter. A fluorescence-activated cell sorter (FACS®)  is an instrument that measures the size, granularity, and fluorescence of cells attributable to bound fluorescent antibodies, as individual cells flow in a stream past photodetectors. Single-cell analysis by this method is termed flow cytometry, and the machine used to make these measurements and/or sort cells is termed a flow cytometer or cell sorter. The flow cytometry

Immunobeads Target cell

Specific antibody against target cell

Figure 28.68  Immunobeads.

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Figure 28.69  Nitroblue tetrazolium test (NBT).

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Orange

Immunological Methods and Molecular Techniques

Time Time Time

Data processor

FSC

Green FSC SSC Green

Time Time

Orange Red

Figure 28.70  Flow cytometry is a fast, accurate way to measure multiple characteristics of a single cell simultaneously. These objective measurements are made one cell at a time, at routine rates of 500 to 4000 particles per second in a moving fluid stream. A flow cytometer measures relative size (FSC), relative granularity or internal complexity (SSC), and relative florescence. Use of three-color flow cytometry to analyze blood cells by size, cytoplasmic granularity, and surface markers labeled with different fluorochromes.

fluorescence detectors are connected to computer-controlled electromagnetic deflector plates that are programmed to deposit a cell with a particular fluorescent signal due to bound fluorochrome-tagged antibody of a particular wavelength (color) and intensity to a specific collection tube. Dim  is an adjective used in flow cytometry to indicate the relative fluorescence intensity of cells being analyzed, with dim representing the lowest intensity and bright designating the greatest intensity of fluorescence. Immunophenotyping  is the use of monoclonal antibodies and flow cytometry to reveal cell surface or cytoplasmic antigens that yield information that may reflect clonality and cell lineage classification. This type of data is valuable clinically in aiding the diagnosis of leukemias and lymphomas through the use of a battery of B cell, T cell, and myeloid markers. However, immunophenotyping results must be used only in conjunction with morphologic criteria when reaching a diagnosis of leukemia or lymphoma. Texas red  is a fluorochrome derived from sulforhodamine 101. It is often used as a second label in fluorescence antibody techniques where fluorescein, an apple-green label, is also used. This provides two-color fluorescence. Chemiluminescence  is the conversion of chemical energy into light by an oxidation reaction. A high-energy peroxide intermediate, such as luminol, is produced by the reaction of a precursor substance exposed to peroxide and alkali. The emission of light energy by a chemical reaction may occur during reduction of an unstable intermediate to a stable form. Chemiluminescence measures the oxidative formation of free radicals such as superoxide anion by polymorphonuclear neutrophils and mononuclear phagocytes. Light is released from these cells after they have taken up

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luminol (5-amino-2,3-dihydro-1,4-phthalazinedione). This is a mechanism to measure the respiratory burst in phagocytes. The oxidation of luminol increases intracellular luminescence. Chronic granulomatous disease may be diagnosed by this technique. Chemotactic assays:  The chemotactic properties of various substances can be determined by various methods. The most popular is the Boyden technique. This consists of a chamber separated into two compartments by a Millipore® filter of appropriate porosity, through which cells can migrate actively but not drop passively. The cell preparation is placed in the upper compartment of the chamber, and the assay solution is placed in the lower compartment. The chamber is incubated in air at 37°C for 3 h, after which the filter is removed and the number of cells migrating to the opposite surface of the filter are counted. Phycoerythrin  is an extensively used label for immunofluorescence. This light-gathering plant protein absorbs light efficiently and emits a brilliant red fluorescence. Light scatter  refers to light dispersion in any direction which can be useful in the study of cells by flow cytometry (Figures 28.71 to 28.85). A cell passing through a laser beam both absorbs and scatters light. Fluorochrome staining of cells permits absorbed light to be emitted as fluorescence. Forward angle light scatter permits identification of a cell in flow and determination of its size. If higher-angle light scatter is added, some specific cell populations may also be identified. Light scatter measured at 90° to the laser beam and flow stream yields data on cell granularity or fine structure. Light scatter depends on such factors as cell size and shape, cell orientation in flow, cellular internal structure, laser beam shape and wavelength, and the angle of light collection.

Right angle light detector α cell complexity

Incident Light Source

Forward light detector α cell surface area

Forward scatter—diffracted light Related to cell surface area Detected along axis of incident light in the forward direction Side scatter—reflected and refracted light Related to cell granularity and complexity Detected at 90° to the laser beam

Figure 28.71  Properties of forward scatter light (FSC) and side scatter light (SSC) are measured by observing how light disperses when a laser hits the cell.

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1000

Side Scatter

800

Neutrophils

600 400 Monocytes

200 0

Lymphocytes 0

200 400 600 800 Forward Light Scatter

1000

Figure 28.72  Each dot represents the data from one cell. The bigger the cell, the larger the FSC signal and the farther to the right the dot will appear on the x-axis. The more complex or granular the cell, the larger the SCC signal and the higher it will appear on the y-axis. It is possible to discern lymphocytes, monocytes, and neutrophils in this plot.

FITC

1000 800 600 400 200 0

400

450

500 550 600 Wavelength (nm)

650

700

Figure 28.73  The absorption and emission spectra for the FITC fluorochrome are shown here. The peak absorption is around 488 nm and the peak emission is around 530 nm.

104

CD 19 PE

103 102 101 100

100

101

102 103 CD3 FITC

104

Figure 28.74  The FITC-positive cells fall in the lower right quadrant and PE-positive cells fall in the upper left quadrant. Cells that are positive for both FITC and PE are in the upper right quadrant.

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Limiting dilution  (Figure 28.86) is a method of preparing aliquots that contain single cells through dilution to a point where each aliquot contains only one cell. The apportionment of cells by this method follows the Poissonian distribution, which yields 37% of aliquots without any cells and 63% with one or more cells. This technique can be used to estimate a certain cell’s frequency in a population. For example, it may be employed to approximate the frequency of helper T lymphocytes, cytotoxic T lymphocytes, or B lymphocytes in a lymphoid cell suspension or to isolate cells for cloning in the production of monoclonal antibodies. The prick test  (Figure  28.87) is an assay for immediate (IgE-mediated) hypersensitivity in humans. The epidermal surface of the skin on which drops of diluted antigen (allergen) are placed is pricked by a sterile needle passed through the allergen. The reaction produced is compared with one induced by histamine or another mast-cell secretogogue. The test is convenient, simple, and rapid and produces little discomfort for the patient in comparison with the intradermal test. It may even be used for infants. The patch test  (Figure 28.88) is an assay to determine the cause of skin allergy, especially contact allergic (type IV) hypersensitivity. A small square of cotton, linen, or paper impregnated with the suspected allergen is applied to the skin for 24 to 48 h. The test is read by examining the site 1 to 2 d after applying the patch. The development of redness (erythema), edema, and formation of vesicles constitutes a positive test. The impregnation of tuberculin into a patch was used by Vollmer for a modified tuberculin test. There are multiple chemicals, toxins, and other allergens that may induce allergic contact dermatitis in exposed members of the population. A skin test  is any one of several assays in which a test substance is either injected into the skin or applied to it to determine the host response. Skin tests have long been used to determine host hypersensitivity or immunity to a particular antigen or product of a microorganism. Examples include the tuberculin test, the Schick test, the Dick test, the patch test, the scratch test, etc. The Dick test  (Figure 28.89) is a skin test to signify susceptibility to scarlet fever in subjects lacking protective antibody against the erythrogenic toxin of Streptococcus pyogenes. A minute quantity of diluted erythrogenic toxin is inoculated intra­dermally in the individual to be tested. An area of redness (erythema) occurs at the injection site 6 to 12 h following inoculation of the diluted toxin in individuals who do not have neutralizing antibodies specific for the erythrogenic toxin and who are therefore susceptible to scarlet fever. A heat-inactivated preparation of the same diluted toxin is also injected intradermally in the same individual as a control against nonspecific hypersensitivity to other products of the preparation.

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104

104

103

103

103

102 101

100 100 101 102 103 104 CD3 APC

CD8 PE

104 CD8 PE

CD4 FITC

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102 101 100

100 101 102 103 104 CD3 FITC

102 101 100

100 101 102 103 104 CD3 APC

Figure 28.75  These are three bivariate plots displaying FITC−, PE−, and APC−stained lymphocytes. All CD3+ cells are stained with APC, the CD4+ cells are stained with FITC, and the CD8+ cells are stained with PE.

Waaler-Rose test:  See Rose-Waaler test.

as the tuberculin test, to evaluate whether an individual has cell-mediated immunity against this microorganism.

The tuberculin test  refers to the 24- to 48-h response to intradermal injection of tuberculin. If positive, it signifies delayed-type hypersensitivity (type IV) to tuberculin and implies cell-mediated immunity to Mycobacterium tuberculosis. The intradermal inoculation of tuberculin or of purified protein derivative (PPD) leads to an area of erythema and induration within 24 to 48 h in positive individuals. A positive reaction signifies the presence of cell-mediated immunity to M. tuberculosis as a consequence of past or current exposure to this microorganism. However, it is not a test for the diagnosis of active tuberculosis.

The histoplasmin test  is a skin test analogous to the tuberculin skin test, which determines whether or not an individual manifests delayed-type hypersensitivity (cell-mediated) immunity to Histoplasma capsulatum, the causative agent of histoplasmosis in man. A positive skin test implies an earlier or a current infection with H. capsulatum.

Stormont test:  A double intradermal tuberculin test.

Johnin  is an extract from culture medium in which Mycobacterium johnei is growing. It can be used in a skin test of cattle for the diagnosis of Johne’s disease. Its preparation parallels the extraction of old tuberculin or purified protein derivative (PPD) used in the tuberculin test.

The tine test  is a human tuberculin test that involves the intradermal inoculation of dried, old tuberculin using a fourpointed applicator that introduces the test substance 2 mm below the surface.

Brucellin  is a substance similar to tuberculin, but derived from a culture filtrate of Brucella abortus that is used to test for the presence of delayed-type hypersensitivity to brucella antigens. The test is of questionable value in diagnosis.

The Vollmer test (historical)  is a tuberculin patch test employing gauze treated with tuberculin.

The Schick test  is a test for susceptibility to diphtheria. Standardized diphtheria toxin is adjusted to contain 1/50 MLD in 0.1 ml, which is injected intracutaneously into the subject’s forearm. Development of redness and induration within 24 to 36 h after administration constitutes a positive test if it persists for 4 d or longer. The presence of 1/500 to 1/250 or more of a unit of antitoxin per milliliter of the patient’s blood will result in a negative reaction because of neutralization of the injected toxin. Neither redness nor induration appears if the test is negative. An individual

Coccidioidin  is a Coccidioides immitis culture extract that is used in a skin test for cell-mediated immunity against the microorganism in a manner analogous to the tuberculin skin test. Histoplasmin  is an extract from cultures of Histoplasma capsulatum that is injected intradermally, in the same manner

H2N

NH2 N+1– (CH2)3

Structure

CH2 N+ 1–

CH2

CH3

CH4 CH2

CH4

Bound to DNA

Figure 28.76  DNA content can be quantified by use of the fluorescent dye propidium iodide (PI). The dye intercalates between the base pairs to stain double-stranded nucleic acids. The amount of DNA that PI binds is proportional to the DNA content.

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G0/G1

Counts

200 150 100 50 S

G2/M

0 0

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600 400 FL2-Area

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Figure 28.77  Staining DNA with PI and analyzing the sample by flow cytometry permits the percentage of cells in each phase of the cell cycle to be determined. Longpass 460 500 540

Shortpass 460 500 540

Bandpass 460 500 540

LP 500

SP 500

BP 500/50

Laser

Voltage

Figure 28.78  The specificity of an optical detector for a particular fluorescent dye is optimized by placing a filter in front of the detector which allows a narrow range of wavelengths to pass through the filter.

Time

Voltage

Time

Laser

Time

Figure 28.79  A pulse is created when the particle enters the laser beam and starts to scatter light. The highest point of the pulse occurs when the particle is in the center of the beam, and the maximum amount of scatter is achieved. As the particle leaves the laser, the pulse comes back down to the baseline.

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The Casoni test  is a diagnostic skin test for hydatid disease in humans induced by Echinococcus granulosus infection. In sensitive individuals, a wheal and flare response develops within 30 min following intradermal inoculation of a tapeworm or hydatid cyst fluid extract. This is followed within 24 h by an area of induration produced by this cell-mediated delayed-hypersensitivity reaction. The Heaf test  is a type of tuberculin test in which an automatic multiple puncture device with six needles is used to administer the test material by intradermal inoculation. The multiple needles advance 2 to 3 mm into the skin. Also called tine test. The Montenegro test  is a diagnostic assay for South American leishmaniasis induced by Leishmania brasiliensis. The intracutaneous injection of a polysaccharide antigen derived from the causative agent induces a delayed hypersensitivity response in the patient. They are not usually found in myositis, scleroderma, or Sjögren’s syndrome. Old tuberculin (OT)  is a broth culture, heat-concentrated filtrate of medium in which Mycobacterium tuberculosis microorganisms were grown. It was developed by Robert Koch for use in tuberculin skin tests nearly a century ago.

Voltage

Laser

with a negative test possesses sufficient antitoxin to protect against infection with Corynebacterium diphtheriae, whereas a positive test denotes susceptibility. A control is always carried out in the opposite forearm. For this test, toxin that has been diluted and heated to 70°C for 15 min is injected intracutaneously. Heating destroys the toxin’s ability to induce local tissue injury; however, it does not affect the components of the diphtheria bacilli or of the medium that might evoke an allergic response in the individual. If the size and duration of the reaction at the injection site in the control approximates the reaction in the test arm, the result is negative. If the reaction is at least 50% larger and of longer duration on the test arm compared to the control, the individual is both allergic to the materials in the bacilli or in the medium and susceptible to the toxin. A positive Schick reaction suggests that diphtheria immunization is needed.

OT (historical)  is old tuberculin. Romer reaction (historical):  Romer in 1909 described erythematous swelling following intracutaneous injection of diphtheria toxin in small quantities. The reaction was found to be neutralized by homologous antitoxin. The smallest amount of diphtheria toxin that produced a definite reaction was defined as the MRD or minimal reaction dose. In general, the MRD of a given toxin is equivalent to about 1/250 to 1/500 of the MLD (minimal lethal dose). The Lr is the smallest amount of toxin which, after mixing with one unit of antitoxin, will produce a minimal skin lesion when injected intracutaneously into a guinea pig.

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List-Mode Data SSC 60

FL1 638

FL2 840

Event 2

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Event 3

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FSC Event 1 30

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638

Figure 28.80  Useful information can be obtained with the dot plot by determining the percentages for each population.

Passive cutaneous anaphylaxis (PCA)  is a skin test that involves the in vivo passive transfer of homocytotropic antibodies that mediates type I immediate hypersensitivity (e.g., IgE in man) from a sensitized to a previously nonsensitized individual by intradermally injecting the antibodies, which become anchored to mast cells through their Fc receptors. This is followed hours or even days later by intravenous injection of antigen mixed with a dye such as Evans Blue. Cross-linking of the cell-fixed (e.g., IgE) antibody receptors by the injected antigen induces a type I immediate hypersensitivity reaction in which histamine and other pharmacological mediators of immediate hypersensitivity are released. Vascular permeability factors act on the vessels to permit plasma and dye to leak into the extravascular space, forming a blue area that can be measured with calipers. In humans, this is called the Prausnitz-Küstner (PK) reaction. 3-D Plot

Test dosing  is a method to determine whether or not an individual has type I anaphylactic hypersensitivity to various drugs, e.g., penicillin, or antisera prior to administration. However, the procedure is not without danger, as even a scratch test with highly diluted penicillin preparations in Charging electrode

Sample injection tube Sheath tube Vent tube

Deflection plates

Multiview Plot FL3 FL2 FL1 FSC

Density Plot

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Contour Plot

104

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100

SSC

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Collection tubes

104

Figure 28.81  The different plots can be used to clearly display and analyze populations of interest.

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Figure 28.82  Particles can be isolated after they are passed through the laser by charging the particle. Depending on the charge, the particle will either travel to the left or right sort tube, repelled from or attracted toward the charged plate. All noncharged particles travel to the waste.

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103 CD19 PE

SSC-Height

856

150 100 50 0

R1 0

102 101 100 100

50 100 150 200 250 FSC-Height

Cells labeled with fluorescent Ab

101

102

103

104

CD3 FITC

Figure 28.83  The conventional method for identifying lymphocyte subsets is light-scatter gating. The lymphocytes are gated, markers are set using a two-color isotype control, then subsequent immunofluorescence analyses of the remaining files are completed.

Fluorescence detector Forword-angle light scatter detector

highly sensitized subjects has been known to produce fatal anaphylactic shock. Histamine release assay:  In 4 to 13% of allergic patients, the IgE-mediated release of histamine from basophils depends on cytokines. These anti-IgE-nonreleasers do respond to f-MetLeu-Pro (FMLP), which bypasses the IgE receptor pathway. Histamine-released assays are valuable when skin testing in RAST function suboptimally, especially in urticaria and atopic dermatitis patients in whom only a weak correlation between IgE in disease is apparent. Histamine-release assays may be informative in urticaria, asthma, and atopic dermatitis patients. Rebuck skin window  (Figure 28.90) is a clinical method for assessing chemotaxis used in vivo by making a superficial abrasion of the skin which is then covered with a glass slide. This is removed several hours later, air dried, and stained for leukocyte content.

250

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SSC-Height

A skin window  is a method to observe the sequential changes in types of cells during the development of acute inflammation. Following superficial abrasion of an area of skin, sterile cover slips are applied, removed at specified intervals thereafter, stained, and observed microscopically for the types of cells present. The first cells to appear are polymorphonuclear

150 100 50 0

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Charged deflection plates

Cells sorted

Figure 28.85  Light scatter.

neutrophils which comprise most of the cell population within 3 to 4 h of the induced injury. By contrast, the cover slip removed after 12 h reveals the presence of mononuclear cells such as lymphocytes, plasma cells, and monocytes. The cover slip removed after 24 h reveals predominantly monocytes and macrophages. Also termed a Rebuck window, named for the individual who perfected the method. The radioallergosorbent test (RAST)  (Figure  28.91) is a technique to detect specific IgE antibodies in a patient’s serum. This solid-phase method involves binding of the allergen–antigen complex to an insoluble support such as dextran particles or Sepharose®. The patient’s serum is then passed over the allergen-support complex, which permits specific IgE antibodies in the serum to bind with the allergen. After washing to remove nonreactive protein, radiolabeled antihuman IgE antibody is then placed in contact with the insoluble support where it reacts with the bound IgE antibody. Both the allergen and the anti-IgE antibody must be present in excess for the test to be accurate. The amount of radioactivity on the beads is proportional to the quantity of serum antibody that is allergen specific.

CD3 FITC

Figure 28.84  Unlike traditional methods of light-scatter gating where lymphocyte gate purity and recovery are concerns, TriTEST allows the CD45-positive lymphocyte population to be gated, providing unambiguous identification.

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Laser beam

Radioallergosorbent test:  See RAST. The radioimmunosorbent test (RIST)  (Figure  28.92) is a solid-phase radioimmunoassay to determine the serum

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Lymphocytes from potential donor

2450 cells

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8750 cells

Fraction of Microcultures that did not Respond

Cells 1250 produce cells clones in culture

1

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Figure 28.86  Limiting dilution.

IgE concentration. A standard quantity of radiolabeled IgE is added to the serum sample to be assayed. The mixture is then combined with Sephadex® or Dextran beads coated with antibody to human IgE. Following incubation and washing, the quantity of radiolabeled IgE bound to the beads is measured. The patient’s IgE competes with the radiolabeled IgE or antibody attached to the beads. Therefore, the decrease in labeled IgE attached to the beads compared to a control in which labeled IgE combines with the beads without competition represents the patient’s serum concentration of IgE. The radioallergosorbent test by comparison assays IgE levels reactive with a specific allergen. RIST:  See radioimmunosorbent test. The paper radioimmunosorbent test (PRIST)  (Figure 28.93) is a technique to assay serum IgE levels. It resembles the radioimmunoabsorbent test except that filter paper discs impregnated with antihuman IgE is used in place of Sephadex® discs.

Immunoradiometry  is a radioimmunoassay method in which the antibody rather than the antigen is radiolabeled. The immunoradiometric assay (IRMA)  (Figure 28.94) is a quantitative method to assay certain plasma proteins based on a “sandwich” technique using radiolabeled antibody, rather than radiolabeled hormone competing with hormone from a patient in the radioimmunoassay (RIA). The hook effect  is an artifact that may be seen in IRMA that occurs when a hormone being assayed is in very high concentration. The excess amount cannot be measured by the detector system since it will have obtained a theoretical limit. The diminished counts with the labeled antibody at the elevated hormone concentration yield spuriously low results. Thus, IRMA is not an appropriate method to assay hormones present in relatively high concentrations, such as gastrin, prolactin, or hCG. The hook effect requires measurement of two separate concentrations to establish linearity.

1. Antigens 1–4 dropped on to skin

Control 2. Skin is lightly pricked (Several minutes later) Wheal and flare reaction to antigen 3

1

4

2

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3

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Suspected allergens (1–6) applied to back under occlusive dressing

Figure 28.87  Prick test.

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(48 hours later) Eczematous reaction to allergen 5

Figure 28.88  Patch test.

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Intracutaneous injection Control

Test

cpm

cpm

Test sample

76% = Amount labeled IgE added to test

Labeled IgE

Erythrogenic toxin inoculation causes the skin to redden in 24–48 hours in susceptible individuals

[IgE standard] Labeled IgE

Anti-IgE

IgE to be tested

Figure 28.92  Radioimmunosorbent test (RIST).

Figure 28.89  Dick test.

Control

Filter paper disc

cpm

Test cpm

76% = Amount labeled IgE added to test

Labeled IgE

Test sample

[IgE standard] Labeled IgE

Anti-IgE

IgE to be tested

Figure 28.93  Paper radioimmunosorbent test (PRIST). Figure 28.90  Rebuck skin window.

1.

Add IgE (test sample)

2.

Add anti-IgE (labeled)

Ag covalently bound to cellulose disc *

*

Labeled anti-IgE etc.

* * * ** Anticytokine antibody

Addition of cytokine

IgE

Addition of iodinated monoclonal antibody

Figure 28.91  Radioallergosorbent test (RAST).

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Figure 28.94  Immunoradiometric assay (IRMA).

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% Injected I*HSA in Total Blood Volume

Equilibration 100

Tumor target cells

Catabolic decay

Effector lymphocytes

+

10 Immune elimination

1

2

4 6 8 10 Days after Injection of I*HSA

12

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Tumor growth inhibited

Tumor growth continues

Figure 28.95  Immune elimination.

Figure 28.96  Winn assay.

Immune elimination  (Figure 28.95) refers to accelerated removal of an antigen from the blood circulation following its interactions with specific antibody and elimination of the antigen–antibody complex through the mononuclear phagocyte system. A few days following antigen administration, antibodies appear in the circulation and eliminate the antigen at a much more rapid rate than occurs in nonimmune individuals. Splenic and liver macrophages express Fc receptors that bind antigen–antibody complexes as well as complement receptors which bind those immune complexes that have already fixed complement. This is followed by removal of immune complexes through the phagocytic action of mononuclear phagocytes. Immune elimination also describes an assay to evaluate the antibody response by monitoring the rate at which a radiolabeled antigen is eliminated in an animal with specific (homologous) antibodies in the circulation.

The reverse genetics approach  is used to determine a gene’s role in a particular disease by investigating a diseased animal’s genome and identifying sustained mutations in its gene.

Immune clearance:  See immune elimination. The Winn assay  (Figure 28.96) is a method to determine the ability of lymphoid cells to inhibit the growth of transplantable tumors in vivo. Following incubation of the lymphoid cells and tumor cells in vivo, the mixture is injected into the skin of X-irradiated mice. Growth of the transplanted cells is followed. T lymphocytes that are specifically immune to the tumor cells will inhibit tumor growth and provide information related to tumor immunity. The forward genetics approach  is a method of mutating a gene in an experimental animal to prove that gene’s role in a particular disease and determining whether or not that mutation produces the disease of interest.

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Strain  refers to genetically identical animals such as mice or rats used in medical research. A transgenic animal  is an animal into whose genome a foreign gene has been introduced. Introduction of the exogenous gene into a mouse can be by either microinjection into a pronucleus of an egg that has been fertilized recently or through retroviruses. The egg that has received the foreign gene is transferred to the oviduct of a pseudopregnant female. If the gene becomes integrated into a chromosome, it is passed on to the progeny through the germ line and will be expressed in all cells. Natural transcriptional promoters and enhancers, or exogenous regulatory elements engineered into it may control expression of a transgene. Transgenes  are genes that are artificially and deliberately introduced in the germ line and are foreign. Foreign gene:  See transgenic mice. A knock-in transgene  is a transgene with a specific mutation that is flanked by sequences that are homologous to the endogenous locus, thereby ensuring that the transgene integrates in its natural position. A dominant negative transgene  is a transgene whose product is a protein that is not functional and disrupts the endo­ genous protein’s function or expression.

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Isolate fertilized eggs

Gene injected into Termination pronucleus Promoter sequences Enhancer

Eggs are reimplanted into pseudopregnant female

Gene

and therefore inherited in Mendelian fashion by succeeding generations. Cloned gene fragment

Study of hybrid gene expression occurs with litters of mated founder animals

Figure 28.97  Transgenic mice.

Transgenic  is a term that describes an organism that has had DNA from another organism put into its genome through recombinant DNA techniques. These animals are usually made by microinjection of DNA into the pronucleus of fertilized eggs, with the DNA integrating at random. Transgenic mice  (Figure  28.97) carry a foreign gene that has been artificially and deliberately introduced into their germ line. The added genes are termed transgenes. Fertilized egg pronuclei receive microinjections of linearized DNA. These are placed in pseudopregnant female oviducts and development proceeds. About one-fourth of the mice that develop following injection of several hundred gene copies into pronuclei are transgenic mice. Transgenic mice have been used to study genes not usually expressed in vivo and alterations in genes that are developmentally regulated to express normal genes and cells where they are not usually expressed. Transgenic mice are also used to delete certain populations of cells with transgenes that encode toxic proteins. They are highly significant in immunologic research. A transgenic mouse  is a mouse developed from an embryo into which foreign genes were transferred. Transgenic mice have provided much valuable information related to immunological tolerance, autoimmune phenomena, oncogenesis, developmental biology, and related topics. The transgene has been introduced and stably incorporated into germ-line cells ensuring that it can be passed on to the progeny. A specific DNA sequence is injected in to the pronuclei of fertilized mouse eggs. Trangenes insert randomly at chromosomal breakpoints and are inherited as simple Mendelian traits. Studies with transgenic mice have yielded much data about cytokines, cell surface molecules, and intracellular signaling molecules. Transgenic organisms  are animals or plants into which foreign genes that encode specific proteins have been inserted. However, controlling the site of gene insertion has not been accomplished yet. Insertion into some positions may even lead to activation of the host’s own structural genes. Transgenic line  refers to a transgenic mouse strain in which the transgene is stably integrated into the germ line

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Transgenics  refers to the transfer of needed genes into an organism for the purpose of providing a missing protein which these genes encode. A germ-free animal  is one such as a laboratory mouse, raised under sterile conditions, where it is free from exposure to microorganisms and is not exposed to larger organisms. Germ-free animals have decreased serum immunoglobulin and lymphoid tissues that are not fully developed. The diet may also be controlled to avoid exposure to food antigens. Most difficult is the ability to maintain a virus-free environment for these animals. Gene knockout  is laboratory jargon for gene disruption by homologous recombination. It may refer to a cell or animal in which a specific gene’s function has been purposely eliminated by replacing the normal gene with an inactive mutant gene. A knockout mouse  is a transgenic mouse in which a mutant allele or disrupted form of the gene replaces a normal gene, leading to the mouse’s failure to produce a functional gene product. Much has been learned about the role of cytokines, cell surface receptors, signaling molecules, and transcription factors in the immune system using knockout mice. A conditional knockout mouse  involves the rendering of a gene of interest nonfunctional, i.e., “knocked out,” exclusively at a precise stage of development in a mutant mouse, or in a designated cell or tissue following induction by a specific stimulus. Knockout gene  is a descriptor for the generation of a mutant organism in which the function of a particular gene has been completely eliminated (a null allele). To successfully knock out a gene, cloned and sequenced genomic DNA and a suitable embryonic (ES) cell line are necessary. A sequence insertion targeting approach may be used. The advantage of an insertion vector is that the frequency of integration is nine-fold higher than with an equal-length replacement-type vector. Homologous recombination techniques can be used to achieve targeted disruption of one or more genes in mice. Knockout mice deprived of functional genes that encode cytokines, cell surface receptors, signaling molecules, and transcription factors are critical for contemporary immunologic research. Genetic knockout  is a technique to introduce precise genetic lesions into the mouse genome to cause “gene disruption” and generate an animal model with a specific genetic defect. Specific defects may be introduced into any murine gene by permitting investigation of this alteration in vivo. Technological advances that have made this possible include using homologous recombination to introduce defined

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changes into the murine genome, and the reintroduction of genetically altered embryonic stem cells into the murine germ line to produce mutant mouse strains. RAG blastocyst complementation  is a method employed to test a gene’s function when its null mutation in the body is embryonic lethal. RAG mice are bereft of both T and B cells. The introduction of embryonic stem cells from a particular mutant whose RAG genes are intact into the eight-cell stage blastocyst of a developing embryo that is RAG -/-, results in new cells that are absorbed by the embryo, which proceeds with normal development following implantation. Different regions of the chimeric embryo are the source of different tissues, leading to some viable reconstituted mice that possess a lymphoid system expressing the specific mutation. This technique permits determination of the effect of the mutation on lymphocyte development and function. Outbreeding  refers to mating of subjects who showed greater genetic differences between themselves than randomly chosen individuals of a population. This process encourages genetic diversity. It is in contrast to inbreeding and random breeding. Isoelectric focusing (IEF)  is an electrophoretic method to fractionate amphoteric molecules, especially proteins, according to their distribution in a pH gradient in an electric field created across the gradient. Molecular distribution is according to isoelectric pH values. The anode repels proteins that are positively charged and the cathode repels proteins that are negatively charged. Thus, each protein migrates in the pH gradient and bands at a position where the gradient pH is equivalent to the isoelectric pH of the protein. A chromatographic column is used to prepare a pH gradient by the electrolysis of amphoteric substances. A density gradient or a gel is used to stabilize the pH gradient. Proteins or peptides focus into distinct bands at that part of the gradient that is equivalent to their isoelectric point. Isoelectric focusing is a technique that permits the separation of protein substances on the basis of their isoelectric characteristics. Thus, this technique can be employed to define heterogeneous antibodies. It may also be employed to purify homogeneous immunoglobulins from heterogeneous pools of antibody. IEF  is an abbreviation for isoelectric focusing. Spectrotype:  In isoelectric focusing analysis, the arrangement of bands in a gel that is characteristic for either one protein or a category of proteins. An antibody spectrotype on an isoelectric focusing gel may signify that it is the product of a particular antibody-synthesizing clone. Isoelectric point (pI)  is the pH at which a molecule has no charge, as the number of positive and negative charges are equal. At the isoelectric pH, the molecule does not migrate in an electric field. The solubility of most substances is minimal at their isoelectric point.

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Viability techniques  are methods employed to determine the viability of cells maintained in vitro. Of the many dyes that have been employed for viability assays, the anionic trypan blue is the one most frequently used. For example, the viability of leukocytes suspended in balanced salt solution is evaluated for their ability to exclude trypan blue form the cell interior. Leukocytes are counted on a hemacytometer and the percentage of viability is calculated by dividing the number of unstained viable cells by the number of all cells counted (stained and unstained). Dye exclusion test  is an assay for the viability of cells in vitro. Vital dyes such as eosin and trypan blue are excluded by living cells; however, the loss of cell membrane integrity by dead cells admits the dye that stains the cell. The dye exclusion principle is used in the microlymphocytotoxicity test employed for HLA typing in organ transplantation. A dye test  is an assay to determine whether an individual has become infected with Toxoplasma. Antibody in an infected patient’s serum prevents living toxoplasma organisms, obtained from an infected mouse’s peritoneum, from taking up methylene blue. Therefore, the organisms do not stain blue if antitoxoplasma antibody is present in the serum. Immunomagnetic technique:  The use of magnetic microspheres to sort, isolate, or identify cells with specific antigenic determinants. Immunonephelometry  is a test that measures light that is scattered at a 90° angle to a laser or light source as it is passed through a suspension of minute complexes of antigen and antibody. Measurement is made at 340 to 360 nm using a spectrophotometer. Plaque-forming cells  are the antibody-producing cells in the center of areas of hemolysis observed microscopically when reading a hemolytic plaque assay. The antibodies they form are specific for red blood cells suspended in the gel medium surrounding them. Once complement is added, the antibody-coated erythrocytes lyse, producing clear areas of hemolysis surrounding the antibody-forming cell. The antibody produced may be specific not only for red blood cell surface antigens but also for soluble antigens deliberately coated on their surfaces for assay purposes. Plaque-forming assay:  See hemolytic plaque assay. The plaque-forming cell (PFC) assay  is a technique for demonstrating and enumerating cells forming antibodies against a specific antigen. Mice are immunized with sheep red blood cells (SRBC). After a specified period of time, a suspension of splenic cells from the immunized mouse is mixed with antigen (SRBC) and spread on a suitable semisolid gel medium. After or during incubation at 37°C, complement is added. The erythrocytes that have anti-SRBC antibody on their surface will be lysed. Circular areas of hemolysis

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appear in the gel medium. If viewed under a microscope, a single antibody-forming cell can be identified in the center of the lytic area. There are several modifications of this assay, since some antibodies other than IgM may fix complement less efficiently. In order to enhance the effects, an antiglobulin antibody called developing antiserum is added to the mixture. The latter technique is called indirect PFC assay. Plaque technique:  See hemolytic plaque assay or phage neutralization assay. Paternity testing  refers to tests performed to ascertain the biological (genetic) parentage of a child. In the past, these have included erythrocyte enzymes, red blood cell antigens, HLA antigens, immunoglobulin allotypes, nonimmunoglobulin serum proteins, and more recently, DNA “fingerprinting” (typing). The demonstration of a genetic marker in a child that is not present in either the father or mother or in cases where none of the paternal antigens are present in the child is enough evidence for direct exclusion of paternity. Another case of direct exclusion of paternity is the failure of a child to express a gene found in both the mother and putative father. When a child expresses a gene that only the man can transmit and which the putative father does not express, this is evidence for indirect exclusion of paternity. When a child is homozygous for a marker not present in the mother or putative father, or if the parent is homozygous for a marker not found in the child, then paternity can be excluded as an indirect exclusion. Also called identity testing. Identity testing:  See paternity testing. A virus neutralization test  is an assay based upon the ability of a specific antibody to neutralize virus infectivity. This assay can be employed to measure the titer of antiviral antibody. This test may be performed in vivo using susceptible animals or chick embryos or it may be done in vitro in tissue culture. Bacteriophage neutralization test:  See phage neutralization assay. The phage neutralization assay  is a laboratory test in which bacteriophage is combined with antibodies specific for it to diminish its capacity to infect a host bacterium. This neutralization of infectivity may be quantified by showing the decreased numbers of plaques produced when the phage, which has been incubated with specific antibody, is plated on appropriate bacteria. The technique is sensitive and can demonstrate even weak antibody activity. The neutralization test  is an assay based on the ability of antibody to inactivate the biological effects of an antigen or of a microorganism expressing it. Neutralization applies especially to inactivation of virus infectivity or of the biological activity of a microbial toxin.

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Buffy coat  refers to the white cell layer that forms between the red cells and plasma when anticoagulated blood is centrifuged. Cell separation methods:  Cell separation techniques were first based, in the early 1960s, on differences such as cell size and density. Subsequently, membrane receptors or surface antigens were found to be differently expressed by lymphocyte subsets. Currently, the most popular lymphocyte separation techniques include immunoselection procedures that employ monoclonal antibodies. The two methods used for lymphocyte separation based on physical differences include sedimentation separation and density gradient separation. Other methods are based on functional properties of the cell such as adhesive or phagocytic properties. Selected mononuclear cell types can adhere to plastic surfaces or to nylon wool. Other techniques employ selective depletion of cells such as lymphocytes undergoing proliferation. Mitogens in culture can be employed to select given lymphocyte populations based on their ability to respond to these stimulants. Rosetting techniques permit the detection or purification of cells expressing a certain surface receptor for antigen. Immunoselection techniques employ either monoclonal or polyclonal antibodies specific for surface antigens on lymphocyte subsets. Immunotoxicity procedures can be used to induce selective cytolysis of cells expressing a certain antigen at the cell surface by reacting the cell with antibodies. Immunoadhesion procedures make use of antibodies against cell surface antigens bound to a solid support that permits the capture of cells by adherence to the support. Immunomagnetic beads to which antibodies have been attached may also be used. Magnetic cell sorting is based on the use of monoclonal antibodies or lectins that bind specifically to surface antigen/ receptor expressed by a certain cell subset. Flow cytometry is a precise and objective method to quantify the number of cells expressing a given surface marker and the extent to which the marker is expressed. Cell surface molecule immunoprecipitation  is a technique to analyze cell surface molecules with monoclonal antibodies and antisera. Immunoprecipitation is based on solubilization of membrane proteins by the use of nonionic detergents; subsequent interaction of specific antibody with solubilized membrane antigen; and recovery of antibody–antigen complexes by binding to an insoluble support which permits washing procedures to remove unbound molecules. Analysis of immunoprecipitates can be accomplished by SDS-page or isoelectric focusing (IEF). Chromium release assay:  The release of chromium (51Cr) from labeled target cells following their interaction with cytotoxic T lymphocytes or antibody and K cells (ADCC) or NK cells. The test measures cell death, which is reflected by the amount of radiolabel released according to the number of cells killed. Cell line  refers to cultured neoplastic cells or normal cells that have been transformed by chemicals or viruses.

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Immunological Methods and Molecular Techniques

Transformed cell lines may be immortal, enabling them to be propagated indefinitely in culture. Cryopreservation  is the technique of freezing tissue or cells or other biological materials to remain genetically stable and metabolically inert. Cryopreservation may involve freezers (–80°C), or preservation with dry ice (–79°C) or liquid nitrogen (–196°C). Ficoll  is a 400-kDa water-soluble polymer comprised of sucrose and epichlorohydrin. It is employed in the manufacture of Ficoll-Hypaque, a density gradient substance used to separate and purify mononuclear cells by centrifugation following removal of the buffy coat. Ficoll-Hypaque  is a density gradient medium used to separate and purify mononuclear cells by centrifugation. Footprints  refer to macrophages filled with Mycobacterium leprae without caseation necrosis. A similar situation may be observed in anergic Hodgkin’s disease patients and in AIDS patients infected with M. avium-intercellulare. Heterokaryon  refers to the formation of a hybrid cell by fusion of two or more separate cells that are not genetically identical, leading to a cell with two nuclei and a single cytoplasm. Cell fusion may be accomplished through the use of polyethylene glycol or ultraviolet light-inactivated Sendai virus. A hybrid cell  is a cell produced when two cells fuse and their two nuclei fuse to form a heterokaryon. Although hybrid cell lines can be established from clones of hybrid cells, they lack stability and delete chromosomes, which is nevertheless useful for gene mapping. Hybrid cell lines can be isolated by using HAT as a selective medium. T cell hybridomas:  The immortalization of normal T lymphocytes by fusion with continuously replicating tumor cells. Fusion randomly immortalizes T lymphocytes regardless of their antigen specificity and genetic restrictions to form a T cell hybridoma. This represents one of two methods to isolate and propagate T cell lines in clones of defined specificity. The other technique is to span clones of normal immune T lymphocytes stimulated with appropriate antigens and antigen-presenting cells. The hybridoma technique holds the advantage over T cell cloning in the relative ease in securing large numbers of T cells of interest and their biologically active products. Lymphokines and other regulatory molecules together with their nRNA and DNA represent T cell hybridoma products. This technology has also facilitated evaluation of T cell receptors and their antigen recognition mechanisms. The adoptive (passive) transfer of autoantigen-specific T cell hybridomas in mice can induce autoimmune diseases. Immune cell cryopreservation  is accomplished by using glycerol and dimethylsufoxide (DMSO) to cryopreserve bone

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marrow for transplantation. It first involved freezing at a constant rate of 1°C min–1 to –79°C, with storage for a 6-month period. Dye exclusion was used to test cells for viability post thaw. Lymphocytes have been cryopreserved for in vitro studies using 15% DMSO and storing in liquid nitrogen (–196°C) for 3 months followed by an assay for viability. Immunocytoadherence  is a method to detect cells with surface immunoglobulin, either synthesized or attached through Fc receptors. Red blood cells coated with the homologous antigen are mixed with the immunoglobulin-bearing cells and result in rosette formation. A laboratory assay employed to identify antibody-bearing cells by the formation of rosettes comprised of red blood cells and antibody-bearing cells. Leukocyte culture:  Whereas mononuclear blood cells have been cultured in vitro in a medium containing serum to support growth in the past, culture of cells to be used for patient reinfusion must be grown in media that is free of serum, endotoxin, and antibiotics. Tissue culture vessels for the large-scale expansion of leukocytes include polystyrene flasks that can be stacked on top of one another in an incubator. Gas-permeable cell-culture bags, 30 of which contain 1500 ml of cell culture each, may be placed in an incubator; tissue culture bioreactors include the hollow fiber cell culture bioreactors and the rotary cell culture system, both of which provide a 3-dimensional growth environment. Clinical applications of leukocyte culture include (1) generation of LAK cells and TILs for adoptive immunotherapy; (2) CD34+ cell culture and in vitro generation of hematopoietic precursors for bone marrow reconstitution; and (3) culture of dendritic cells for use in active immunization. Antisperm antibody  is an antibody specific for any one of several sperm constituents. Antisperm agglutinating antibodies are detected in blood serum by the Kibrick sperm agglutination test, which uses donor sperm. Sperm-immobilizing antibodies are detected by the Isojima test. The subject’s serum is incubated with donor sperm, and motility is examined. Testing for antibodies is of interest to couples with infertility problems. Treatment with relatively small doses of prednisone is sometimes useful in improving the situation by diminishing antisperm antibody titers. One-half of infertile females manifest IgG or IgA sperm-immobilizing antibodies which affect the tail of the spermatozoa. By contrast, IgM antisperm head agglutinating antibodies may occur in homosexual males. Plasmapheresis  is a technique in which blood is withdrawn from an individual, the desired constituent is separated by centrifugation, and the cells are reinjected into the patient. Thus, plasma components may be removed from the circulation of an individual by this method. The technique is also useful to obtain large amounts of antibodies from the plasma of an experimental animal.  lasmapheresis is used therapeutically to rid the body of P toxins or autoantibodies in the blood circulation. Blood

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taken from the patient is centrifuged, the cells are saved, and the plasma is removed. Cells are resuspended in albumin, fresh normal plasma, or albumin in saline, and returned to the patient. The ill effects of a toxin or of an autoantibody may be reduced by 65% by removing approximately 2500 ml of plasma. Removal of twice this amount of plasma may diminish the level of a toxin or of an autoantibody by an additional 20%. This procedure has been used to treat patients with myasthenia gravis, Eaton-Lambert syndrome, Goodpasture syndrome, hyperviscosity syndrome, posttransfusion purpura, and acute Guillain-Barré syndrome.

In situ transcription  is a method in which mRNA acts as a template for complementary DNA for reverse transcription in tissues that have been fixed.

Acridine orange  is a fluorescent substance that binds nonspecifically to RNA with red fluorescence and to DNA with green fluorescence. It also interacts with polysaccharides, proteins, and glycosaminoglycans. It is a nonspecific tissue stain that identifies increased mitoses and shows greater sensitivity but less specificity than the Gram stain. It is carcinogenic and of limited use in routine histology.

A plasmid  is an extrachromosomal genetic structure that consists of a circular, double-stranded DNA molecule which permits the host bacterial cell to resist antibiotics and produce other effects that favor its survival. Plasmid replication is independent of the bacterial chromosome. Plasmids have been used widely in recombinant DNA technology.

Cloned DNA  is a DNA fragment or gene introduced into a vector and replicated in eukaryotic cells or bacteria. Concatamer integration  occurs when the entire genome of vector including the bacterial plasmid is integrated into the host genome. Electroporation  is a technique to insert molecules into cells through use of brief high-voltage electric pulses. It can be used to insert DNA into animal cells. The electrical discharge produces tiny pores that are nanometers in diameter in the plasma membrane. These pores admit supercoiled or linear DNA. The Feulgen reaction  is a standard method that detects DNA in tissues. Methyl green pyronin stain  is a stain used in histology or histopathology that renders DNA green and RNA red. It has been widely used to demonstrate plasma cells and lymphoblasts that contain multiple ribosomes containing RNA in their cytoplasm. Footprinting  is a method to ascertain the DNA segment (or segments) that binds to a protein. Radiolabeled doublestranded DNA is combined with the binding protein to yield a complex that is exposed to an endonuclease that cuts the molecules once and at random. The digested DNA is electrophoresed in polyacrylamide gel together with a control DNA sample (which has been treated similarly, but without added protein) to permit separation of fragments differing in length by one nucleotide. Autoradiography of the material reveals a series of bands representing the DNA fragments. In the area of protein binding, the DNA is spared from digestion, and no corresponding bands appear compared to the control. The protected area’s specific location can be ascertained by running a DNA sequencing gel in parallel.

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Minisatellite  refers to DNA regions comprised of tandem repeats of DNA short sequences. Nick translation  is a technique used to make a radioactive probe of a DNA segment. Nick translation signifies the movement of a nick, i.e., single-stranded break in the doublestranded helix, along a duplex DNA molecule.

Protoplast fusion  is a technique for DNA transfer from one group of bacteria to others, to myeloma cells, or other animal cells in culture. The exposure of plasmid-bearing Escherichia coli microorganisms to lysozyme and EDTA yields protoplasts that may be fused with myeloma cells by polyethylene glycol treatment. Recombinant DNA technology  is the technique of isolating genes from one organism and purifying and reproducing them in another organism. This is often accomplished through ligation of genomic or cDNA into a plasmid or viral vector where replication of DNA takes place. RNAse protection assay  is a technique to detect and quantify messenger RNA (mRNA) copies of specific genes based on nRNA hybridization to radiolabeled RNA probes followed by digestion of the unhybridized RNA with RNAse. Doublestranded RNA duplexes formed as a result of the hybridization are resistant to RNAse degradation. Their size depends on the probe length. Gel electrophoresis is used with their separation. Radioautography is employed for their detection and quantification. Slot blot analysis  is a quick technique to detect gene amplification by determining a solution’s DNA content by electrophoresis. The technique is closely similar to dot blot analysis, except that a slot instead of a punched-out hole is cut in the agar. The TUNEL assay (TdT-dependent dUTP-biotin nick end labeling)  is a technique that identifies apoptotic cells in situ by fragmentation of their DNA. Immunohistochemical staining with enzyme-linked streptavidin identifies biotin-tagged dUTP added to the free 3′ ends of the DNA fragments by the enzyme TdT. In TUNEL-based assays:  DNA fragments can be stained in situ by using terminal deoxynucleotidyl transferase (TdT)

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to polymerize labeled nucleotides onto the ends of nicked DNA (TUNEL, TdT-mediated d UTP nick end labeling). For example, following TdT labeling, biotinylated nucleotides may be detected with a chromogenic or fluorometricconjugated streptavidin, or brominated nucleotides may be detected with a highly sensitive, biotinylated anti-BrdU antibody and chromogenic-conjugated streptavidin. Transcription  refers to RNA synthesis using a DNA template. Transduction  is the use of a virus to transfer genes, such as the use of a bacteriophage to convey genes from one bacterial cell to another one. Other viruses such as retroviruses may also transfer genes from one cell type to another. Transfection  is the transfer of double-stranded DNA extracted from neoplastic cells for the purpose of producing phenotypic alterations of malignancy in the recipient cells. Spectratyping  refers to selected types of DNA gene segments that give a repetitive spacing of three nucelotides, or one codon. SRY  is the protein encoded for by the sex-determining region of the Y chromosome termed the sry gene in man. It is equivalent to the Y chromosome’s testis-determining gene. The corresponding protein in mice is termed Sry. The murine sry gene can cause transgenic female mice to become phenotypic males when the gene is inserted into them. The shift assay  is a useful method to identify protein-DNA interactions that may mediate gene expression, DNA repair, or DNA packaging. The assay can also be used to determine the affinity abundance, binding constants, and binding specificity of DNA-binding proteins. The gel shift assay is performed by annealing two labeled oligonucleotides that contain the test binding sequence, then incubating the duplex with the binding protein. The mixture is then separated on a nondenaturing polyacrylamide gel. Duplexes that are bound by protein migrate more slowly than unbound duplexes and appear as bands that are shifted relative to the bands from the unbound duplexes. Also called gel mobility shift assay or gel shift assay or gel retardation or band shift assay. Zygosity  refers to characterization of an individual’s heredity traits in terms of gene pairing in the zygote from which it develops. RNA splicing  is the method whereby RNA sequences that are nontranslatable (known as introns) are excised from the primary transcript of a split gene. The translatable sequences (known as exons) are united to produce a functional gene product. Apheresis  is the technique whereby blood is removed from the body, its components are separated, and some are

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retained for therapeutic or other use, and the remaining elements are recombined and returned to the donor. Also called hemapharesis. Leukapheresis  is a method that removes circulating leukocytes from the blood of healthy individuals for transfusion to recipients with decreased immunity or who are leukopenic. Leukapheresis is also used in leukemia patients who have too many white cells. The procedure leads to temporary relief of symptoms attributable to hyperleukocytosis. Conditioning of a donor with cytokines to mobilize hematopoietic stem cells from the bone marrow into the peripheral blood may be employed to enrich the donor leukocyte preparation for hematopoietic stem cells. Site-directed mutagenesis  is a laboratory procedure that involves the substitution of amino acids in a protein whose function is defined for the purpose of localizing a certain activity. Capsule swelling reaction:  Pneumococcus swelling reaction. See Quellung reaction. Lethal dose  is the amount of a toxin, virus, or any other material that produces death in all members of the species receiving it within a specified period of time following administration. Minimum lethal dose (MLD)  is that dose of a substance or agent that will kill 100% of the population being tested. The Ramon test (historical)  was an imprecise method for assaying the activity of any given preparation of diphtheria (or tetanus) toxin. Varying quantities of antitoxin are combined with a constant quantity of toxin in vitro. The tubes are placed in a 44 to 46°C water bath and are observed often. The test is read by noting the tube where flocculation occurs first. This is the point of equivalence where antitoxin has neutralized the homologous toxin. However, this assay is based on antigenicity of the toxin with which the antitoxin combines, in contrast to toxicity. Therefore, it is a measure of combining power and provides an indirect idea of toxicity only insofar as toxicity and antigenicity are positively correlated. Since the two are not always closely correlated, this method is less reliable than the in vivo technique of Ehrlich that measures the actual toxic effect of the toxin and the ability of antitoxin to combat it. The Ramon test measures toxin in Lf (flocculating) units. The Lf unit is defined as the amount of toxin which flocculates most rapidly with one unit of antitoxin. The Lf value, in contrast to other L values described, must be calculated. To determine the Lf value for a given toxin, the following formula is used:

L f /ml toxin =

antitoxin units/ml × ml of antitoxin ml of toxin

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 hus, the Lf content of a toxin may be determined if the following T values are known: (1) antitoxin units per milliliter of antitoxin; (2) milliliter of antitoxin required for most rapid flocculation with toxin; and (3) milliliter of toxin employed. Although the Ramon flocculation test was classically used to determine the Lf value of toxin, it may be carried out in reverse to assay the antitoxin units in each milliliter of antitoxin which has not been previously standardized. The same formula is applicable. L f /ml toxin =

antitoxin units/ml × ml of antitoxin ml of toxin

Antitoxin units/ml =

L f /ml toxin × ml of toxin ml off antitoxin

Varying quantities of toxin of known Lf value are combined with a constant amount of antiserum. The tube where

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flocculation first occurs is the point of equivalence. Therefore the amount of toxin in a milliliter is substituted into the formula together with the known values, which include the Lf per milliliter of toxin and the number of milliliters of antitoxin held constant. By simple arithmetic, the antitoxin units per milliliter may then be calculated. In this quantitative precipitin test, antibody dilutions are varied, but antigen dilutions are kept the same. The first tube where precipitation occurs is considered the end point. Serial passage  is a method to attenuate a pathogenic microorganism but retain its immunogenicity by transfer through several animal hosts, growth media, or tissue culture cells. Stochastic models:  Designs characterized by chance events without external direction or regulation.

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Diagnostic Immunohistochemistry

Immunohistochemistry  is a method to detect antigens in tissues that employs an enzyme-linked antibody specific for antigen. The enzyme degrades a colorless substrate to a colored insoluble substance that precipitates where the antibody and, therefore, the antigen are located. Identification of the site of the colored precipitate and the antigen in the tissue section is accomplished by light microscopy. Diagnostic pathology services routinely offer approximately 100 antigens, identified by immunoperoxidase technology, which are used in diagnosis. Immunoperoxidase method:  Nakene and Pierce, in 1966, first proposed that enzymes be used in the place of fluorochromes as labels for antibodies. Horseradish peroxidase (HRP) is the enzyme label most widely employed. The immunoperoxidase technique permits the demonstration of antigens in various types of cells and fixed tissues. This method has certain advantages that include the following: (1) the use of conventional light microscopy; (2) the stained preparations may be kept permanently; (3) the method may be adapted for use with electron microscopy of tissues; and (4) counterstains may be employed. The disadvantages include the following: (1) the demonstration of relatively minute positively staining areas is limited by the resolution of the light microscope; (2) endogenous peroxidase may not have been completely eliminated from the tissue under investigation; and (3) diffusion of products results from the enzyme reaction away from the area where antigen is localized. The peroxidase–antiperoxidase (PAP) technique  (Figure  29.1) employs unlabeled antibodies and a PAP reagent. This has proven highly successful for the demonstration of antigens in paraffin-embedded tissues as an aid in surgical pathologic diagnosis. Tissue sections preserved in paraffin are first treated with xylene, and after deparaffinization they are exposed to a hydrogen peroxide solution that destroys the endogenous peroxidase activity in tissue. The sections are next incubated with normal swine serum, which suppresses nonspecific binding of immunoglobulin molecules to tissues containing collagen. Thereafter, the primary rabbit antibody against the antigen to be identified is reacted with the tissue section. Primary antibody that is unbound is removed by rinsing the sections, which are then covered with swine antibody against rabbit immunoglobulin. This so-called linking antibody will combine with any primary rabbit antibody in the tissue. It is added in excess, which will result in one of its antigen-binding sites remaining free. After washing, the PAP reagent is placed on the section, and

the antibody portion of this complex, which is raised in rabbits, will be bound to the free antigen-binding site of the linking antibody on the sections. The unbound PAP complex is then washed away by rinsing. To read the sections microscopically, it is necessary to add a substrate of hydrogen peroxide and aminoethylcarbazole (AEC), which permits the formation of a visible product that may be detected with the light microscope. The AEC is oxidized to produce a reddish-brown pigment that is not water-soluble. Peroxidase catalyzes the reaction. Because peroxidase occurs only at sites where the PAP is bound via linking antibody and primary antibody to antigen molecules, the antigen is identified by the reddish-brown pigment. The tissue sections can then be counterstained with hematoxylin or other suitable dye, covered with mounting medium and cover slips, and read by conventional light microscopy. The PAP technique has been replaced, in part, by the avidin–biotin complex (ABC) technique (Figure 29.2). Streptavidin  is a protein isolated from streptomyces that binds biotin. This property makes streptavidin useful in the immunoperoxidase reaction that is employed extensively in antigen identification in histopathologic specimens, especially in surgical pathologic diagnosis. Decorate  is a term used by immunologists to describe the reaction of tissue antigens with monoclonal antibodies, described as “staining,” in the immunoperoxidase reaction. Thus, a tissue antigen stained with a particular antibody is said to be decorated with that monoclonal antibody. Immunoperoxidase techniques give a reddish-brown color to the reaction product that is read by light microscopic observation. Immunodiagnosis  involves the use of antibody assays, immunocytochemistry, the identification of lymphocyte markers, and other techniques to diagnose infectious diseases and malignant neoplasms.

Epithelium/Carcinoma Anti-BRST-3 (B72.3) monoclonal antibody  recognizes TAG-72 (Figure 29.3), a tumor-associated oncofetal antigen expressed by a wide variety of human adenocarcinomas. This antigen is expressed by 84% of invasive ductal breast carcinoma and 85 to 90% of colon, pancreatic, gastric, esophageal, lung (non-small cell), ovarian, and endometrial adenocarcinomas. It is not expressed by leukemias, lymphomas, sarcomas, mesotheliomas, melanomas, or benign tumors. TAG-72 867

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Tissue Ag

ABC Complex Tissue Ag

+ Rabbit Sheep antirabbit immunoglobulin primary [linking antibody] antibody [in excess]

Reagent A

Reagent B

+

Rabbit primary Avidin DH antibody Biotinylated secondary antibody (in excess) (e.g., goat antirabbit)

Biotinylated Peroxidase H

Peroxidase Peroxidase

+

+

Peroxidase-antiperoxidase (PAP)

Development in chromogenic hydrogen donor and hydrogen peroxide. (The reaction product is seen as a reddish brown or brown granular deposit depending upon the chromogenic hydrogen donor used.)

Figure 29.1  The peroxidase–antiperoxidase (PAP) technique.

is also expressed on normal secretory endometrium but not on other normal tissues. Anti-BRST-2 (GCDFP-15) monoclonal antibody  is specific for BRST-2 antigen expressed by apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic epithelium of the breast, benign sweat gland tumors of the skin, and the serous cells of the submandibular gland. Breast carcinomas (primary and metastatic lesions) with apocrine features express the BRST-2 antigen. BRST-2 is positive in extramammary Paget’s disease. Other tumors are negative.

Avidin (with four binding sites for biotin) Biotin Peroxidase Development in chromogenic hydrogen donor and hydrogen peroxide. (The reaction product is seen as a reddish brown or brown granular deposit depending upon the chromogenic hydrogen donor used.)

Figure 29.2  The avidin–biotin complex (ABC) technique.

BRST-2 (GCDFP-15) monoclonal antibody (murine)  detects BRST-2 antigen expressed by apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic epithelium of the breast, benign sweat gland tumors of the skin, and the serous cells of the submandibular gland. Breast carcinomas (primary and metastatic lesions) with apocrine features express the BRST-2 antigen. BRST-2 is positive in extramammary Paget’s disease. Other tumors tested are negative. GCDFP-15 (23A3), mouse:  Gross cystic disease fluid protein-15 is a 15,000-Da glycoprotein that was localized in the apocrine metaplastic epithelium lining breast cysts and in apocrine glands in the axilla, vulva, eyelid, and ear canal.

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Figure 29.3  Tag 72—carcinoma of the breast.

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Figure 29.4  CA125—papillary carcinoma of the ovary.

Figure 29.6  Caldesmon.

Approximately 70% of breast carcinomas stain positive with antibody to GCDFP-15. Colorectal carcinomas, as well as mesotheliomas, do not stain with this antibody. Lung adenocarcinoma rarely stains with this antibody.

CA-125 antibody  is a mouse monoclonal antibody that reacts with malignant ovarian epithelial cells. The antigen is formalin resistant, permitting the detection of ovarian cancer by immunohistochemistry, although serum assays for this protein are widely used to monitor ovarian cancer. CA-125 also reacts with antigens in seminal vesicle carcinoma and anaplastic lymphoma.

Gross cystic disease fluid protein 15 (GCDFP-15) antigen  is a 15-kDa glycoprotein that is demonstrable with immunoperoxidase staining and expressed by primary and metastatic breast carcinomas with apocrine features and extramammary Paget’s disease. Normal apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic breast epithelium, benign sweat gland tumors of skin, and submandibular serous cells express GCDFP-15 antigen. CA-125  (Figure 29.4) is a mucinous ovarian carcinoma cell surface glycoprotein detectable in blood serum. Increasing serum concentrations portend a grave prognosis. It may also be found in the blood sera of patients with other adenocarcinomas, such as breast, gastrointestinal tract, uterine cervix, and endometrium.

Figure 29.5  CEA—carcinoma of the colon.

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Caldesmon:  (Figure 29.6) Anti-caldesmon rabbit monoclonal antibody is specific for a regulatory protein present in smooth muscle and selected other tissues. It interacts with actin, myosin, tropomyosin and calmodulin. This antibody interacts with smooth muscle and neoplasia of smooth muscle, myofibroblastic and myoepithelial differentiation. It has been of use in the differentiation of epithelioid mesothelioma from serous papillary carcinoma of the ovary. Calponin  (Figure  29.7) A 34kD polypeptide restricted to smooth muscle that is interactive with actin, tropomyosin

Figure 29.7  Calponin.

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and calmodulin. It participates in smooth muscle contraction. Anti-calponin is a mouse monoclonal antibody used to differentiate benign sclerosing breast lesions from breast carcinoma. It may also be positive in malignant myothepithelioma, pleomorphic adenoma of the salivary glands, and angiomatoid malignant fibrous histiocytoma. Carcinoembryonic antigen (CEA)  (Figure  29.5) is a 200kDa membrane glycoprotein epitope that is present in the fetal gastrointestinal tract in normal conditions. However, tumor cells, such as those in colon carcinoma, may reexpress it. CEA was first described as a screen for identifying carcinoma by detecting nanogram quantities of the antigen in serum. It was later shown to be present in certain other conditions as well. CEA levels are elevated in almost one-third of patients with colorectal, liver, pancreatic, lung, breast, head and neck, cervical, bladder, medullarythyroid, and prostatic carcinoma. However, the level may be elevated also in malignant melanoma, lymphoproliferative disease, and smokers. Regrettably, CEA levels also increase in a variety of nonneoplastic disorders, including inflammatory bowel disease, pancreatitis, and cirrhosis of the liver. Nevertheless, determination of CEA levels in the serum is valuable for monitoring the recurrence of tumors in patients whose primary neoplasm has been removed. If the patient’s CEA level reveals a 35% elevation compared to the level immediately following surgery, this may signify metastases. This oncofetal antigen is comprised of one polypeptide chain with one variable region at the amino terminus and six constant region domains. CEA belongs to the immunoglobulin superfamily. It lacks specificity for cancer, thereby limiting its diagnostic usefulness. It is detected with a mouse monoclonal antibody directed against a complex glycoprotein antigen present on many human epithelial derived tumors. This reagent may be used to aid in the identification of cells of epithelial lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti CEA antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surround heavily stained tissue and/or cells which show immunoreactivity. Clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. Collagen type IV (CIV22):  Anti-collagen type IV is a mouse monoclonal antibody that detects collagen type IV, the major component of the basal lamina. Antibodies to this molecule confirm its presence and reveal the morphological appearance of the structure. Normal tissue stains with this antibody in a fashion consistent with the sites of mesenchymal elements and epithelial basal laminae. Collagen IV can also be useful in the classification of soft tissue tumors, Schwannomas, and leiomyomas, and their well-differentiated malignant counterparts usually immunoreact to this antibody. The vascular nature of neoplasma, hemangiopericytoma, angiosarcoma, and epitheliod hemangioendothelioma can be revealed by this antibody

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with greater reliability than nonspecific stains (e.g., silver reticulum). Antibroad-spectrum cytokeratin  is a mouse monoclonal antibody that may be used to identify cells of normal and abnormal epithelial lineage and as an aid in the diagnosis of anaplastic tumors. The cytokeratins are a group of intermediate filament proteins that occur in normal and neoplastic cells of epithelial origin. The 19 known human cytokeratins are divided into acidic and basic subfamilies. They occur in pairs in epithelial tissues, the composition of pairs varying with the epithelial cell type, stage differentiation, cellular growth, environment, and disease state. The pankeratin cocktail recognizes most of the acidic and all of the basic cytokeratins, making it a useful general stain for nearly all epithelial tissues and their tumors. This antibody binds specifically to antigens located in the cytoplasmic region of normal simple and complex epithelial cells. The antibody is used to qualitatively stain cytokeratins in sections of formalin-fixed paraffin-embedded tissue. Antipankeratin primary antibody contains a mouse monoclonal antibody raised against an epitope found on human epidermal keratins. It reacts with 56.5-kDa, 50-kDa, 48-kDa, and 40-kDa cytokeratins of the acidic subfamily, and 65- to 67-kDa, 64-kDa, 59-kDa, 58-kDa, 56-kDa, and 52-kDa cytokeratins of the basic subfamily. In anaplastic tumors, the percentage of tumor cells showing cytokeratin reactivity may be small (less than 5%). Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. Anti-high molecular weight human cytokeratin antibodies  are mouse monoclonal antibodies that identify keratins of approximately 66 kDa and 57 kDa in extracts of the stratum corneum. The antibody labels squamous, ductal, and other complex epithelia. It is reactive with both squamous and ductal neoplasms and variably with those derived from simple epithelium. Consistently positive are squamous cell carcinomas and ductal carcinomas, most notably those of the breast, pancreas, bile duct, and salivary gland; transitional cell carcinomas of the bladder and nasopharynx; and thymomas and epithelioid mesotheliomas. Ade­nocarcinomas are variably positive. The antibodies are largely unreactive with adenomas of endocrine organs, carcinomas of the liver (hepatocellular carcinoma), endometrium, and kidney. Mesenchymal tumors, lymphomas, melanomas, neural tumors, and neuroendocrine tumors are unreactive. Cytokeratin (34betaE12), mouse:  Anticytokeratin (34beta E12) mouse monoclonal antibody detects cytokeratin 34betaE12, a high-molecular-weight cytokeratin that reacts with all squamous and ductal epithelium and stains carcinomas. This antibody recognizes cytokeratins 1,5,10, and 14 that are found in complex epithelia. Cytokeratin 34betaE12 shows no reactivity with hepatocytes, pancreatic acinar

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cells, proximal renal tubes, or endometrial glands; there has been no reactivity with cells derived from simple epithelia. Mesenchymal tumors, lymphomas, melanomas, neural tumors, and neuroendocrine tumors are unreactive with this antibody. Cytokeratin 34betaE12 has been shown to be useful in distinguishing prostatic adenocarcinoma from hyperplasia of the prostate.

from other sites were not positive using the antibody to cytokeratin 20, e.g., adenocarcinomas of the breast, lung, and endometrium, and nonmucinous tumors of the ovary. Merkel cell carcinomas of the skin stain normally with the anticytokeratin 20 antibody. There was a lack of positivity in small-cell lung carcinomas and in intestinal and pancreatic neuroendocrine tumor cells.

Anti-low molecular weight cytokeratin  is a mouse monoclonal antibody directed against an epitope found on human cytokeratins. It may be used to aid in the identification of cells of epithelial lineage. The antibodies are intended for qualitative staining in sections of formalin-fixed paraffin-embedded tissue. Antikeratin primary antibody specifically binds to antigens located in the cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show apparent immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

Antihuman cytokeratin 7 antibody  (Figure 29.9) is a mouse monoclonal antibody directed against the 54-kDa cytokeratin intermediate filament protein identified as cyto­keratin 7, a basic cytokeratin found in most glandular epithelia and in transitional epithelia. The antibody reacts with a large number of epithelial cell types including many ductal and glandular epithelia. In general, the antibody does not react with stratified squamous epithelia but is reactive with transitional epithelium of the urinary tract. The antibody reacts with many benign and malignant epithelial lesions. Keratin 7 is expressed in specific subtypes of adenocarcinomas from ovary, breast, and lung, whereas carcinomas from the gastrointestinal tract remain negative. Transitional cell carcinomas express keratin 7, whereas prostate cancer is generally negative. The antibody does not react with squamous cell carcinomas, rendering it a rather specific marker for adenocarcinoma and transitional cell carcinoma. In cytological specimens, the antibody permits ovarian carcinoma to be distinguished from colon carcinoma.

Antihuman cytokeratin-20 monoclonal antibody  (Fig­ ure 29.8) reacts with the 46-kDa cytokeratin intermediate filament protein. It reacts with intestinal epithelium, gastric foveolar epithelium, a number of endocrine cells of the upper portions of the pyloric glands, as well as with the urothelium and Merkel’s cells in the epidermis. The antibody has been tested on a series of carcinomas including primary and metastatic lesions. There is a marked difference in expression of cytokeratin 20 among various carcinoma types. Neoplasia expressing cytokeratin 20 are derived from normal epithelia expressing cytokeratin 20. Colorectal carcinomas consistently express cytokeratin 20, whereas adenocarcinomas of the stomach express cytokeratin 20 to a lesser degree. Adenocarcinomas of the gall bladder and bile ducts, ductal cell adenocarcinomas of the pancreas, mucinous ovarian tumors, and transitional-cell carcinomas have been found to stain positively with the antibody. Most of the carcinomas

Cytokeratin 7 (K72), mouse:  Anticytokeratin 7 (K72) mouse monoclonal antibody reacts with proteins that are found in most ductal, glandular, and transitional epithelium of the urinary tract and bile duct epithelial cells. Cytokeratin 7 distinguishes between lung and breast epithelium that stain positive, and colon and prostate epithelial cells that are negative. This antibody also reacts with many benign and malignant epithelial lesions, e.g., adenocarcinomas of the ovary, breast, and lung. Transitional cell carcinomas are positive and prostate cancer is negative. This antibody does not recognize intermediate filament proteins.

Figure 29.8  Cytokeratin 20—adenoma of the colon.

Figure 29.9  Cytokeratin 7—adenocarcinoma of the lung.

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Figure 29.10  Cytokeratin cocktail—prostate.

AE1/AE3 pan-cytokeratin monoclonal antibody  (Fig­ ure 29.10) provides the broadest spectrum of keratin reactivity among the 19 catalogued human epidermal keratins and produces positive staining in virtually all epithelia. Antihuman cytokeratin (CAM 5.2) (cytokeratin 8,18)  (Figure 29.11) is a monoclonal antibody against cytokeratins which are polypeptide chains that form structural proteins within the epithelial cell cytoskeleton. Nineteen different molecular forms of cytokeratin have been identified in both normal and malignant epithelial cell lines. Because specific combinations of cytokeratin peptides are associated with different epithelial cells, these peptides are clinically important markers for classifying carcinomas (tumors of epithelial origin) and for distinguishing carcinomas from malignant tumors of nonepithelial origin such as lymphomas, melanomas, and sarcomas. The identification of cytokeratin has gained increasing importance in immunopathology. E-Cadherin (ECH-6), mouse:  Anti-E-Cadherin mouse monoclonal antibody detects E-Cadherin, an adhesion protein

Figure 29.11  Cytokeratin 18—salivary gland.

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Figure 29.12  Epithelial carcinoma.

membrane

antigen—squamous

expressed in cells of epithelial lineage. It stains positively in glandular epithelium as well as adenocarcinomas of the lung and GI tract and ovary. It has been useful in distinguishing adenocarcinoma from mesothelioma. It has also been shown to be positive in some thyroid carcinomas. Epithelial membrane antigen (EMA)  (Figure  29.12) is a marker that identifies, by immunoperoxidase staining, most epithelial cells and tumors derived from them, such as breast carcinomas. However, various nonepithelial neoplasms, such as selected lymphomas and sarcomas, may express EMA also. Thus, it must be used in conjunction with other markers in tumor identification and/or classification. Antiepithelial membrane antigen (EMA) antibody  is a mouse monoclonal antibody directed against a mucin epitope present on most human epithelial cells. This antibody reacts with epithelial mucin, a heavily glycosylated molecule with a molecular weight of circa 400 kDa. Epithelial membrane antigen is widely distributed in epithelial tissues and tumors arising from them. Normal glandular epithelium and tissue from nonneoplastic diseases stain in lumen membranes and cytoplasm. Malignant neoplasms of glandular epithelium frequently show a change in pattern with the appearance of adjacent cell membrane staining. EMA is of value in distinguishing both large-cell anaplastic carcinoma from diffuse histiocytic lymphoma and small-cell anaplastic carcinoma from well- and poorly differentiated lymphocytic lymphomas. Estrogen/progesterone receptor protein:  Monoclonal antibodies against estrogen receptor protein and against progesterone receptor protein permit identification of tumor cells by their preferential immunoperoxidase staining for these markers, whereas stromal cells remain unstained. This method is claimed by some to be superior to cytosol assays in evaluating the clinical response to hormones. (Figure 29.13)

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Diagnostic Immunohistochemistry

Figure 29.13  Estrogen receptor—carcinoma of the breast.

Antiestrogen receptor antibodies  are mouse monoclonal specific for estrogen receptors. The estrogen receptor (ER) content of breast cancer tissue is an important parameter in the prediction of prognosis and response to endocrine therapy. Monoclonal antibodies to ER permit the determination of receptor status of breast tumors to be carried out in routine histopathology laboratories. Although monoclonal antibodies that recognize ER were only effective on frozen sections initially, currently available monoclonal antibodies are effective on formalin-fixed, paraffin-embedded tissues to allow the determination of ER in routinely processed and archival material. In diagnostic immunology, estradiol  is a marker identifiable in breast carcinoma tissue by monoclonal antibody and the immunoperoxidase technique that correlates, to a limited degree, with estrogen receptor activity in cytosols from the same preparation. Her2/Neu(c-erb-B-2):  (Figure  29.14) A c-erb-B-2 protein expressed by cell membranes of normal and neoplastic human

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tissues. Both mouse and rabbit monoclonal antibodies have been used for immunohistochemical staining and anti-Her2/ Neu (c-erb-B-2) preparations. The rabbit monoclonal antibody is specific for the internal domain of the c-erb-B-2 oncoprotein (Her2) and is useful for the semi-quantitative detection of Her2 antigen in formalin-fixed, paraffin embedded sections. It facilitates the evaluation of breast cancer patients who are candidates for Herceptin® treatment. Intense staining of cell membranes for c-erb-B-2 protein has been demonstrated in tumor cells of invasive breast carcinoma. Cytoplasmic staining without membrane staining is not considered to be clinically relevant. Precise criteria govern the interpretation of positive and negative results in breast carcinoma. Results considered positive for Her2 protein overexpression require membrane staining of 2+ or greater intensity on a scale of 0–3+. In addition, the percentage of positive tumor cells must be greater than 10%. Cell membrane staining rather than cytoplasmic staining is considered to be requisite for positive results. Anti-Ki-67 (MIB)  is a mouse monoclonal antibody directed against the Ki-67 nuclear antigen. This reagent may be used to aid in the identification of proliferating cells in normal and neoplastic cell populations. It is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue (some form of antigen enhancement is required for paraffin-embedded samples), frozen tissue, and cytologic preparations. Ki-67 antibody specifically binds to nuclear antigen(s) associated with cell proliferation which is present throughout the active cell cycle (G1, S, G2, and M phases) but absent in resting (G0) cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissues and or cells will show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. Ki-67 or -780  are nuclear antigens expressed by both normal and neoplastic-proliferating cells. They are demonstrable by immunoperoxidase staining (Figure  29.15). A relatively high percentage of positive cells in a neoplasm implies an unfavorable prognosis. MLH1:  (Figure  29.16) A mismatch repair gene that is decreased in many patients with microsatellite instability (MSI-H). This condition is associated with hereditary nonpolyposis colon cancer (HNPCC), which has an autosomal dominant mode of inheritance. Anti-MLH1 is a mouse monoclonal antibody that is beneficial in screening patients and families for this condition. Microsatellite instability in colon cancer portends a better prognosis than expected in those cases that are microsatellite stable.

Figure 29.14  Her2/Neu(c-erb-B-2).

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MSH2:  (Figure 29.17) This match repair gene has the same characteristics and incidence as described for MSH1. AntiMSH2 mouse monoclonal antibody is used to identify the same conditions as described for MSH1.

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(a)

Figure 29.17  MSH2.

high affinity for calcium. It also has a strong binding capacity for apatite. Only 60–90% of de novo synthesized osteocalcin is incorporated into the bone matrix binding to hydroxy apatite during matrix mineralization. The remainder enters the circulation where it may be assayed as a sensitive marker for bone formation. Serum osteocalcin is derived almost exclusively from bone formation, rather than resorption. Serum osteocalcin is increased in diseases marked by elevated bone turnover, such as osteoporosis, hyperparathyroidism, and Paget disease, and is decreased in conditions associated with low bone turnover, including hypoparathyroidism and growth hormone deficiency. (b) Figure 29.15  Ki-67—Carcinoma of the breast.

Osteocalcin antibody:  An osteocalcin/matrix Gla protein comprising 1–2% of the total bone protein. This 49-amino acid single-chain vitamin K-dependent protein synthesized by fibroblasts is a principal constituent of the noncollagenous bone matrix. Posttranslational alteration by vitamin K-dependent carboxylase yields three gamma carboxyglutamic acid residues at positions 17, 21, and 24, affording it a

Figure 29.16  MLH1.

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P63 (ap53 Homolog at 3q27–29) Ab-4 (cocktail) mouse monoclonal antibody  recognizes a 63-kDa protein, identified as p63. Ths p63 gene, a homolog of the tumor-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Protein p63 shows remarkable structural similarity to p53 and to the related p73 gene. Unlike p53, the p63 gene encodes multiple isotypes with remarkable divergent abilities to transactivate p53 reporter genes and induce apoptosis. NeoMarkers’ Ab-4 recognizes all known isotypes of p63. Antiprogesterone receptor antibody  is a mouse monoclonal antibody against human progesterone receptor. A mouse monoclonal antihuman progesterone receptor antibody that specifically recognizes the A and B forms of the receptor in Western blot purified recombinant receptor, normal endometrium, and cell lysates of the progesterone receptor-rich T47D human breast carcinoma cell line. No reactivity was observed with lysate of the progesterone receptor-negative MDA-MB-231 breast carcinoma cells. No cross-reactivity was found with androgen receptor, estrogen receptor, or glucocorticoid receptor. The antibody binds an epitope found between amino acids 165 and 534, in the N-terminal transactivation domain of the progesterone receptor molecule. Various tumors of the female reproductive tract have been shown to express progesterone receptor. Immunoreactivity has been demonstrated in breast carcinoma, uterine papillary

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Figure 29.18  Prostatic acid phosphatase (PSAP)—prostate.

Figure 29.19  Prostate-specific antigen (PSA)—prostate.

serous carcinoma, endometrial carcinoma, ovarian serous borderline tumor, endometrial stromal sarcoma, uterine adenomatoid tumor, and ovarian thecoma. Other tumors that have been shown to stain positively include medullary carcinoma of the thyroid and meningioma.

is a 34-kDa glycoprotein found exclusively in benign and malignant epithelium of the prostate. Men with PSA levels of 0 to 4.0 ng/ml and a nonsuspicious digital rectal examination are generally not biopsied for prostate cancer. Men with PSA levels of 10.0 ng/ml and above typically undergo prostate biopsy. About one-half of these men will be found to have prostate cancer. Certain kinds of PSA, known as bound PSA, link themselves to other proteins in the blood. Other kinds of PSA, known as free PSA, float by themselves. Prostate cancer is more likely to be present in men who have a low percentage of free PSA relative to the total amount of PSA. This finding is especially valuable in helping to differentiate between cancer and other, benign conditions, thus eliminating unnecessary biopsies among men in that diagnostic gray zone, who have total PSA levels between 4.0 and 10.0 ng/ml. The PSA molecule is smaller than prostatic acid phosphatase (PAP). In patients with prostate cancer, preoperative PSS serum levels are positively correlated with the disease. PSA is more stable and shows less diurnal variation than does PAP. PSA is increased in 95% of new cases of prostatic carcinoma compared with 60% for PAP. It is increased in 97% of recurrent cases compared with 66% of PAP. PAP may also be increased in selected cases of benign prostatic hypertrophy and prostatitis, but these elevations are less than those associated with adenocarcinoma of the prostate. It is inappropriate to use either PSA or PAP alone as a screen for asymptomatic males. Transurethral resection (TUR), urethral instrumentation, prostatic needle biopsy, prostatic infarct, or urinary retention may also result in increased PSA values. PSA is critical for the prediction of recurrent adenocarcinoma in postsurgical patients. PSA is also a useful immunocytochemical marker for primary and metastatic adenocarcinoma of the prostate.

Antihuman prostatic acid phosphatase (PSAP)  (Fig­ ure 29.18) is a rabbit antibody that reacts with prostatic ductal epithelial cells—normal, benign hypertrophic, and neoplastic. This antibody labels the cytoplasm of prostatic epithelium, secretions, and concretions. Prostatic acid phosphatase (PAP)/prostatic epithelial antigen  are prostate antigens, identifiable by immunoperoxidase staining, that are prostate specific and prostate sensitive. Used together, they detect approximately 99% of prostatic adenocarcinomas. PSA (prostate-specific antigen)  (Figure  29.19) is a substance secreted only by the prostate epithelium and is a 34-kDa glycoprotein serine protease that lyses seminal coagulum. Individuals with benign prostatic hypertrophy have a 30 to 50% elevation in PSA levels, whereas those with prostatic carcinoma have a 25 to 92% elevation. It is a more reliable indicator of prostatic carcinoma than is serum prostatic acid phosphatase (PAP). PSA levels are also valuable in signifying recurrence of prostatic adenocarcinoma. Prostate cancer may occur in 22% of the individuals with PSA levels greater than 4.0 μg/l and 60% of the individuals with PSA levels greater than 10 μg/l. Antiprostate specific antigen (PSA) antibody  is a rabbit antibody that reacts with prostatic ductal epithelial cells— normal, benign hypertrophic, and neoplastic. This antibody labels the cytoplasm of prostatic epithelium, secretions, and concretions. Prostate-specific antigen (PSA)  is a marker in serum or tissue sections for adenocarcinoma of the prostate. PSA

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Antihuman thyroglobulin  is a rabbit antibody that reacts with human thyroglobulin. It labels the cytoplasm of normal and neoplastic thyroid follicle cells. Some staining of colloid may also be observed.

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TTF-1 (8G7G3/a), mouse:  Thyroid transcription factor-1 is useful in differentiating primary adenocarcinoma of the lung from metastatic carcinomas from the breast and malignant mesothelioma. It can also be used to differentiate small-cell lung carcinoma from lymphoid infiltrates.

Germ Cell Alpha-fetoprotein:  (Figure 29.20) An antigen detectable in hepatocytes of fetal liver and hepatoma using anti-AFP rabbit polyclonal antibody. Adult serum contains only traces of AFP. Increased levels suggest a benign or malignant liver lesion, a carcinoma of the yolk sac, as well as selected other tumors. It has been detected in yolk sac carcinomas of gonadal and extragonadal occasions, hepatic neoplasia, and selected other neoplasms.

Figure 29.21  Placental alkaline phosphatase (PLAP)—placenta.

Antihuman chorionic gonadotropin (HCG) antibody  is an antibody that reacts with the beta chain of human chorionic gonadotropin (HCG). HCG is a polypeptide hormone synthesized in the syncytiotrophoblastic cells of the placenta and in certain trophoblastic tumors. HCG is a marker for the biochemical differentiation of trophoblastic cells, which often precedes their morphological differentiation. The antibody aids detection of HCG in trophoblastic elements of germ cell tumors of the ovaries, testes, and extragonadal sites. It crossreacts with luteinizing hormone.

tumors appear to be universally reactive for PLAP, whereas somatic tumors show only 15 to 20% reactivity.

Antiplacental alkaline phosphatase (PLAP) antibody  (Figure  29.21) is normally produced by syncytiotrophoblasts after the 12th week of pregnancy. Human placental alkaline phosphatase is a member of a family of membranebound alkaline phosphatase enzymes and isoenzymes. It is expressed by both malignant somatic and germ-cell tumors. PLAP immunoreactivity can be used in conjunction with epithelial membrane antigen (EMA) and keratin to differentiate between germ cell and somatic tumor metastases. Germ cell

Hepatitis B virus surface antigen:  (Figure 29.22) A marker demonstrated mainly in the cytoplasm of hepatocytes of hepatitis B virus-infected patients using an HBsAg monoclonal antibody.

Figure 29.20  Alpha-fetoprotein.

Figure 29.22  HBsAg.

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Infectious Agents Antihepatitis B virus core antigen (HBcAg) antibody  labels the nuclei and occasionally cytoplasm of virus infected cells. HBcAg is expressed predominantly in the nuclei of infected liver cells, although variable staining may also be seen in the perinuclear cytoplasm.

Herpes simplex virus I and II:  (Figure  29.23) These viral antigens are detected through use of a rabbit polyclonal antibody. Common infections with herpes simplex virus (HSV) is governed by many factors, such as age, the subject’s immune

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Figure 29.25  Cytomegalovirus (CMV)—placenta. Figure 29.23  HSV I and II.

status, the antigenic type of infecting virus, i.e., HSV type I or HSV type II, and the site of infection. HSV type I causes acute necrotizing encephalitis and can be found in the temporal lobe of an infected brain. Herpes simplex virus type II identifies HSV type II strains in infected tissues. HHV-8:  (Figure 29.24) Human herpesvirus type 8 (HHV-8) is believed to the etiologic agent of Kaposi’s sarcoma as DNA sequences of this virus have been detected in Kaposi’s sarcoma tissue, primarily effusion lymphoma and multi-centric Castleman disease using the polymerase chain reaction and in situ hybridization. Latent nuclear antigen (LNA-1, LNA, LANA-1), also designated as ORF73, is a 222- or 234-kD protein frequently expressed in HHV-8 infected cells. AntiHHV-8 is a mouse monoclonal antibody that interacts with latent nuclear antigen protein demonstrable by the immunoperoxidase technique. Antipapillomavirus  is a rabbit antibody against papillomavirus. The structural antigens on this virus can be detected in a

Figure 29.24  HHV-8.

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variety of proliferative squamous lesions. Only 50 to 60% of lesions caused by papillomavirus will express the structural antigens. This antibody staining is predominantly intranuclear in a focal or diffuse pattern, although perinuclear cytoplasmic staining of koilocytotic cells may also be seen. Anticytomegalovirus antibody  (Figure  29.25) is a mouse monoclonal antibody that reacts with CMV-infected cells giving a nuclear staining pattern with early antigen and a nuclear and cytoplasmic reaction with the late viral antigen. The antibody shows no cross-reactivity with other herpesviruses or adenoviruses. EBV:  (Figure  29.26) Epstein–Barr virus (latent membrane antigen) is a 60kD latent membrane protein (LMP-1) encoded by the BNLF1 gene of Epstein–Barr virus (EBV). It is identified using a four-mouse monoclonal antibody cocktail, which permits identification of different epitopes on the hydrophilic C terminus of the LMP-1 cytoplasmic domain. This antibody reacts strongly with EBV-infected B cell immunoblasts in infectious mononucleosis. It also interacts with 25–50% of EBV-associated nasopharyngeal carcinomas that

Figure 29.26  EBV.

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myofibroblasts. In surgical pathologic diagnosis, monoclonal antibodies against desmin are useful in identifying muscle tumors.

Figure 29.27  Parvovirus B19.

are undifferentiated, as well as with Reed Sternberg cells in about 90% of EBV-associated Hodgkin lymphoma cases. Parvovirus B19:  (Figure 29.27) This virus has been postulated to be associated with spontaneous abortion in humans. Anti-Parvovirus 19 mouse monoclonal antibody is used to detect viral antigens, VP1 (84kD) and VP2 (58kD) in tissues that have been aborted. Parvovirus B19 infection should be suspected in cases of intrauterine fetal death. Toxoplasma gondii:  (Figure  29.28) A protozoan parasite that may induce either asymptomatic infections of healthy humans or may induce severe congenital defects in the fetus and life-threatening consequences in immunocompromised hosts. It expresses a P30 antigen, also called SAG-1, on the principal surface antigen of Toxoplasma gondii tachyzoites in the initial of invasion. It is a highly conserved antigen among most T. gondii strains rendering it a useful target for detection of this protozoan parasite using polyclonal rabbit antibodies.

Stroma/Sarcoma Desmin  is a 55-kDa intermediate filament molecule found in mesenchymal cells that include both smooth and skeletal muscle, endothelial cells of the vessels, and probably

Desmin (D33), mouse:  Desmin antibody detects a protein that is expressed by cells of normal smooth, skeletal, and cardiac muscles. The light microscope has suggested that desmin is primarily located at or near the periphery of Z lines in striated muscle fibrils. In smooth muscle, desmin interconnects cytoplasmic dense bodies with membrane-bound dense plaques. Desmin antibody reacts with leiomyomas, rhabdomyomas, and perivascular cells of glomus tumors of the skin (if they are of myogenic nature). This antibody is basically used to demonstrate the myogenic components of carcinosarcomas and malignant mixed mesodermal tumors. Antidesmin antibody  is a mouse monoclonal antibody (clone DE-R-11) raised against purified porcine desmin that reacts with the 53-kDa intermediate filament protein desmin. This reagent may be used to aid in the identification of cells of myocyte lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Antidesmin primary antibody specifically binds to antigens located in the cytoplasm of myocytic cells. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. Anti-Ewing’s sarcoma marker (CD99)  is a mouse monoclonal antihuman MIC2 gene product. (Ewing’s sarcoma marker) antibody reacts only with glioblastoma and ependymoma of the central nervous system and certain islet cell tumors of the pancreas. Because the MIC2 gene products are most strongly expressed on the cell membrane of Ewing’s sarcoma (ES) and primitive peripheral neuroectodermal tumors (pPNET), demonstration of the gene products allows for the differentiation of these tumors from other round-cell tumors of childhood and adolescence. CD99 (HO36-1.1):  Anti-CD99 mouse monoclonal antibody reacts with MIC-2 antigen present on the cell membrane of Ewing’s sarcoma and primitive peripheral neuroectodermal tumors (PNET). It is also present on some bone marrow, lymph nodes, spleen, cortical thymocytes, granulosa cells of the ovary, most beta cells, CNS ependymal cells, Sertoli’s cells of the testis, and a few endothelial cells. MIC-2 has also been identified in lymphoblastic lymphoma, rhabdomyosarcoma, mesenchymal chondrosarcoma, and thymoma. Factor VIII  (Figure  29.29) is a coagulation protein produced by endothelial cells, which makes it a useful marker for vascular tumors. It is demonstrable by immunoperoxidase staining. Megakaryocytes and platelets also stain for factor VIII.

Figure 29.28  Toxoplasma gondii.

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Antifactor VIII  is a mouse monoclonal antibody that gives positive staining in the cytoplasm of normal vascular

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Figure 29.29  Factor VIII—placenta.

Figure 29.30  S-100—metastatic melanoma—lymph node.

endothelial cells of arteries, veins, capillaries, and endocardial cells. Factor VIII related antigen is also present in megakaryocytes and platelets.

S-100  (Figure  29.30) is a heterodimeric protein comprised of α and β chains. It is present in a variety of tissues and is especially prominent in nervous system tissue including brain, neural crest, and Schwann cells. It also is positive in breast ducts, sweat and salivary glands, bronchial glands and Schwann cells, serous acini, malignant melanomas, myoepithelium, and neurofibrosarcomas.

Actin  the principal muscle protein, which together with myosin causes muscle contraction, is used in surgical pathology as a marker for the identification of tumors of muscle origin. Actin is identified through immunoperoxidase staining of surgical pathology tissue specimens. Antimuscle actin primary antibody  is a mouse monoclonal antibody (clone HUC1-1) directed against an actin epitope found on muscle actin isoforms. This reagent may be used to aid in the identification of cells of myocytic lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Antimuscle actin antibody specifically binds to antigens located in the cytoplasmic regions of normal muscle cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells will show immunoreactivity. Clinical interpretation must be complemented by morphological studies and the evaluation of appropriate controls. Myogenin (F5D), mouse:  Anti-myogenin monoclonal antibody labels the nuclei of myoblasts in developing muscle tissue and is expressed in tumor cell nuclei of rhabdomyosarcoma. Positive nuclear staining may occur in Wilms’ tumor, as well as in some myopathies. Myoglobin  is an oxygen-storing muscle protein that serves as a marker of muscle neoplasms, demonstrable by immunoperoxidase staining for surgical pathologic diagnosis. Myoglobin antibody  is a reagent that stains normal striated muscle and striated muscle-containing tumor. Using immunohistochemical procedures on formalin-fixed, paraffinembedded tissues, this antibody stains human skeletal and cardiac muscle.

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S-100 protein  is a marker, demonstrable by immunoperoxidase staining, that is extensively distributed in both central and peripheral nervous systems and tumors arising from them, including astrocytomas, melanomas, Schwannomas, etc. Most melanomas express S-100 protein. Such nonneuronal cells as chondrocytes and histiocytes are also S-100 positive. S-100 protein antibody  is a mouse monoclonal antibody specific for S-100 protein that is found in normal melanocytes; Langerhans cells; histiocytes; chrondrocytes; lipocytes; skeletal and cardiac muscle; Schwann cells; epthelial and myoepithelial cells of the breast, salivary, and sweat glands; and glial cells. Neoplasms derived from these cells also express S-100 protein, albeit nonuniformly. A large number of welldifferentiated tumors of the salivary gland, adipose and cartilaginous tissue, and Schwann cell-derived tumors express S-100 protein. Almost all malignant melanomas and cases of histiocytosis X are positive for S-100 protein. Despite the fact that S-100 protein is a ubiquitous substance, its demonstration is of great value in the identification of several neoplasms, particularly melanomas. Antihuman α-smooth muscle actin  is a mouse monoclonal antibody that reacts with the α-smooth muscle isoform of actin. The antibody reacts with smooth muscle cells of vessels and different parenchyma without exception, but with different intensity, according to the amount of α-smooth muscle actin present in smooth muscle cells, myoepithelial cells, pericytes, and some stromal cells in the intestine, testes, breast, and ovary. The antibody also reacts with myofibroblasts in

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benign and reactive fibroblastic lesions and perisinusoidal cells of normal and diseased human livers. Vimentin  is a 55-kDa intermediate filament protein synthesized by mesenchymal cells such as vascular endothelial cells, smooth muscle cells, histiocytes, lymphocytes, fibroblasts, melanocytes, osteocytes, chondrocytes, astrocytes, and occasional ependymal and glomerular cells. Malignant cells may express more than one intermediate filament. For example, immunoperoxidase staining may reveal vimentin and cytokeratin in breast, lung, kidney, or endometrial adenocarcinomas Antivimentin antibody  is a mouse monoclonal antibody raised against purified bovine eye lens vimentin. This antibody reacts with the 57-kDa intermediate filament protein, vimentin. This reagent may be used to aid in the identification of cells of mesenchymal origin. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffinembedded tissue. It binds specifically to antigens located in the cytoplasm of mesenchymal cells. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

Hematopoietic/Lymphoid α-1 antichymotrypsin  is a histiocytic marker. By immunoperoxidase staining, it is demonstrable in tumors derived from histiocytes. It may also be seen in various carcinomas. A-1-Antitrypsin:  (Figure  29.31) A polyclonal rabbit antibody is used to investigate inherited AAT deficiency, hepatic neoplasia that are both benign and malignant, yolk sac carcinomas, as well as histiocytic lesions that are benign or malignant. The antibody has also been used to screen patients with cryptogenic cirrhosis or other liver diseases with portal fibrosis of uncertain etiology.

Atlas of Immunology, Third Edition

Anti-bcl-2 primary antibody  is a mouse monoclonal antibody. The bcl-2 oncoprotein expression is inhibited in germinal centers where apoptosis forms a part of the B cell production pathway. In 90% of follicular lymphomas, a translocation occurs which justaposes the bcl-2 gene at 19q21 to an immunoglobulin gene, with subsequent deregulation of protein synthesis and cell proliferation. The bcl-2 product is considered to act as an inhibitor of apoptosis. This observation has turned out to have clinical implications. Distinction of follicular hyperplasia from follicular lymphoma is a common problem in histopathology. Reactive follicles show no staining for bcl-2, whereas the cells in neoplastic follicles exhibit membrane staining. Anti-BCL-6 (PG-B6p) mouse monoclonal antibody:  This is a transcriptional regulator gene which codes for a 706-amino-acid nuclear zinc finger protein. Antibodies to this protein stain the germinal center cells in lymphoid follicles, the follicular cells and interfollicular cells in follicular lymphoma, diffuse large B cell lymphomas, and Burkitt lymphoma, and the majority of the Reed-Sternberg cells in nodular lymphocyte predominant Hodgkin disease. In contrast, anti-BCL-6 rarely stains mantle cell lymphoma, and MALT lymphoma bcl-6 expression is seen in approximately 45% of CD30+ anaplastic large cell lymphomas but is consistently absent in other peripheral T cell lymphomas. CD10  is a mouse monoclonal antibody that reacts with common acute lymphoblastic leukemia antigen (CALLA/CD10) as a useful marker for the characterization of childhood leukemia and B cell lymphomas. This antibody reacts with antigen of lymphoblastic, Burkitt, and follicular lymphomas; and chronic myelocytic leukemia. Also, CD10 detects the antigen of glomerular epithelial cells and the brush border of the proximal tubules. This characteristic may be helpful in interpreting renal ontogenesis in conjunction with other markers. Other nonlymphoid cells that are reactive with CD10 are breast myoepithelial cells, bile canaliculi, neutrophils, and small population of bone marrow cells, fetal small intestine epithelium, and normal fibroblasts. Common acute lymphoblastic leukemia antigen (CALLA/ CD10)  is a useful marker for the characterization of childhood leukemia and B cell lymphomas. This antibody reacts with antigen of lymphoblastic, Burkitt, and follicular lymphomas; and chronic myelocytic leukemia. Also, CD10 detects the antigen of glomerular epithelial cells and the brush border of the proximal tubules; this characteristic may be helpful in interpreting renal ontogenesis in conjunction with other markers. Other nonlymphoid cells that are reactive with CD10 are breast myoepithelial cells, bile canaliculi, neutrophils, and small population of bone marrow wells, fetal small intestine epithelium, and normal fibroblasts.

Figure 29.31  A-1-Antitrypsin.

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CD117 (c-kit) (polyclonal), rabbit:  Anti-CD117 is a purified immunoglobulin fraction of rabbit antiserum that detects CD117, a tyrosine kinase receptor found on interstitial cells

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Figure 29.32  Syndecan-1.

of Cajal, germ cells, bone marrow stem cells, melanocytes, breast epithelium, and mast cells. This receptor is found on a wide variety of tumor cells (follicular and papillary carcinoma of thyroid; adenocarcinomas from endometrium, lung, ovary, pancreas, and breast; and malignant melanoma, endodemal sinus tumor, and small cell carcinoma) but has been particularly useful in differentiating gastrointestinal stromal tumors from Kaposi’s sarcoma and tumors of smooth muscle origin. CD138/syndecan-1:  (Figure  29.32) This marker is present in both normal and neoplastic plasma cells and plasmacytoid lymphomas. Various types of Hodgkin lymphoma may also be positive. Anti-CD138/syndecan-1 is a mouse monoclonal antibody used for immunohistochemical demonstration of this antigen. CD15 (Leu M1)  is a monoclonal antibody that recognizes the human myelomonocytic antigen lacto-N fucopentose III. It is present on greater than 95% of mature peripheral blood eosinophils and neutrophils and is present at low density on circulating monocytes. In lymphoid tissue, CD15 reacts with Reed-Sternberg cells of Hodgkin disease and with granulocytes. CD15 reacts with few tissue macrophages and does not react with dendritic cells.

Figure 29.33  Hodgkin disease.

CD20 primary antibody  (Figure 29.34) is a mouse monoclonal antibody (Clone L26) directed against an intracellular epitope of the CD20 antigen present on human B lymphocytes. This reagent may be used to aid in the identification of cells of B lymphocytic lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-CD20 antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal B lymphocytes which may also be expressed in Reed-Sternberg cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells may show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. The CD21 antigen  is a restricted B cell antigen expressed on mature B cells. The antigen is present at high denisty on follicular dendritic cells (FDC), the accessory cells of the B zones. It shows moderate labeling of B cells and a strong

Leu-M1 (CD15)  (Figure 29.33) is a granulocyte-associated antigen. Immunoperoxidase staining detects this marker on myeloid cells but not on B or T cells, monocytes, erythrocytes, or platelets. It can be detected in Hodgkin cells and Reed-Sternberg cells. Anti-CD1a  is a murine monoclonal antibody that reacts with CD1a, a nonpolymorphic MHC class I-related cell surface glycoprotein, expressed in association with β2 microglobulin. In normal tissues the antibody reacts with cortical thymocytes, Langerhans cells, and interdigitating reticulum cells. It also reacts with thymomas, Langerhans histiocytosis cells (histiocytosis X), and some T cell lymphomas and leukemias. The staining is localized on the membrane.

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Figure 29.34  CD20—tonsil.

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labeling of FDC in cryostat sections, whereas the staining of B cells is reduced or abolished in paraffin sections. However, the labeling of FDC in paraffin sections is as strong as on cryostat sections.  he antibody reacts with FDC meshwork in normal and T hyperplastic lymph nodes and tonsils. Sharply defined, dense meshwork of FDC in germinal centers is revealed. Follicular mantles of secondary and primary follicles show a loosely textured and ill-defined meshwork of FDC. I mmunohistological analysis of FDC in paraffin sections of non-Hodgkin lymphomas demonstrates a nodular and usually a dense and sharply defined FDC meshwork in follicular lymphomas (e.g., centroblastic/centrocytic lymphoma) and a loose, ill-defined FDC meshwork of varying size in some diffuse lymphoma types (e.g., centrocytic lymphoma). Precursor B cell lymphomas (lymphoblastic lymphomas), Burkitt lymphomas, plasmacytomas, and hairy cell leukemias constantly lack FDC. FDC in non-Hodgkin lymphomas is mainly restricted to peripheral T cell lymphomas of angioimmunoblastic lymphadenopathy (AILD) type and some cases of pleomorphic T cell lymphomas. The FDC meshwork in AILD contains constant hyperplastic venules in contrast to pleomorphic T cell lymphomas. In contrast to B cell lymphomas, the FDC meshworks in T cell lymphomas and AILD contain only a relatively small number of B cells. CD23(1B12):  Anti-CD23 mouse monoclonal antibody is a B cell antibody that is useful in differentiating between B-CLL and B-SLLs that are CD23 positive from mantle cell lymphomas and small cleaved lymphomas that are CD23 negative. This antibody reacts with the antigen that is found on a subpopulation of peripheral blood cells, B lymphocytes, and on EBV transformed B-lymphoblastoid cell lines. CD3  antibody has been considered the best all-around T cell marker. This antibody reacts with an antigen present in early thymocytes. The positive staining of this marker may represent a sign of early commitment to the T cell lineage. Antihuman Ki-1 antigen, CD30  is a mouse monoclonal antibody that reacts with a 595-amino acid transmembrane, 121-kDa glycoprotein. It contains six cysteine-rich motifs in the extracellular domain and is homologous to members of the nerve growth factor receptor superfamily. The CD30 gene was assigned to the short arm of chromosome 1 at position 36. The CD30 antigen was initially designated Ki-1. The antibody detects a formalin-resistant epitope on the 90-kDa precursor molecule. This molecule is processed in the Golgi system into the membrane-bound phosphorylated mature 120-kDa glycoprotein and into the soluble 85-kDa form of CD30, which is released from the supernatant and appears in serum at detectable levels in conditions such as infectious mononucleosis or neoplastically amplified CD30-positive blasts. The CD30 antigen is expressed by Hodgkin and ReedSternberg cells in Hodgkin disease, by the tumor cells of a

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Figure 29.35  CD30—Hodgkin disease.

majority of anaplastic large-cell lymphomas, and by a varying proportion of activated T and B cells. It is also expressed on embryonal carcinomas. Ki-1 (CD30 antigen)  is a marker of Reed-Sternberg cells found in Hodgkin disease of the mixed-cellularity, nodular-sclerosing, and lymphocyte-depleted types and in selected cases of large-cell non-Hodgkin lymphomas (Figure 29.35). CD31 (JC/70A):  Anti-CD31 mouse monoclonal antibody detects CD31 expressed by stem cells of the hematopoietic system and is primarily used to identify and concentrate these cells for experimental studies as well as for bone marrow transplantation. Endothelial cells also express this marker; therefore, antibodies to CD31 have been used as a tool to identify the vascular origin of neoplasms. CD31 has shown to be highly specific and sensitive for vascular endothelial cells. Staining of nonvascular tumors (excluding hematopoietic neoplasms) has not been observed. Anti-CD34  (Figure 29.37) is a murine monoclonal antibody, raised by immunization with human placental endothelial

Figure 29.36  CD4.

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Figure 29.37  CD34—highly vascular tumor.

Figure 29.38  CD45—tonsil.

cells, that has a specificity for the CD34 glycoprotein, which is considered the earliest known CD marker and is expressed on virtually all human hematopoietic progenitor cells.

binds to antigens located predominantly in the plasma membrane and to a lesser degree in the cytoplasm of lymphocytes, with variable reactivity to monocytes/histiocytes, and polymorphonuclear leukocytes. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasional stromal elements surrounding heavily stained tissues and/or cells would show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological features and evaluation of proper controls.

CD4:  (Figure  29.36) A 55kD glycoprotein found on surfaces of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells. Previous names for this antigen include leu-3 and T4. Anti-CD4 is a rabbit monoclonal antibody employed for immunophenotyping of reactive lymphocytes and lymphoproliferative disorders. Most peripheral T cell lymphomas are of helper T cell subset origin. Most postthymic T cell neoplasms are CD4+ CD8–. Neoplastic T cells undergo antigenic deletion in which the T cell antigen CD4 is aberrantly eliminated. CD4 is detected immunohistochemically using a rabbit monoclonal antibody. Anti-CD43  is a murine monoclonal antibody directed against an epitope present on human monocytes, granulocytes, and lymphocytes. This reagent may be used to aid in the identification of cells of lymphoid lineage. It is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-CD43 antibody specifically binds to antigen located in the plasma membrane and cytoplasmic regions of normal granulocytes or T lymphocytes. Common leukocyte antigen (LCA) (CD45)  is an antigen shared in common by both T and B lymphocytes and expressed, to a lesser degree, by histiocytes and plasma cells. By immunoperoxidase staining, it can be demonstrated in sections of paraffin-embedded tissues containing these cell types. Thus, it is a valuable marker to distinguish lymphoreticular neoplasms from carcinomas and sarcomas (Figure 29.38). Anti-CD45R (leukocyte common antigen)  is a mouse monoclonal antibody specific for an epitope present on the majority of human leukocytes. This reagent may be used to aid in the identification of cells of lymphocytic lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. It specifically

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Anti-T cell (CD45RO)  antibody reacts with CD45RO determinant of leukocyte common antigen. It reacts with most T lymphocytes, macrophages, and Langerhans cells of normal tissues. It also reacts with peripheral T cell lymphomas, T cell leukemia, histiocytosis, and monocytic leukemia with mature phenotype. It reacts very rarely with B cell lymphoma and leukemia. UCHL1 antihuman T cell, CD45RO  is a mouse monoclonal antibody that recognizes specifically the 180-kDa isoform of CD45 (leukocyte common antigen). The 180-kDa glycoprotein occurs on most thymocytes and activated T cells, but only a proportion of resting T cells. This antibody and antibodies to the high-molecular-weight form of CD45 (CD45R) seem to define complementary, largely nonoverlapping populations in resting peripheral T cells demonstrating heterogeneity within the CD4 and CD8 subsets. The antibody labels most thymocytes, a subpopulation of resting T cells within both the CD4 and CD8 subsets, and mature activated T cells. Cells of the myelomonocytic series, e.g., granulocytes and monocytes, are also labeled, whereas most normal B cells and NK cells are consistently negative. Weak cytoplasmic staining is however seen in cases of centroblastic and immunoblastic lymphoma. Anti-CD5 monoclonal antibody  detects CD5 antigen, which is expressed in 95% of thymocytes and 72% of peripheral blood lymphocytes. In lymph nodes, the main reactivity

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Figure 29.39  CD56.

is observed in T cells. CD5 antigen is expressed by many T cell leukemias, lymphomas, and activated T cells. CD5 antigen is also expressed on a subset of B cells. CD5 is recommended for the identification of mantle cell lymphomas. Antibodies to CD5 may prove to be of particular use in the detection of T cell acute lymphocytic leukemias (T-ALL), some B cell chronic lymphocytic leukemias (B-CLL), as well as B and T cell lymphomas. CD5 does not react with granulocytes or monocytes. CD56:  (Figure  29.39) Two neural cell adhesion molecule proteins. The basic molecule is expressed on most neuroectodermally derived cell lines, tissues, and neoplasms, including retinoblastoma, medulloblastomas, astrocytomas, neuroblastomas, and small cell carcinomas. Selected tumors of mesodermal derivation such as rhabdomyosarcoma, as well as natural killer cells and NK lymphomas, may also express the antigen. It is detectable by immunohistochemical staining using an anti-CD56 mouse monoclonal antibody. CD61:  (Figure  29.40) The IIIa subunit of the noncovalently linked glycoprotein heterodimer IIb/IIIa complex found on human platelets and the cells giving rise to them. Anti-CD61 is a mouse monoclonal antibody used to identify

Figure 29.40  CD61.

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Figure 29.41  CD68—tonsil.

megakaryoblastic differentiation as occurs in megakaryoblastic leukemia. Anti-CD68 (human macrophage marker)  (Figure 29.41) is a murine monoclonal antibody that stains macrophages and a wide variety of human tissues, including Kupffer cells and macrophages in the red pulp of the spleen, in the lamina propria of the gut, in lung alveoli, and in bone marrow. Antigen-presenting cells, such as Langerhans cells, are either negative or show weak and/or restricted areas of reactivity, e.g., interdigitating reticulum cells. Resting microglia in the normal white matter of the cerebrum and microglia in areas of infarction react with the antibody. Peripheral blood monocytes are also positive, with a granular staining pattern. The antibody reacts with myeloid precursors and peripheral blood granulocytes. The antibody also reacts with the cell population known as “plasmacytoid T cells” which are present in many reactive lymph nodes and which are believed to be of monocyte/macrophage origin. The antibody stains cases of chronic and acute myeloid leukemia, giving strong granular staining of the cytoplasm of many cells, and also reacts with rare cases of true histiocytic neoplasia. The positive staining of normal and neoplastic mast cells is seen with the antibody as well as staining of a variable number of cells in malignant melanomas. Neoplasms of lymphoid origin are usually negative, although some B cell neoplasms, most frequently small lymphocytic lymphoma and hairy cell leukemia, show weak staining of the cytoplasm, usually in the form of a few scattered granules. CD7:  (Figure 29.42) A 40kD cell surface glycoprotein found on the surfaces of immature and mature T cells, as well as natural killer cells. It belongs to the immunoglobulin gene superfamily and is the first T cell lineage-associated antigen to appear in T cell ontogeny. It is demonstrable in pre-thymic T cell precursors prior to CD2 expression, as well as in myeloid precursors in fetal liver and bone marrow. It is demonstrable in circulating T cells. It has been postulated to serve as an Fc receptor for IgM. CD7 is expressed more consistently

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Figure 29.42  CD7.

than any other T cell antigen in lymphoblastic lymphomas and leukemias rendering it a useful marker to identify these neoplastic proliferations. In mature post-thymic T cell neoplasm, the pan-T cell antigen was commonly found to be aberrantly absent, which may signify neoplastic conversion. This antigen, which is demonstrable with an anti-CD7 mouse monoclonal antibody is present on 85% of mature peripheral T cells, most post-thymic T cells, NK cells, selected myeloid cells, T cell acute lymphoblastic leukemia/lymphoma, acute myelogenous leukemia, and chronic myelogenous leukemia. Curiously, CD7 is absent in adult T cell leukemia/lymphoma and in Sézary cells. CD79a:  (Figure 29.43) A B cell antigen often used in association with CD20. Anti CD79a is a mouse monoclonal antibody used in immunohistochemistry to stain many of the same lymphomas that are reactive with CD20, yet it is more likely to stain precursor B lymphoid leukemias than is CD20 antibody. It is also more effective in staining plasma cell myeloma, as well as selected types of endothelial cells. It is frequently positive in promyelocytic leukemia (FAB-M3), but is rarely detectable in other types of myeloid leukemia. CD8:  (Figure  29.44) A T cell antigen expressed by cytotoxic/suppressor T cell subset, NK cells, most thymocytes,

Figure 29.43  CD79a.

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Figure 29.44  CD8.

a subpopulation of null cells and bone marrow cells. AntiCD8 is a mouse monoclonal antibody that can be used in conjunction with other markers to distinguish reactive from neoplastic T cells. Cyclin D1 (polyclonal), rabbit:  Anti-Cyclin D1 is a rabbit polyclonal antibody that detects Cyclin D1, one of the key cell-cycle regulators that is a putative protooncogene overexpressed in a wide variety of human neoplasms. Cyclins are proteins that govern transitions through distinct phases of the cell cycle by regulating the activity of the cyclin-dependent kinases. In mid to late G1, Cyclin D1 shows a maximum expression following growth factor stimulation. Cyclin D1 has been successfully employed and is a promising tool for further studies in both cell-cycle biology and cancer-associated abnormalities. This antibody is useful for separating mantle cell lymphomas (Cyclin D1-positive) from SLLs and small cleaved-cell lymphomas (Cyclin D1-negative). Macrophage (HAM-56):  (Figure  29.45) Anti-macrophage or anti-HAM-56 is a mouse monoclonal antibody used to identifiable tingible macrophages, interdigitating macrophages of lymph nodes, and tissue macrophages, such as Kupffer cells of the liver and alveolar macrophages of the lung. This antibody also interacts with a subpopulation of endothelial cells such as those of capillaries and small blood

Figure 29.45  Macrophage (HAM-56).

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Figure 29.46  IgA.

vessels. Anti-HAM-56 antibody reacts with monocytes but not with B and T lymphocytes. Antihuman hemoglobin  is a rabbit antibody against hemoglobin A, isolated from erythrocytes of normal adults, that reacts with hemoglobin A and, due to a common alpha chain, also with hemoglobin A2 and hemoglobin F. IgA:  (Figure 29.46) Anti-IgA is a rabbit polyclonal antibody preparation that interacts with surface immunoglobulin IgA alpha chains. It serves as an aid in the identification of leukemias, plasmacytomas, and B cell lineage-derived Hodgkin lymphoma. This antibody facilitates the identification of B cell lymphomas, plasmacytoma in which there is restricted expression of heavy and light chains. IgG:  (Figure  29.47) Anti-IgG rabbit polyclonal antibody interacts with surface immunoglobulin IgG gamma chains. It facilitates the identification of leukemias, plasmacytomas, and B cell lineage-derived Hodgkin lymphoma. Clonal gene rearrangement is useful in the demonstration of B cell lymphomas in which there is heavy- and light-chain expression. IgM:  (Figure  29.48) Anti-IgM rabbit polyclonal antibody interacts with surface immunoglobulin IgM mu chains. IgM is the predominant immunoglobulin on the surfaces of B lymphocytes. Anti-IgM polyclonal antibody facilitates identification of lymphomas, plasmacytomas, and B cell lineage-

Figure 29.47  IgG.

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Figure 29.48  IgM.

derived Hodgkin lymphoma. Clone gene rearrangement studies facilitate the demonstration of B cell lymphomas because of restricted expression of heavy- and light-chain genes in these diseases. Immunoglobulin  is demonstrable by immunoperoxidase staining of plasma cell and B lymphocyte cytoplasm in frozen or paraffin-embedded sections. B-5 fixative is preferable to formalin for demonstration of intracellular IgG or light chains in paraffin sections. Monoclonal cytoplasmic staining for either κ or λ light chains aids the diagnosis of B cell lymphomas. Antihuman kappa light chain  (Figure  29.49) is a rabbit antibody that reacts with free kappa light chains as well as kappa chains in intact immunoglobulin molecules. This antibody may be used for typing of free and bound monoclonal light chains by immunoelectrophoresis and immunofixation. It may also be used for immunohistochemistry. Antihuman lambda light chain  is a rabbit antibody that reacts with free lambda light chains as well as the lambda light chains in intact immunoglobulin molecules.

Figure 29.49  Kappa light chain—tonsil.

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Figure 29.50  Myeloperoxidase—bone marrow.

Lysozyme:  (Figure  29.51) Anti-lysozyme is rabbit polyclonal antibody that interacts with myeloid cells, histiocytes, granulocytes, macrophages, and monocytes in human tonsil, colon, and skin. It identifies the myeloid or monocytic nature of acute leukemia. Anti-lysozyme antibody restrictive staining suggests that reactive histiocytes rather than resting unstimulated phagocytes may represent the predominant sites of synthesis of lysozyme. Anti-lysozyme rabbit polyclonal antibody is useful in the identification of histiocytic neoplasms, large lymphocytes and in the classification of lymphoproliferative disorders. Antihuman myeloperoxidase antibody  (Figure  29.50) is an antibody that is used to discriminate between lymphoid leukemias and myeloid leukemias in formalin-fixed, paraffin-embedded tissues. PAX-5:  (Figure  29.52) PAX-5 encodes for B cell-specific activator protein (BSAP), an antigen for B cells, including B-lymphoblastic neoplasms and maturation stage. It is positive in the majority of cases of mature and precursor B cell non-Hodgkin lymphoma/leukemias. Reed-Sternberg cells express PAX-5 in approximately 97% of classic Hodgkin lymphoma cases. It is not demonstrable in multiple myeloma

Figure 29.51  Lysozyme.

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Figure 29.52  PAX-5.

and solitary plasmacytoma, rendering it useful for such differentiation. Diffuse large B cell lymphomas express PAX-5 except for those with terminal B cell differentiation. Yet, T cell neoplasms do not interact with anti-PAX-5. There is a strong association between CD20 and PAX-5 expression. TdT:  (Figure 29.53) Anti-TdT rabbit polyclonal antibody identifies an enzyme present in the nucleus of normal hematopoietic cells and normal cortical thymocytes and in the cytoplasm of bone marrow megakaryocytes. It interacts with normal cortical thymocytes and primitive lymphocytes. TdT expression occurs in over 90% of acute lymphoblastic lymphoma/ leukemia cases other than pre-B cell ALL. Normal mature T or B lymphocytes do not express TdT. Since about one-third of chronic myeloid leukemia cases are positive for TdT, it is a good indicator of a better response to chemotherapy.

Pituitary Immunoperoxidase staining of pituitary adenomas with antibodies to the pituitary hormones ACTH, GH,

Figure 29.53  TdT.

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thyroid stimulating hormone (TSH) in thyrotrophic cells and in certain pituitary tumors.

Neuroendocrine/Brain Calcitonin  (Figure  29.55) is a hormone that influences calcium ion transport. Immunoperoxidase staining demonstrates calcitonin in thyroid parafollicular or C cells. It serves as a marker characteristic of medullary thyroid carcinoma and APUD neoplasms. Lung and gastrointestinal tumors may also form calcitonin.

Figure 29.54  Follicle-stimulating hormone (FSH)—pituitary.

prolactin, FSH, and LH facilitates definition of their clinical phenotype. Adrenocorticotrophic hormone (ACTH) antibody  is a polyclonal antibody preparation useful in immunoperoxidase procedures to stain corticotroph cells of the pituitary gland and benign and malignant tumors arising from these cells, in formalin-fixed, paraffin-embedded tissue biopsies. Antihuman follicle-stimulating hormone (FSH) antibody  (Figure  29.54) is a rabbit antibody that labels gonadotropic cells in the pituitary. Positive staining for adenohypophyseal hormones assists in the classification of pituitary tumors. FSH is an adenohypophyseal glycoprotein hormone found in gonadotropic cells of the anterior pituitary gland of most mammals. Gonadotropic cells average about 10% of anterior pituitary cells. This antibody can be used for immunohistochemical staining. Antigrowth hormone (GH) antibody  is a rabbit polyclonal antibody against human growth hormone that positively stains the growth hormone-producing cells and somatotrophs of the pituitary gland and malignant and benign neoplasms arising from these cells.

Chromogranin monoclonal antibody  (Figure  29.56) is used to recognize chromogranin A (68 kDa) and other related chromogranin polypeptides from human, monkey, and pig. It is designed for the specific and quantitative localization of human chromogranin in paraffin-embedded and frozen tissue sections. It aids the localization of secretory storage granules in endocrine cells. Chromogranin A is a large, acidic protein present in catecholamine-containing granules of bovine adrenal medulla. It may be widely distributed in endocrine cells and tissues, which share some common characteristics and are known as APUD cells. Dispersed throughout the body, they are also referred to as the diffuse neuroendocrine system (DNES). Chromogranin has been demonstrated in several elements of the DNES, including anterior pituitary, thyroid parafollicular C cells, parathyroid chief cells, pancreatic islet cells, intestinal enteroendocrine cells, and tumors derived from these cells. Chromogranin immunoreactivity has also been observed in the thymus, spleen, lymph nodes, fetal liver, neurons, the inner segment of rods and cones, the submandibular gland, and the central nervous system. Chromogranin is a widespread histological marker for polypeptide producing cells (APUD) and the tumors derived from them. Fascin (55k-2), mouse:  Fascin is a very sensitive marker for Reed-Sternberg cells and variants in nodular sclerosis, mixed cellularity, and lymphocyte depletion Hodgkin disease. It is

Antihuman luteinizing hormone (LH)  is a rabbit antibody that labels gonadotropic cells of the pituitary. Positive staining for adenohypophyseal hormones assists in classification of pituitary tumors. Luteinizing hormone (LH) is an adenohypophyseal glycoprotein hormone found in gonadotropic cells of the anterior pituitary gland of most mammals. Gonadotropic cells average about 10% of anterior pituitary cells. Antiprolactin antibody  is a rabbit antibody that gives positive staining of the prolactin cells of the anterior pituitary and benign and malignant neoplasms derived from these cells. Antihuman thyroid-stimulating hormone (TSH)  is a rabbit antibody used for the immunochemical detection of

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Figure 29.55  Calcitonin—medullary carcinoma of the thyroid.

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Figure 29.56  Chromogranin—pancreas.

Figure 29.57  Glial fibrillary acidic protein—brain.

uniformly negative in lymphoid cells, plasma cells, and myeloid cells. Fascin is positive in dendritic cells. This marker might be helpful in distinguishing between Hodgkin disease and non-Hodgkin lymphoma in difficult cases. Also, the lack of expression of fascin in the neoplastic follicles in follicular lymphoma can be helpful in distinguishing these lymphomas from reactive follicular hyperplasia in which the number of follicular dendritic cells is normal or increased.

Inhibin, alpha (R1), mouse:  Anti-Inhibin alpha is an antibody against a peptide hormone which has demonstrated utility in differentiation between adrenal cortical tumors and renal cell carcinoma. Sex cord stromal tumors of the ovary, as well as trophoblastic tumors, also demonstrate cytoplasmic positivity with this antibody.

Antihuman gastrin  is a rabbit antibody that labels G-cells of antropyloric mucosa of the stomach. It permits immunohistochemical detection of gastrin-secreting tumors and G-cell hyperplasia. Antiglial fibrillary acidic protein (GFAP) antibody  is a rabbit polyclonal antibody directed against glial fibrillary acidic protein present in the cytoplasm of most human astrocytes and ependymal cells. This reagent may be used to aid in the identification of cells of glial lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-GFAP antibody specifically binds to the glial fibrillary acidic protein located in the cytoplasm of normal and neoplastic glial ells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. Glialfibrillary acidic protein (GFAP)  (Figure  29.57) is an intermediate filament protein constituent of astrocytes, which is also abundant in glial cell tumors. The immunoperoxidase technique employing monoclonal antibodies against the GFAP is used in surgical pathologic diagnosis to identify tumors based on their histogenetic origin. Antihuman glucagon antibody  is a rabbit antibody that labels A cells of the endocrine mammalian pancreas.

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Monoclonal antiinsulin antibody  is an antibody used for the immunohistochemical localization of the polypeptide hormone insulin that is the most reliable means to accurately characterize the functional repertoire of islet cell tumors. Islet cell neoplasms of the pancreas appear as solitary or multiple circumscribed lesions that contrast sharply with the neighboring pancreatic parenchyma. These tumors are grouped on the basis of their predominant secretory hormone. This monoclonal antibody is used for the specific and qualitative localization of insulin in routinely fixed paraffin-embedded or frozen tissue sections. A neurofilament  is a marker, demonstrable by immunoperoxidase staining, for neural-derived tumors as well as selected endocrine neoplasms with neural differentiation. Neurofilament (2F11), mouse antibody:  Neurofilament antibody stains an antigen localized in a number of neural, neuroendocrine, and endocrine tumors. Neuromas, ganglioneuromas, gangliogliomas, ganglioneuroblastomas, and neuroblastomas stain positively for neurofilament. Neurofilament is also present in paragangliomas and adrenal and extra-adrenal pheochromocytomas. Carcinoids, neuroendocrine carcinomas of the skin, and oat cell carcinomas of the lung also express neurofilament. Neuron-specific enolase (NSE) antibody  is a murine monoclonal antibody directed against γ-γ enolase present on most human neurons, normal and neoplastic neuroendocrine cells, and some megakarocytes. This reagent may be used to aid in the identification of cells of neural or neuroendocrine lineage. The antibody is intended for qualitative staining in

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Figure 29.58  Neuron-specific enolase (NSE)—pancreas.

Figure 29.59  Synaptophysin—pancreas.

sections of formalin-fixed, paraffin-embedded tissue. AntiNSE antibody specifically binds to the γ-γ enolase located in the cytoplasm of normal and neoplastic neuroendocrine cells. Unexpected antigen expression or loss of expression may occur, especially neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells will show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

by the mononuclear phagocyte system. Immunoperoxidase staining for 5-HT, which is synthesized by various neoplasms, especially carcinoid tumors, is a valuable aid in surgical pathologic diagnosis of tumors producing it.

Neuron-specific enolase (NSE)  (Figure 29.58) is an enzyme of neurons and neuroendocrine cells, as well as their derived tumors, e.g., oat cell carcinoma of lung, demonstrable by immunoperoxidase staining. NSE occurs also in some neoplasms not derived from neurons or endocrine cells. Neurofilament (2F11), mouse:  Neurofilament antibody stains an antigen localized in a number of neural, neuroendocrine, and endocrine tumors. Neuromas, ganglioneuromas, gangliogliomas, ganglioneurobla­stomas, and neuroblastomas stain positively for neurofilament. Neurofilament is also present in paragangliomas and adrenal and extra-adrenal pheochromocytomas. Carcinoids, neuroendocrine carcinomas of the skin, and oat cell carcinomas of the lung also express neurofilament. Serotonin (5-hydroxytryptamine [5-HT])  is a 176-mol-wt catecholamine found in mouse and rat mast cells and in human platelets that participates in anaphylaxis in several species such as the rabbit but not in humans. It induces contraction of smooth muscle, enhances vascular permeability of small blood vessels, and induces large blood vessel vasoconstriction. 5-HT is derived from tryptophan by hydroxylation to 5-hydroxytryptophan and decarboxylation to 5-hydroxytryptamine. In man, gut enterochromaffin cells contain 90% of 5-HT, with the remainder accruing in blood platelets and the brain. 5-HT is a potent biogenic amine with wide species distribution. 5-HT may stimulate phagocytosis by leukocytes and interfere with the clearance of particles

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Antisomatostatin antibody  is a rabbit antibody that can be used for the immunohistochemical staining of somatostatin in tumors and hyperplasias of pancreatic islets. Synaptophysin  (Figure 29.59) is a neuroendocrine differentiation marker that is detectable by the immunoperoxidase technique used in surgical pathologic diagnosis. Tumors in which it is produced include ganglioneuroblastoma, neuroblastoma, ganglioneuroma, paraganglioma, pheochromocytoma, medullary carcinoma of the thyroid, carcinoid, and tumors of the endocrine pancreas. Antihuman synaptophysin antibody  is a rabbit antibody that reacts with a wide spectrum of neuroendocrine neoplasms of neural type including neuroblastomas, ganglio­ neuroblastomas, ganglioneuromas, pheochro­mocytomas, and chromaffin and nonchromaffin paragangliomas. The antibody also labels neuroendocrine neoplasms of epithelial type including pituitary adenomas, islet cell neoplasms, medullary thyroid carcinomas, parathyroid adenomas, carcinoids of the bronchopulmonary and gastrointestinal tracts, neuroendocrine carcinomas of the bronchopulmonary and gastrointestinal tracts, and neuroendocrine carcinomas of the skin. Tau antibody:  A neuronal microtubule associated protein present mainly on axons. Its function is to facilitate polymerization of tubulin and stabilize microtubules. However, it also links selected signaling pathways to the cytoskeleton. In its hyperphosphorylated form, tau is a major component of pair helical filaments (PHF) and neurofibrillary lesions in Alzheimer’s disease (AD) brain. Hyperphosphorylation blocks the microtubule binding capability of tau, leading to destabilization of microtubules in AD brains, ultimately

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Diagnostic Immunohistochemistry

leading to neuron degeneration. Hyperphosphorylated tau may also occur in other central nervous system diseases. Multiple serine/threonine kinases, such as GSK# beta, PKA, Cdk5, and casein kinase II can phosphorylate tau. Recombinant human tau protein expressed by E. coli microorganisms is useful for the generation of antibodies reactive with phosphorylated and non-phosphorylated varieties of tau protein. The antibody generated identifies tau protein in Alzheimer neurofibrillary tangles. Ubiquitin antibody:  A highly conserved 8.5 kDa protein, which has an ATP-dependent function in targeting proteins for proteolytic degradation. The protein destined for degradation is first covalently linked to the C terminus of ubiquitin and the ubiquitinated complex is then identified by a complex of degradative enzymes. Ubiquitin also binds covalently to numerous pathological inclusions, which are resistant to normal degradation. Thus, these antibodies are valuable for investigation of these inclusions, including neurofibrillary tangles and paired helical filaments diagnostic of AD, Lewy bodies associated with Parkinson’s disease, and Pick bodies discovered in Pick disease, are all heavily ubiquitinated, which is revealed using ubiquitin antibodies. The antibody used for immunization can be isolated from bovine erythrocytes and coupled to chicken gamma globulin. Anti-GM1 antibodies  are antibodies found in 2 to 40% of Guillain-Barré syndrome (GBS) patients. They are mainly IgG1, or IgA, rather than IgM, even though IgM anti-GM1 antibodies have been found in a few GBS cases. Anti-GM1 are more frequent in GBS patients who experienced C. jejuni infection (up to 50% of cases). Titers are highest initially and fall as the disease progresses. These antibodies are present in spinal fluid, apparently due to disruption of the blood–nerve barrier. Anti-GM1 antibodies recognize surface epitopes on Campylobacter bacteria, stains, and possibly a saccharide identical to the terminal tetrasaccharide of GM1 that has been found in Campylobacter lipopolysaccharide. IgG anti-GM1 has been postulated to selectively injure motor nerves. ALZ-50  is a monoclonal antibody that serves as an early indicator of Alzheimer’s disease by reacting with Alzheimer’s brain tissue, specifically protein A-68.

Muscle Dysferlin:  A protein product of the 2p13 gene that is defective in Limb-Girdle Muscular Dystrophy type 2B (LGMD2B) and Miyoshi Myopathy (MM) patients. Dysferlin is a normal constituent of muscle plasma membrane, but in LGMD2B and MM patients its expression is severely diminished or lost, depending on the type of mutation. Dysferlin expression is normal in patients with other neuromuscular conditions, which makes its identification in the characterization of LGMD2B and MM. Critical immunohistochemical control is the use of an antibody to beta-spectrin to monitor membrane integrity.

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Dystrophin (Rod Domain) 1:  Duchenne muscular dystrophy (DMD) is the most devastating of the muscular dystrophies leading to progressive muscular wasting and death. Dystrophin is a 427 kD protein product of the DMB/BMD gene situated on the X chromosome at the position Xp21. Abnormalities of dystrophin expression muscle biopsies can be demonstrated using Western blotting and immunohistochemistry. While dystrophin abnormalities occur in all patients with DMD/BMD, genetic abnormalities may be detectable in up to 65% of cases. Severe DMD patients have marked dystrophin deficiency, yet patients with the milder form of Becker muscular dystrophy have less pronounced abnormalities of protein expression. Dystrophin (C-terminus)2 and Dystrophin (N-terminus)3:  Intragenic deletions may eliminate the antibody binding site of DYS1 and DYS3. Emerin:  Emery-Dreifuss muscular dystrophy (EDMD) is late an onset X-linked recessive disorder marked by slowly progressing contractures, skeletal muscle wasting, and cardiomyopathy, often manifested as heart block. Contractures may occur in the elbows, Achilles tendons, and post cervical muscles with humeroperoneal distribution early in the disease. The STA gene at Xq28 locus encodes emerin, a serine-rich 34 kD protein that is ubiquitous in tissues but is most concentrated in skeletal and cardiac muscle. It is found in the nuclear membrane of normal muscle cells. Emerin deficiency has a critical role in the pathology of EDMD. Anti-emerin antibody is useful to detect the normal STA gene product and possibly of help in the diagnosis of Xq-linked EDMD. Merosin:  The muscle-specific form of laminin, which is comprised of three chains, alpha 2, beta 1, and gamma 1. Mutations in the gene for the laminin alpha 2 chain of merosin encoded by chromosome 6 lead to a form of congenital muscular dystrophy (CMD). CMD cases that are merosin negative are marked by severe clinical phenotype in which patients rarely become capable of independent ambulation. It is associated with changes in the white matter on brain imaging. Anti-merosin antibody is useful to stain muscles, skin, or placenta. It reacts specifically with the 300 kD fragment of merosin. A critical immunohistochemical control is the use of an antibody against beta-spectrin to monitor membrane integrity. Spectrin:  (Figure  29.60) A cytoskeletal protein present in muscle, erythrocytes, and erythrocyte precursors. Antispectrin mouse monoclonal antibody facilitates the diagnosis of erythroid leukemias. Spectrin shares some cytoskeletal protein homology with dystrophin. It is the defective protein in Duchenne and Becker muscular dystrophy. There is a certain amount of subtle membrane injury that accompanies excision and freezing of muscle biopsies. Antibodies against spectrin must be also used to monitor membrane integrity. Fibers that are negative for both dystrophin and spectrin likely reflect injury, whereas fibers that are negative for dystrophin but

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Atlas of Immunology, Third Edition

Figure 29.60  Spectrin.

positive for spectrin represent abnormal dystrophin expression and signify a diagnosis of Xp21-linked muscular dystrophy. Thus, both spectrin and dystrophin antibodies must be used in immunohistochemical methods to identify Duchenne and Becker muscular dystrophy. Alpha Synuclein antibody:  Alpha Synuclein has a role in the regulation of dopamine release and transport. The soluble protein is expressed mainly in the brain and is also found in low concentrations in all tissues except liver. Alpha Synuclein in the nervous system is found mainly at presynaptic terminals and bound to membranes in dopaminergic neurons. It may be present as filamentous aggregates that represent the principal nonamyloid component of intracellular inclusions in various neurodegenerative diseases (synucleinopathies), such as Parkinson disease. Alpha Synuclein facilitates fribrillization of microtubule associated protein tau, and diminishes neuronal responsiveness to apoptotic stimuli resulting in diminished caspase 3 activation. Alpha Synuclein protein is phosphorylated mainly on serine residues.

Miscellaneous ALK Protein:  (Figure  29.61) ALK-1 is a fusion protein found in 60% of anaplastic large cell lymphomas and portends an improved prognosis in the ALK-1(+) group. This protein is detected using anti-ALK protein mouse monoclonal antibody. Normal lymphocytes and most other types of lymphomas, including Hodgkin lymphoma are negative for ALK protein. CA-19-9  is a tumor-associated antigen found on the Lewis A blood group antigen that is sialylated or in mucin-containing tissues. In individuals whose serum levels exceed 37 U/ml, 72% have carcinoma of the pancreas. In individuals whose levels exceed 1000 U/ml, 95% have pancreatic cancer. Anti-CA-19-9 monoclonal antibody is useful to detect the recurrence of pancreatic cancer following surgery and to distinguish between neoplastic and benign conditions of

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Figure 29.61  ALK protein.

the pancreas. However, it is not useful for pancreatic cancer screening. Polyclonal rabbit anti-calretinin  is intended to qualitatively detect normal and malignant mesothelial cells in formalinfixed, paraffin-embedded tissue sections using light micro­ scopy. Calretinin, a calcium-binding protein with a mol wt of 29 kDa, is a member of the large family of EF-hand proteins that also include S-100 protein. EF-hand proteins are characterized by a helix–loop–helix fold that acts as the calciumbinding site. Calretinin contains six such EF-hand stretches. It is abundantly expressed in central and peripheral neural tissues, especially in the retina and neurons of the sensory pathways. Calretinin is also consistently expressed in normal and reactive mesothelial cell lining of all serosal membranes, eccrine glands of skin, convoluted tubules of kidney, Leydig and Sertoli cells of the testis, endometrium and ovarian stromal cells, and adrenal cortical cells. Calretinin is also a sensitive and specific indicator of normal and reactive mesothelial cells in effusion cytology. This antibody is useful as part of an immunohistochemical marker panel to distinguish mesothelioma from adenocarcinoma. The combination of calretinin and E-Cadherin was shown to have high sensitivity and specificity in differentiating malignant mesothelioma from metastatic adenocarcinoma to the pleura in one study. CDX-2  (Figure 29.62) A caudal-related homeobox transcription factor expressed mainly in the adult intestinal epithelium. It is important for the development and maintenance of the intestinal mucosa. It is demonstrable by immunohistochemistry in normal intestinal epithelium nuclei. In colorectal cancer with loss of differentiation, there is also loss of CDX-2 protein expression. Anti-CDX-2 rabbit monoclonal antibody is of value in distinguishing gastrointestinal origin of metastatic adenocarcinomas and carcinoids. Many mucinous carcinomas of the ovary also stain positive for this antibody as do carcinomas of the upper gastrointestinal tract.

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Diagnostic Immunohistochemistry

Figure 29.62  CDX-2.

Figure 29.64  Hepatocyte specific antigen.

Epidermal growth factor receptor (EGFR):  EGFR represents the prototype of the type I tyrosine kinases. Its overexpression in neoplasia portends a poor prognosis and is found in tumors of the head and neck, brain, bladder, stomach, breast, lung, endometrium, cervix, vulva, ovary, esophagus, and stomach and in squamous cell carcinoma.

neoplasms as hepatoblastoma, hepatocellular carcinoma, and hepatic adenoma. It interacts with both normal adult and fetal liver tissue yielding a granular cytoplasmic staining pattern. This antibody facilitates the differentiation of hepatocellular carcinomas with adenoid features from adenocarcinomas, either primary in the liver or metastatic to the liver. It facilitates the recognition of hepatoblastoma and differentiates it from other small round-cell tumors.

Factor XIIIa:  (Figure  29.63) A proenzyme in the blood that is found in platelets, megakaryocytes, and fibroblastlike mesenchymal or histiocytic cells in the placenta, uterus, and prostate. Monocytes, macrophages, and dermal dendritic cells may also express it. Antifactor XIIIa rabbit monoclonal antibody may prove useful to differentiate dermatofibroma (90% +), dermatofibrosarcoma protuberans (25% +), and desmoplastic malignant melanoma (0% +). Positivity for this factor may also be demonstrated in capillary hemangioblastoma (100% +), hemangioendothelioma (100% +), hemangiopericytoma (100% +), xanthogranuloma (100% +), xanthoma (100% +), hepatocellular carcinoma (93% +), glomus tumor (80% +), and meningioma (80% +). Hepatocyte specific antigen:  (Figure  29.64) Antihepatocyte specific antigen mouse monoclonal antibodies are useful by immunohistochemistry in identifying both benign and malignant tumors derived from the liver, including such

Figure 29.63  Factor XIIIa.

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MART-1 (M2-7C10), mouse:  MART-1 (also known as Melan A) is a melanocyte differentiation antigen. It is present in melanocytes of normal skin and retina, nevi, and in more than 85% of melanomas. This antibody is very useful in establishing the diagnosis of metastatic melanomas. Antimelanoma primary antibody  is a mouse monoclonal antibody (clone HMB-45) raised against an extract of pigmented melanoma metastases from lymph nodes directed against a glycoconjugate present in immature melanosomes (Figure 29.65). This antibody may be used to aid in the identification of cells of melanocytic lineage. The antibody is for qualitative staining in sections of formalin-fixed, paraffinembedded tissue. This antibody binds specifically to antigens

Figure 29.65  HMB-45. Melanoma in lymph node.

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located on immature melanosomes. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Clinical interpretation must be complemented by morphological studies and evaluation of proper controls. Anti-p53 primary antibody (clone Bp53-11)  is a mouse monoclonal antibody directed against both the mutant and wild-type of the p53 nuclear phosphoprotein. Very rare normal cells express p53, but alterations in the p53 suppressor gene result in an overproduction of this protein in malignancies. This reagent may be used to aid in the identification of abnormally proliferating cells in neoplastic cell populations. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue on a Ventana automated slide staining device. Some form of antigen enhancement is required for paraffin-embedded samples. The p53 antibody specifically binds to nuclear antigen(s) associated with the normal downregulation of cell division. Increased expression of p53 in actively dividing cells is an indication of loss of function due to mutation of the p53 gene. Renal cell carcinoma:  (Figure 29.66) Anti-renal cell carcinoma is a mouse monoclonal antibody that identifies a 200kD glycoprotein found in the brush border of the proximal renal tubules. It interacts with 90% of primary renal cell carcinomas and about 85%  of metastatic renal cell carcinomas. It may also be reactive with parathyroid adenoma, rare breast carcinoma cases. Staining with this antibody is not positive in nephroblastoma, oncocytoma, mesoblastic nephroma, transitional cell carcinoma, and angiomyolipoma. Tyrosinase:  (Figure 29.67) An enzyme that has a role in the biosynthesis of melanin. Anti-tyrosinase mouse monoclonal antibody used in immunoperoxidase techniques identifies melanotic lesions, including malignant melanoma and melanotic neurofibroma. Carcinomas are negative for this marker.

Figure 29.67  Tyrosinase.

cystadenocarcinoma, gonadoblastoma, nephroblastoma, and desmoplastic small round-cell tumor. Adenocarcinomas of the lung are only rarely positive with this stain. 200-4 nuclear matrix protein  is a marker expressed preferentially by malignant cells rather than by normal cells. It is demonstrated by immunoperoxidase staining. An epithelial cell adhesion molecule (EpCAM)  is considered a pan-carcinoma antigen. It is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon, and lung, whereas its expression in normal tissue is very limited. Anti-LN1  is a mouse monoclonal antibody against a sialoglycan antigen (CDw75) on cell membranes. In lymphoid tissues, the antibody reacts strongly with the B lymphocytes in the germinal centers, but only faintly with B lymphocytes of the mantle zone. No reaction is observed with T lymphocytes. LN1 also reacts with certain epithelial cells, including cells of the distal renal tubules, breast, bronchus, and prostate.

WT1:  (Figure 29.68) A suppressor gene found on chromosome 11p13. Wilms tumor protein (WT1) is found in proliferative mesothelial cells, malignant mesothelioma, ovarian

Antipancreatic polypeptide (PP) antibody  is a polyclonal antibody that detects pancreatic polypeptide in routinely fixed paraffin embedded or frozen tissue sections. Hyperplasia of pancreatic polypeptide-containing cells (PP cells) is often seen in patients with juvenile diabetes, chronic pancreatitis, and islet cell tumors. Hyperplasia of PP cells (greater than 10% of

Figure 29.66  Renal cell carcinoma.

Figure 29.68  WT1.

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Diagnostic Immunohistochemistry

895

staining correlates with gene amplification. In the case of breast cancer, c-erb-B2 expression has been shown to be associated with poor prognosis. Between 15 and 30% of invasive ductal cancers are positive for c-erb-B2. Almost all cases of Paget’s disease and approximately 70% of cases of in situ ductal carcinoma are positive. Colon–ovary tumor antigen (COTA)  is a type of mucin demonstrable by immunoperoxidase staining in all colon neoplasms and in some ovarian tumors. COTA occurs infrequently in other neoplasms. Normal tissues express limited quantities of COTA. Cu-18  is a glycoprotein of breast epithelium. Immunoper­ oxidase staining identifies this marker in most breast tumors and a few tumors of the ovary and lung. Stomach, pancreas, and colon tumors do not express this antigen.

Figure 29.69  P16.

the islet cell population) in the nontumoral pancreas has been observed in nearly 50% of islet cell tumors. Demonstration of increased numbers of cells secreting pancreatic polypeptide found both within the islets and between the islets is characteristic of type II hyperplasia of pancreatic islets. Antiparathyroid hormone (PTH) antibody  is a polyclonal antibody against parathyroid hormone (PTH). PTH controls the concentration of calcium and phosphate ions in the blood. A decrease in blood calcium stimulates the parathyroid gland to secrete PTH, which acts on cells of bone, increasing the number of osteoclasts and leading to absorption of the calcified bone matrix and the release of calcium into the blood. Hyperparathyroidism may be caused by adenomas, rarely by carcinomas and by ectopic PTH production. PTH is released by renal adenocarcinomas as well as by squamous cell cancers of the bronchus. Anti-Ri antibody  is an antibody found in serum and spinal fluid of patients with opsoclonus without myoclonus that occurs in conjunction with gait ataxia in women with breast cancer. The anti-Ri antibody reacts with 55-kDa and 80-kDa proteins present in the nuclei of CNS neurons and breast tumor cells. The condition may remit, manifest exacerbations and remissions, and occasionally respond to steroids or other immune interventions. CA-15-3  is an antibody specific for an antigen frequently present in the blood serum of metastatic breast carcinoma patients. c-erb-B2 murine monoclonal antibody  is specific for c-erb-B2 oncoprotein which is expressed by tumor cell membranes at a level detectable by immunohistochemistry in up to 20% of adenocarcinomas from various sites including ovary, gastrointestinal tract, and breast. Immunohistochemical

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Hanganitziu-Deicher antigen  is an altered ganglioside present in certain human neoplasms (CD3, GM1, and terminal 4NAcNeu). Human milk-fat globulin (HMFG)  is a human milk glycoprotein on secretory breast cell surfaces. Many breast and ovarian carcinomas are positive for HMFG. Intermediate filaments  are 7- to 11-nm diameter intracellular filaments observed by electron microscopy that are lineage specific. They are intermediate in size between actin microfilaments, which are 6 nm in diameter, and microtubules, which are 25 nm in diameter. They are detected in cell and tissue preparations by monoclonal antibodies specific for the filaments and are identified by the immunoperoxidase method. The detection of various types of intermediate filaments in tumors is of great assistance in determining the histogenetic origin of many types of neoplasms. Lactalbumin  is a breast epithelial cell protein demonstrable by immunoperoxidase staining that is found in approximately one-half to two-thirds of breast carcinomas for which it is relatively specific. More than 50% of metastatic breast tumors and some salivary gland and skin appendage tumors stain positively for lactalbumin. Nonsquamous keratin (NSK)  is a marker, demonstrable by immunoperoxidase staining, that is found in glandular epithelium and adenocarcinomas but not in stratified squamous epithelium. O125 (ovarian celomic)  is a nonmucinous ovarian tumor antigen demonstrable with homologous antibody by immunoperoxidase staining. Selected mesotheliomas express this antigen as well. Anti-Purkinje cell antibody  has been detected in the circulation of subacute cerebellar degeneration patients and in those with ovarian neoplasms and other gynecologic malignancies.

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Index (Phe,G)AL, 171 (TG)AL, 171 1,3,dimethylxanthine, 423 1,3-bis (2-carboxychromon-5-yloxy-2-hydroxypropane), 414 1-chloro-2,4,6-trinitrobenzene (picryl chloride), 433 13-26 Fd’ piece. See Fd’ fragment 17-hydroxycorticosteroids (17-OHCS), 653 19 S antibody, 250 2,4-dinitro-1-fluorobenzene, 171 2,4-dinitrophenyl (DNP) group, 170 2-mercaptoethanol agglutination test, 675, 831 200-4 nuclear matrix protein, 894 33THOT, 123 4-1BB, 83 4-1BB ligand (4-1BBL), 83 4-butyl-1,2-diphenyl-3,4-pyrazolidene-dione. See Phenylbutazone 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone), 433 5-hydroxytryptamine (5-HT), 416 6-Mercaptopurine (6-MP), 659 8,8’-(carbonyl-bis-[imino-3,1-phenylenecarbonylimino])-bis-1,3, 5-naphthalene trisulfonic acid, 766. See also Suramin

A α chain, 252 α heavy-chain disease, 587 α helix, 94 α naphthyl acetate esterase, 122 α-1 antichymotrypsin, 880 α-1 antitrypsin, 94 α-2-plasmin inhibitor-plasmin complexes (α2PIPC), 475, 482 α2 macroglobulin (α2M), 562 αβ cells, 108 αβ T cell receptor (αβ TCR), 330 αβ T cells, 330 αβ TCR checkpoint, 313 A blood group, 507 A-1-Antitrypsin, 880 A1AT, 94 AB blood group, 506 Abatacept, 795 ABC method, 838 Abciximab, 790 Abelson murine leukemia virus (A-MuLV), 230, 748 Ablastin, 768 ABO blood group antigen, 506 substances, 505 system, 505–506 Abortive infection, 717 Abrin, 163, 712 Absorption, 815 elution test, 507 ABVD chemotherapy, 796 Abzyme, 273 Acanthosis nigricans, 464, 559 Accessory cell, 119 Accessory molecules, 119 Acetaldehyde adduct autoantibodies, 458 Acetylcholine receptor (AChR) antibodies, 467, 545 Acetylcholine receptor (AChR) binding autoantibodies, 467 Acetylsalicylic acid, 419 Acoelomate, 802

Acquired agammaglobulinemia, 622 Acquired B antigen, 507 Acquired C1 inhibitor deficiency, 634 Acquired Immune Deficiency Syndrome (AIDS). See AIDS Acquired immunity, 152, 153, 729 Acquired immunodeficiency, 636, 637 Acquired tolerance, 439 Acridine orange, 864 ACT-2, 94 Actin, 879 Activated dendritic cell. See Dendritic cells (DC) Activated leukocyte cell adhesion molecule. See ALCAM Activated lymphocytes, 103, 337 Activated macrophages, 115–116 Activation, 106 Activation phase, 106 Activation protein-1 (AP-1), 106 Activation unit, 384, 390 Activation-induced cell death (AICD), 107 Activation-induced deaminase (AID), 279 Active anaphylaxis, 413 Active immunity, 153 Active immunization, 175 Active kinins, 410 Active site, 254 Acute basophilic leukemia, 596 Acute cellular rejection, 692 Acute disseminated encephalomyelitis, 567–568 Acute erythroid leukemia, 594 immunophenotype, 595 Acute febrile neutrophilic dermatosis (Sweet’s syndrome), 540 Acute graft rejection, 692 Acute graft-vs.-host reaction, 695 Acute humoral rejection, 693 Acute inflammation, 149 Acute inflammatory response, 149–150 Acute leukemias of ambiguous disease, 597–598 Acute lymphoblastic leukemia (ALL), 527 Acute megakaryoblastic leukemia, immunophenotype, 596 Acute monoblastic and monocytic leukemia, immunophenotype, 594 Acute myelogenous leukemias (AML), 531–532 Acute myeloid leukemia (AML), 591 with minimal differentiation, 591 Acute myeloid leukemia with maturation, immunophenotype, 591 Acute myeloid leukemia without maturation, immunophenotype, 591 Acute myelomonocytic leukemia, immunophenotype, 594 Acute poststreptococcal glomerulonephritis, 561 Acute promyelocytic leukemia with t(15;17)(q22;q12);PML-RARA, 592–593 immunophenotype, 593 Acute rejection, 692 Acute-phase proteins, 151 reactants, 151 response (APR), 150–151 serum, 729 Acyclic adenosine monophosphate (cAMP), 130 Acyclic guanosine monophosphate (cGMP), 130 Acyclovir 9 (2-hydroxyethoxy-methylguanine), 755 ADA. See Adenosine deaminase Adalimumab, 662, 791 Adaptive differentiation, 313 Adaptive immune response, 730 Adaptive immunity, 730

897

K10141_IDX.indd 897

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898 Adaptor proteins, 96, 337 Addison’s disease, 492 Addressin, 91 Adenoids, 133 Adenosine, 130 Adenosine deaminase (ADA), 311 deficiency, 627 Adenoviruses, infection and immunity, 756 ADEPT, 792 Adherent cell, 116 Adhesins, 94 Adhesion molecules, 81 assays, 82 Adhesion receptors, 83 Adjuvant, 178–179 Adjuvant disease, 578 Adjuvant granuloma, 179 Adoptive immunity, 683 Adoptive immunization, 684 Adoptive immunotherapy, 712 Adoptive tolerance, 438 Adoptive transfer, 684 Adrenal autoantibodies (AA), 463–464 Adrenal, autoimmunity and, 46 Adrenergic receptor agonists, 368–369 Adrenergic receptors, 109 Adrenocorticotrphic hormone (ACTH) antibody, 888 Adsorption, 815 chromatography, 815 Adult respiratory distress syndrome (ARDS), 548 Adult T cell leukemia-lymphoma (ATLL), 548 immunophenotype, 610 AE1/AE3 pan-cytokeratin monoclonal antibody, 872 AET rosette test (historical), 828 Afferent lymphatic vessels, 137 Affinity, 287 chromatography, 816 Affinity constant, 287 Affinity maturation, 272 Agammaglobulinemia, 618 Agar gel, 816 Agarose, 826 Agglutination, 302–303 inhibition, 303 titer, 303 Agglutinin, 303 Agglutinogen, 303 Aggregate anaphylaxis, 414 Aging, immunity and, 474–475 Agonist ligand, 166, 655 Agonist peptides, 106 Agonists, 109 Agranulocytosis, 525 Agretope, 210 Agrin, 205 AH50, 402 AICD. See Activation-induced cell death AIDS (acquired immune deficiency syndrome), 62, 637–638 acute, 638 belt, 638 dementia complex, 645 embryopathy, 641 enteropathy, 645 experimental vaccines, 778 pediatric, 648 serology, 638 treatment, 645–646 AIDS (acquired immune deficiency syndrome) virus. See Human immunodeficiency virus (HIV) AIDS encephalopathy. See AIDS dementia complex AIDS-related complex (ARC), 644–645

K10141_IDX.indd 898

Index AILA. See Angioimmunoblastic lymphadenopathy AIRE, 492 Airway hyper-responsiveness, 422, 550 Airway remodeling, 422, 550 Alanyl-tRNA synthetase autoantibodies, 468 Albumin agglutinating antibody, 513 ALCAM, 80–81 Aldesleukin, 664 Alemtuzumab, 791 Alexine (alexin), 384–385 ALG. See Antilymphocyte globulin ALK protein, 892 ALL. See Acute lymphoblastic leukemia Allele, 158–159 Allelic dropout, 159 Allelic exclusion, 226, 280 Allelic exclusion (TCR locus), 227 Allergen, 419 Allergen immunotherapy, 413 Allergenic extracts, 792–793 Allergic alveolitis. See Farmer’s lung Allergic asthma, 423 Allergic conjunctivitis, 419 Allergic contact dermatitis, 433, 538 Allergic disease immunotherapy, 413 Allergic granulomatosis, 407 Allergic orchitis, 407 Allergic reaction, 420 Allergic response, 420 Allergic rhinitis, 419 Allergoids, 420 Allergy, 30–34, 420 Alloantibody, 259 Alloantigen, 163 Alloantiserum, 688 Allogeneic, 688 bone marrow transplantation, 681 disease, 688 effect, 688 graft, 681 inhibition, 681 Allogenic, 688 Allograft, 680 fetus, 680–681 Allogroup, 259 Alloimmune hemolytic anemia, 526 Alloimmune thrombocytopenia, 525 Alloimmunization, 523, 688 Allophenic mouse, 160 Alloreactive, 688 T cell, 688 Alloreactivity, 688 Allorecognition, 688 indirect, 690 Allotope, 259, 260 Allotransplant, 680 Allotype, 258–259 suppression, 259–260 Allotypic determinant, 260 Allotypic marker. See Allotope Allotypic specificities, 261 Allotypy, 261 Alopecia areata, 475, 575 Alpha synuclein antibody, 892 Alpha-beta T cells, 108 Alpha-fetoprotein, 705, 876 ALS. See Antilymphocyte serum Altered peptide ligands (APL), 792 Altered self, 448 Alternaria species, 408 Alternative C5 convertase, 392

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899

Index Alternative complement pathway, 394–395 Alternative pathway, 395 Alternative pathway C3 convertase, 396 Alum granuloma, 179 Alum-precipitated antigen, 179 Aluminum adjuvant, 180 Aluminum hydroxide gel, 180 Alums, 179 ALVAC, 648 Alveolar basement membrane autoantibodies (ARM autoantibodies), 463 Alveolar macrophage, 120 ALZ-50, 891 Am allotypic marker, 261 Amboceptor (historical), 236 Amebocytes, 801 American Association of Immunologists (AAI) founding of, 67–69 Journal of Immunology, 69–70 support of immunology education by, 70 Amino acyl tRNA synthetases, 468 Aminoethylcarbazole (AEC), 840 Aminophylline. See Theophylline AML. See Acute myelogenous leukemias Ammonium sulfate method, 294 precipitation, 819 Amphibian immune system, 806 Amphipathic, 97 Amphiphysin autoantibodies, 470 Amphiregulin, 702 Amyloid, 582–583 Amyloid β fibrillosis, 583 Amyloid P component, 583 Amyloidosis, 581–582 ANA. See Antinuclear antibodies ANAE (a-naphthyl acetate esterase). See Nonspecific esterase AnaINH. See Anaphylatoxin inhibitor Anakinra (injection), 795 Anamnesis, 177 Anamnestic, 177–178 immune response, 177 Anaphylactic shock, 409 Anaphylactoid reaction, 415 Anaphylatoxin inactivator, 418 Anaphylatoxin inhibitor (AnaINH), 394, 418 Anaphylatoxins, 394, 417–418 Anaphylaxis, 28–30, 409 Anaplastic, 700 Anaplastic large cell lymphoma (ALCL), ALK-negative, immunophenotype, 614 Anaplastic large cell lymphoma (ALCL), ALK-positive, 612 immunophenotype, 613–614 Anavenom, 772 Ancestral haplotype, 199, 675 Anchor residues, 203 Anemia autoimmune hemolytic, 41–42 pernicious, 46–47 Anergic B cells, 441–442 Anergize, 445 Anergy, 432–433, 445 Angiodema, 404–405 Angiogenesis, 146, 405 factor, 146 Angiogenic factors, 146 Angioimmunoblastic lymphadenopathy (AILA), 533 Angiopoietins, 146 Anglogenin, 146 Angry macrophage, 116 Animal reservoir, 718 Ankylosing spondylitis, 578

K10141_IDX.indd 899

ANNA, 542 Annexin V binding, 134 Annexins, 94 Antagonist ligand, 656 Antagonists, 106 Anti-allotypic antibodies, 234 Anti-B cell receptor idiotype antibodies, 223, 662–663 Anti-bcl-2 primary antibody, 880 Anti-BCL-6 (PG-B6p) mouse monoclonal antibody, 880 Anti-BRST-2 (GCDFP-15) monoclonal antibody, 868 Anti-BRST-3 (B72.3) monoclonal antibody, 867 Anti-C1q antibody, 387 Anti-CD34, 882 Anti-CD43, 883 Anti-CD45R (leukocyte common antigen), 883 Anti-CD5 monoclonal antibody, 883 Anti-CD68 (human macrophage marker), 884 Anti-D, 511 Anti-DEX antibodies, 236 Anti-double-stranded DNA, 477, 575 Anti-dsDNA, 575 Anti-Ewing’s sarcoma marker (CD99), 878 Anti-GM antibodies, 891 Anti-high molecular weight human cytokeratin antibodies, 870 Anti-Hu antibodies, 470 Anti-I, 519 Anti-immunoglobulin antibodies, 239 Anti-intrinsic factor autoantibodies, 456 Anti-Ki-67 (MIB), 873 Anti-Ku autoantibodies, 468 Anti-La/SS-B autoantibodies, 488 Anti-LN1, 894 Anti-low molecular weight cytokeratin, 871 Anti-microbial peptides, 800 Anti-neutrophil cytoplasmic autoantibodies (pANCA), 455 Anti-p24, 641 Anti-p53 primary antibody (clone Bp53-11), 894 Anti-PCNA, 477 Anti-phospholipid antibodies. See Lupus anticoagulant Anti-PM/Scl autoantibodies, 468 Anti-Purkinje cell antibody, 895 Anti-RA-33, 483 Anti-retroviral drugs (HIV), 787 Anti-rRNP, 492 Anti-scRNP (Ro/SS-A, La/SS-B), 488 Anti-sense oligonucleotide, 156 Anti-Sm (Smith) autoantibodies, 477–478 Anti-snRNP (Sm, U1-RNP, U2-RNP), 477 Anti-SS-A, 488 Anti-SS-B, 488 Anti-T cell (CD45RO), 883 Anti-T cell receptor idiotype antibodies, 330, 662–663 Anti-target antigen antibodies, 663, 690 Anti-tau antibodies, 568 Anti-topoisomerase I (Scl 70), 580 Anti-Toxoplasma gondii antibody, 834 Anti-U1 RNP autoantibodies, 490 Antiagglutinin, 235 Antianaphylaxis, 413 Antiantibody, 239 Antibodies, 233. See also specific antibodies assays of, 62–67 immune complex-coated (iccosomes), 177 measurement of, 815 to histidyl t-RNA synthetase (anti-HRS), 489 Antibodies to Mi-1 and Mi-2, 464 Antibody absorption test, 815 Antibody affinity, 272, 287 Antibody deficiency syndrome, 619 Antibody detection, 236 Antibody excess immune complexes (ABICs), 306

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900 Antibody feedback, 273–274 Antibody fragment, 254 Antibody half-life, 271 Antibody humanization, 238 Antibody repertoire, 236 Antibody screening, 671–672 Antibody specificity, 234 Antibody synthesis, 237 Antibody titer, 236 Antibody units. See Titer Antibody-antigen intermolecular forces, 288 Antibody-binding site, 234 Antibody-dependent cell-mediated cytotoxicity (ADCC), 423, 709, 725, 726 Antibody-directed enzyme prodrug therapy (ADEPT), 709 Antibody-mediated suppression, 238 Antibody-secreting cells, 222 Antibroad-spectrum cytokeratin, 870 Anticardiolipin antibody syndrome, 475 AntiCD1a, 881 Anticentriole antibodies, 485 Anticentromere autoantibody, 487 Anticomplementary, 402 Anticytomegalovirus antibody, 756, 877 Antidesmin antibody, 878 Antiendothelial cell autoantibodies, 490 Antiepithelial membrane antigen (EMA) antibody, 872 Antiestrogen receptor antibodies, 873 Antifactor VIII, 878–879 Antifibrillarin antibodies, 485 Antigen binding site, 172 Antigen capture assay, 819 Antigen clearance, 172 Antigen excess, 305 Antigen masking, 766 Antigen presentation, 201 Antigen processing, 201–202 Antigen receptors, 110 Antigen recognition activation motif, 329 Antigen unmasking, 172 Antigen-antibody complex, 294–295 Antigen-binding capacity, 285 Antigen-binding cell (ABC) assay, 819 Antigen-binding site, 236–237 Antigen-presenting cell (APC), 205–206 Antigen-specific cells, 110 Antigen-specific suppressor cells, 176 Antigenic, 167 Antigenic competition, 172 Antigenic determinant, 161, 167 Antigenic drift, 173 Antigenic mosaicism, 173 Antigenic peptide, 164, 202 Antigenic profile, 164 Antigenic shift, 173 Antigenic variation, 172–173, 753 Antigenicity, 167 Antigens, 163. See also specific antigens assays of, 62–67 measurement of, 815 Antigliadin antibodies (AGA), 554 Antiglial fibrillary acidic protein (GFAP) antibody, 889 Antiglobulin, 512 antibodies, 512 consumption test, 829 inhibition test, 513 test, 512–513 Antiglutinin, 814 Antigranulocyte antibodies, 521 Antigrowth hormone (GH) antibody, 888 Antiheat shock protein antibodies, 96

K10141_IDX.indd 900

Index Antihepatitis B virus core antigen (HBcAg) antibody, 876 Antihistamine, 415, 795 Antihistone antibodies, 841 Antihuman chorionic gonadotropin (HCG) antibody, 876 Antihuman cytokeratin (CAM5.2), 872 Antihuman cytokeratin 7 antibody, 871 Antihuman cytokeratin-20 monoclonal antibody, 871 Antihuman follicle-stimulating hormone (FSH) antibody, 888 Antihuman gastrin, 889 Antihuman glucagon antibody, 889 Antihuman hemoglobin, 886 Antihuman kappa light chain, 886 Antihuman Ki-1 antigen, 882 Antihuman lambda light chain, 886 Antihuman luteinizing hormone (LH), 888 Antihuman myeloperoxidase antibody, 887 Antihuman α-smooth muscle actin, 879 Antihuman prostatic acid phosphatase (PSAP), 875 Antihuman synaptophysin antibody, 890 Antihuman thyroglobulin, 875 Antihuman thyroid-stimulating hormone (TSH), 888 Antiidiotypic antibody, 274 Antiidiotypic vaccine, 274, 772 Antilymphocyte globulin (ALG), 660, 690 Antilymphocyte serum (ALS), 660, 690 Antimalignin antibodies, 714–715 Antimelanoma primary antibody, 893 Antimetabolite, 658 Antimuscle actin primary antibody, 879 Antimyelin-associated glycoprotein (MAG) antibodies, 568 Antineutrophil cytoplasmic antibodies (ANCA), 542, 568 Antineutrophil cytoplasmic autoantibodies (p-ANCA), 555 Antinuclear antibodies (ANA), 476–477, 574–575 Antinucleosome antibodies, 478 Antioxidants, immunity and, 785 Antipancreatiic polypeptide (PP) antibody, 894 Antipapillomavirus, 877 Antiparathyroid hormone (PTH) antibody, 895 Antiphospholipid antibodies, 480 Antiphospholipid syndrome, 480 Antiplacental alkaline phosphatase (PLAP) antibody, 465, 876 Antiplatelet antibodies, 456, 521 Antiprogesterone receptor antibody, 874–875 Antiprolactin antibody, 888 Antiproliferative agents, 656–658 Antiprostate specific antigen (PSA) antibody, 875 Antiseptic paint, 500 Antiserum, 234–235 Antisomatostatin antibody, 890 Antisperm antibody, 570, 863 Antistreptolysin O (ASO), 819 Antithymocyte globulin (ATG), 660, 690 Antithymocyte serum (ATS), 660 Antitoxin, 235, 239 Antitoxin assay (historical), 236 Antitoxin unit, 236 Antivenin, 793 Antivenom, 237 Antivimentin antibody, 880 Antiviral state, 370 Antrypol, 766 Antrypol. See Suramin AP-1, 106 APC. See Antigen-presenting cell APC licensing, 206 APECED. See Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy Apheresis, 865 Apical, 500 Aplastic anemia, 526 APO-1, 136

3/25/10 12:57:06 PM

901

Index APO-E, 94 Apolar bonding, 286 Apolipoprotein E, 116 Apolipoprotein (APO-E), 94 Apoptosis, 134, 136, 340–341 caspase pathway, 135 immunotoxin-induced, 135–136 suppressors, 136 APT. See Alum-precipitated antigen Aquaphor, 180 Aqueous adjuvants, 180 Arachidonic acid (AA), 409 Arcitumomab, 711 Arenavirus immunity, 756 Arginine, immunity and, 785–797 Arlacel A, 180 ARM autoantibodies. See Alveolar basement membrane autoantibodies Armed cytotoxic T lymphocyte (armed CTL), 108 Armed effector T cells, 334 Armed macrophages, 118 Armed mast cells, 410 Arrhenius, Svante, 20 Artemis SCID, 625 Arthritis, 552 Arthritis panel, collagen disease and, 481 Arthropods, 803 Arthus reaction, 426–427 Arthus, Nicolas Maurice, 31 Artificial antigen, 166 Artificial passive immunity, 153 Artificially acquired immunity, 153, 731 Artificially acquired passive immunity, 731 Ascaris immunity, 763 Aschoff bodies, 552 Ascites fluid, 146 Ascoli’s test, 823–824 Asialoglycoprotein receptor (ASGP R) autoantibodies, 458 ASLT, 819 ASO, 819 Aspergillus species, 551 Aspirin (ASA), 419 sensitivity reactions, 419–420 Association costant (K A), 293 Asthma, 422, 550 Ataxia telangiectasia, 629–630 ATG. See Antithymocyte globulin Athymic nude mice, 339 Atopic, 418 Atopic allergy, 419 Atopic dermatitis, 419, 538 Atopic hypersensitivity. See Atopy Atopic rhinitis, 419 Atopy, 30–34, 418 ATRA, 796 Attenuate, 769 Attenuated, 769 Attenuated pathogen, 769 AtxBm, 230 Atypical antineutrophil cytoplasmic antibodies, 557 Auer’s colitis, 425 Autoagglutination, 447 Autoallergy, 447 Autoantibodies against lamin, 459 Autoantibodies against pepsinogen, 458 Autoantibody, 447 Autoantibody assays, 820 Autoantigens, 447 Autobody, 263 Autochthonous, 699 Autocrine, 345 Autocrine factor, 345

K10141_IDX.indd 901

Autofluorescence, 834 Autogenous vaccine, 770 Autograft, 685 Autoimmune adrenal failure, 464 Autoimmune and lymphoproliferative syndrome, 452–453 Autoimmune cardiac disease, 469 Autoimmune complement fixation reaction, 447–448 Autoimmune disease, 450 animal models of, 450 spontaneous animal models of, 450 Autoimmune gastritis, 456 Autoimmune hemolytic anemia, 41–42 warm-antibody and cold-antibody types of, 453 Autoimmune hemophilia, 453 Autoimmune hepatitis, 458 Autoimmune lymphoproliferative syndrome (ALPS), 453 Autoimmune myocarditis, 463 Autoimmune neutropenia, 453, 525 Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), 492 Autoimmune polyglandular syndromes, 464 Autoimmune response, 448 Autoimmune skin diseases, 473 Autoimmune thrombocytopenia, 453 Autoimmune thrombocytopenia purpura, 453 Autoimmune thyroiditis, 45, 451 Autoimmune tubulointerstitial nephritis, 462 Autoimmune uveoretinitis, 474 Autoimmunity, 38–39, 447 Autoinflammatory syndromes, 624 Autologous, 685 Autologous bone marrow transplantation (ABMT), 685 Autologous graft, 685 Autolymphocyte therapy (ALT), 792 Autoradiography, 840 Autoreactivity, 448 Autosensitization, 448 Avian (bird) immunity, 808 Avidity, 271, 287, 289 Avidity hypothesis, 271, 287 Avionics, 794 Avr-R system, 800 Azathioprine, 657–658 Azidothymidine, 646 Azoprotein, 172 AZT, 647

B β barrel. See β-pleated sheet β cells, 94 β lysin, 133 β propiolactone, 770 β selection, 313 β-adrenergic receptor antibodies, 474 β-pleated sheet, 94, 583 β-quinine, 796 β1A globulin, 389 β1C globulin, 389 β1E globulin, 390 β1F globulin, 391 β1H. See Factor H β2 microglobulin (β2M), 188 B allotype, 259 B cell activation, 222 B cell antigen receptor, 219 B cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), 528–529 B cell coreceptor, 219 B cell corona, 144 B cell differentiation factors (BCDF), 230

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902 B cell growth factors (BCGF), 230, 354. See also Interleukin-4; Interleukin-5; Interleukin-6 B cell leukemias, 620 B cell lymphoproliferative disorder (BCLD), 696 B cell lymphoproliferative syndrome (BLS), 663 B cell mitogens, 229 B cell receptor (BCR) complex, 219 B cell tolerance, 218, 442 B cell tyrosine kinase (Btk), 230 B cell-specific activator protein (BSAP), 230 B cell-stimulating factor 1 (BSF-1). See Interleukin-4 B cells, 109, 217 anergic, 441–442 bystander, 283 effector, 109 marginal zone, 145 B complex, 186 B genes, 186, 197, 388 B lymphocyte antigen receptor (BCR), 223 B lymphocyte hybridoma, 218, 268 B lymphocyte receptor, 218 B lymphocyte Stimulation (BlyS), 794 B lymphocyte stimulatory factors, 230 B lymphocyte tolerance, 442 B lymphocytes, 215, 217 B symptoms, 536 B-1 cells, 217 B-2 cells, 218 B-cell growth factor I (BCGF-1). See Interleukin-4 B-cell growth factor II (BCGF-2). See Interleukin-5 B-cell-stimulating factor 2 (BSF-2). See Interleukin-6 B-lymphocyte tolerance, 218 B-type virus, 756 B1a B cells (CD5), 218 B220, 100 B4, 283 B5, 283 B6, 283 B7, 107, 209 B7-2, 209 B7.1 costimulatory molecule, 107 B7.2 costimulatory molecule, 107 B9, 283 Babesiosis immunity, 763 Bacille Calmette-Guérin, 663, 781 Bacillus anthracis immunity, 738 Back typing, 509 Backcross, 687 Bacteria, 731, 800 Bacterial agglutination, 303 Bacterial allergy, 431, 732 Bacterial hypersensitivity. See Delayed-type hypersensitivity Bacterial immunity, 731 Bacterial immunoglobulin-binding proteins, 737–738 Bacterial vaccine, 771 Bactericidin, 738 Bacterin, 771 Bacteriolysin, 732 Bacteriolysis, 732 Bacteriophage neutralization test, 862 Bacteroides immunity, 737 BAFF, 230 Bagassosis, 427, 549 Baka, 521 Balancing selection, 199 balb/c mice, 812 BALT. See Bronchial-associated lymphoid tissue Band test, 572 Bare lymphocyte syndrome (BLS), 628–629 bas, 809

K10141_IDX.indd 902

Index Basement membrane, 152 antibody, 564–565 Basiliximab, 790 Basiliximab (injection), 660 Basophil-derived kallikrein (BK-A), 129 Basophilic, 129 Basophils, 129, 414 BCDF. See B cell differentiation factors BCG (Bacille Calmette-Guérin), 663, 780–781 BCGF. See B cell growth factors bcl-2, 231 bcl-2 proteins, 231 bcl-X L, 231 BDB. See Bis-diazotized benzidine Behcet’s disease, 590 Beige mice, 634 Benacerraf, Baruj, 57–58 Bence-Jones (B-J) proteins, 588 Benign lymphadenopathy, 141 Benign lymphoepithelial lesion, 488 Benign monoclonal gammopathy, 586 Benign tumor, 700 Bentonite (Al203-4SiO2-H2O), 830 Bentonite flocculation test, 303 Berger’s disease, 563–564 Berylliosis, 552 Besredka, Alexandre, 30 Beta-2 glycoprotein-1 autoantibodies, 480 Beta-adrenergic receptor autoantibodies, 492 Beta-gamma bridge, 590 Bevacizumab, 791 BFPR. See Biological false-positive reaction BI-RG-587, 647 Biclonality, 536 Bifunctional antibody, 273 Binding constant. See Dissociation constant Binding protein, 284 Binding site, 254 Biochemical sequestration, 164 Biogenic amines, 410 Biolistics, 830 Biological agents, 660–662 Biological false-positive reaction (BFPR), 736 Biological response modifiers (BRM), 381, 710, 793 Biotin-avidin system, 840 Biovin antigens, 165 BiP, 286 Birbeck granules, 122 Bird fancier’s lung, 550 Bird immunity, 808 Bis-diazotized benzidine, 828 Bispecific antibody, 272 Björkman, Pamela J., 37 BLA-36, 535 Blast cells, 132 large pyroninophilic, 140 Blast transformation, 107 Blastogenesis, 106, 338 Blastomas, 699 Blk. See Tyrosine kinase Blocking, 274–275 Blocking antibody, 275 Blocking factors, 714 Blocking test, 820 Blood group antigens, 505 Blood grouping, 505 Blood, immunological diseases and immunopathology of, 525–538 Blood-thymus barrier, 310 Bloom syndrome (BS), 618 Blot, 842

3/25/10 12:57:07 PM

903

Index BLR-1/MDR-15, 536 BlyS, 372 Bm mutants, 186 BMT. See bone marrow, transplantation Bombay phenotype (Ob), 507 Bombesin, 794 Bone marrow, 133, 684–685 cells, 133, 685 chimera, 685 transplantation, 685 Bony fish, 805 Booster, 177 Booster injection, 177 Booster phenomenon, 177 Booster response, 175 Bordet, Jules Jean Baptiste Vincent, 25–26 Bordetella immunity, 738 Bordetella pertussis, 738 Borrelia immunity, 738–739 Botulinum toxin, 794 Botulism immune globulin IV (human) (BIG-IV), 787 Bovet, Daniel, 30 Bovine serum albumin (BSA), 163 Boyden chamber, 93 Bradykinin, 413 Brain death, 683 Brambell receptor (FcRB), 270 BrdU labeling, 828 Brequinar sodium (BQR), 659 Bretscher−Cohn theory, 59–60, 438 Bright, 850 BRMs. See Biological response modifiers Bromelin, 513 Bronchial asthma, 423 Bronchial-associated lymphoid tissue (BALT), 500 Bronchiectasis, 550 Bronchodilators, 550 BRST-2 (GCDFP-15) monoclonal antibody (murine), 868 Brucella immunity, 739 Brucella vaccine, 784 Brucellin, 853 Brush border, 499 autoantibodies, 462 Bruton’s disease, 617 Bruton’s X-linked agammaglobulinemia, 619 BSA. See Bovine serum albumin Btk, 619 Bubble boy, 628 Buchner, Hans, 25 Buffy coat, 862 Bullous pemphigoid, 538 Bullous pemphigoid antigen, 538 Bungarotoxin, 467 Bunyaviridae immunity, 756 Burkitt lymphoma, 536 immunophenotype, 609 Burkitt lymphoma receptor-1/monocyte-derived receptor-15, 536–537 Burnet, Frank Macfarlane, 15 Bursa equivalent, 215 Bursa of Fabricius, 215 Bursacyte, 215 Bursectomy, 215 Busulfan (1,4-butanediol dimethanesulfonate), 659 Butterfly rash, 573–574 BXSB mice, 482–483 Byssinosis, 549 Bystander activation, 105 Bystander B cells, 283 Bystander effects, 80 Bystander lysis, 80

K10141_IDX.indd 903

C C gene, 229 C gene segment, 229, 327 C region (constant region), 245 C segment, 229 C-C subgroup, 347 C-erb-B2 murine monoclonal antibody, 895 C-kit ligand, 374 C-myb, 537 C-myb gene, 310 C-reactive protein (CRP), 151–152 C-terminus, 245 C-type lectin, 95 C-X-C subgroup, 347 C1 deficiencies, 404, 634 C1 esterase inhibitor, 386 C1 inhibitor (C1 INH) deficiencies, 404, 634 C10, 347 C1q, 386–387 C1q autoantibodies, 387, 481 C1q binding assay for circulating immune complexes (CIC), 387, 832 C1q deficiency, 405, 634–635 C1q receptors, 387 C1r, 387 C1s, 387 C2, 388 C2 (complement component 2), 387–388 C2 deficiency, 405, 635 C2 genes, 197 C2a, 388 C2b, 388 C3 (complement component 3), 389 C3 convertase, 384, 385, 389 C3 deficiency, 405, 635 C3 nephritic factor (C3NeF), 397 C3 PA (C3 proactivator), 397 C3 tickover, 396 C3a, 389 C3a receptor (C3a-R), 389 C3a/C4a receptor (C3a/C4a-R), 389–390, 391 C3b, 390. See also Complement receptor 1(CR1) C3b (inactivated C3b), 396 C3b inactivator. See Factor I C3bi (iC3b), 396 C3c, 397 C3d, 397 C3dg, 397 C3e, 397 C3f, 397 C3g, 397 C3H/HeJ mice, 812 C4 (complement component 4), 390 C4 allotypes, 390 C4 deficiency, 405, 635 C4A, 391, 520 C4a, 391 C4B, 391, 520 C4b, 391 C4b inactivator. See Factor I C4b-binding protein (C4bp), 398 C4bi (iC4b), 399 C4c, 399 C4d, 399 C5 (complement component 5), 391 C5 convertase, 391 C5 deficiency, 405, 635 C5a, 391 C5a receptor (C5a-R), 391 C5a74des Arg, 391

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904 C5aR (C5 anaphylatoxin receptor), 394 C5b, 391 C6 (complement component 6), 392 C6 deficiency, 405, 635 C7 (complement component 7), 392 C7 deficiency, 405, 635 C8 (complement component 8), 392 C8 deficiency, 405, 635 C9 (complement component 9), 392 C9 deficiency, 405, 635 CA-125, 714 antibody, 869 CA-15-3, 714, 895 CA-19-9, 714, 892 Cachectin, 370 Cadaveric organ, 683 Cadherins, 91 Caecal tonsils, 146 Calcineurin, 335, 655 Calcineurin inhibitors, 653–656 Calcitonin, 714 Calcitonin, 888 Calcivirus immunity, 756 Caldesmon, 869 CALLA, 702, 714. See also Common acute lymphoblastic leukemia antigen Calmette, Albert, 32 Calnexin, 183 Calponin, 869 Calreticulin, 183 CAM. See Cell adhesion molecules CAMP. See Acyclic adenosine monophosphate Campath-1 (CD52), 661, 787 CAMPATH-1M, 661, 787 Campylobacter immunity, 739–740 Canale-Smith syndrome, 453 Cancer, 699 Cancer-testis antigens, 703 Candida immunity, 761 Canine distemper, 756 Canine immunity, 810 Canine parvovirus vaccine, 778 Canonical structure, 94 Capillary leak syndrome, 353 Caplin’s syndrome, 552 Capping, 224 Capping phenomenon, 224 Caprinized vaccine, 771 Capromab pendetide, 703 Capsid, 745 Capsular polysaccharide, 164, 752–753 Capsule. See Capsular polysaccharide Capsule swelling reaction, 732, 865 Capture assays, 820 Carbohydrate antigens, 164–165 Carcinoembryonic antigen (CEA), 704, 870 Carcinogen, 699 Carcinogenesis, 699 Carcinoma, 699 Carcinoma-associated antigens, 703 Carcinomatous neuropathy, 470 Cardiolipin, 730, 833 Cardiolipin autoantibodies, 481 Carrel, Alexis, 53–54 Carriage (HIV), 639 Carrier, 171 Carrier (person), 718 Carrier effect, 171 Carrier specificity, 171 Cartilage, immunological diseases and immunopathology of, 570 Cartilaginous fish immunity, 805

K10141_IDX.indd 904

Index Cartilaginous fishes, 805 Carwheel nucleus, 111 Cascade reaction, 402 Caseation necrosis, 742 Caseous necrosis, 741–742 Casoni test, 854 Caspase substrates, 135 Caspases, 134 Castleman disease, 535–536 Cat scratch disease, 732 Catalase, 127 Catalytic antibodies, 239 Catch-up vaccine, 771 Cathepsins, 206 Cationic proteins, 127 CBA mouse, 812 CBA/N mouse, 812 CC chemokine receptor 1 (CC CKR-1), 348 CC chemokine receptor 2 (CC CKR-2), 348 CC chemokine receptor 3 (CC CKR-3), 348 CC chemokine receptor 4 (CC CKR-4), 348 CCL2, 347 CCL21, 347 CD. See Cluster of differentiation CD1, 203, 318 CD10, 530, 702, 880 CD11, 89 CD117 (c-kit) (polyclonal), rabbit, 880 CD11a, 89 CD11b, 396 CD13, 119 CD138/syndecan-1, 881 CD14, 530 CD15, 531, 881 CD16, 113 CD19, 220 CD1a, 318 CD1b, 319 CD1c, 319 CD2, 206, 318, 882 CD20, 206, 220 CD20 primary antibody, 881 CD21, 142, 206–207, 220 CD21 antigen, 221, 881 CD22, 110, 207, 220 CD23, 531 CD23(1B12), 882 CD230, 724 CD25, 353–354 CD28, 207 CD29, 319–320 CD2R, 318 CD3, 326 CD3 complex, 326 CD30, 531, 882 CD30 antigen, 882 CD31, 86, 882 CD33, 119, 531 CD34, 90–91, 531 CD35, 400 CD4, 198, 319, 883 CD4 molecule, 198, 319 CD4 T cells, 319 CD40, 106 ligand, 107, 332 CD40-L, 107 CD41, 531 CD42a, 132 CD42b, 132 CD42c, 132 CD42d, 132

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Index CD43, 310 CD44, 89–90, 531 CD45, 100, 332, 883 CD45R, 101 CD45RA, 101 CD45RB, 100 CD45RO, 101, 883 CD5, 217, 331 CD5 B cells, 217 CD56, 113, 531, 884 CD57, 113, 531 CD59, 393 CD61, 884 CD62E, 90, 332 CD62L, 91 CD62P, 90–91 CD7, 332, 884 CD79a, 885 CD8, 198, 206, 322, 885 CD8 molecule, 199, 322 CD8 T cells, 206, 322 CD9, 118–119 CD99 (HO36-1.1), 878 CDX-2, 892 CEA. See Carcinoembryonic antigen Cecropin, 730 Celiac disease, 554 Celiac sprue, 554 Cell adhesion molecules (CAMs), 77 Cell line, 862 Cell separation methods, 862 Cell surface ligands, 81 Cell surface molecule immunoprecipitation, 862 Cell surface receptors, 81 Cell tray panel, 672–673 Cell-bound antibody (cell-fixed antibody), 250 Cell-mediated hypersensitivity, 430 Cell-mediated immune response, 339 Cell-mediated immunity (CMI), 79, 339 Cell-mediated immunodeficiency syndrome, 623 Cell-mediated lympholysis (CML) test, 847 Cell-surface immunoglobulin, 250 Cellular allergy, 430 Cellular and humoral metal hypersensitivity, 431 Cellular hypersensitivity, 430 Cellular immunity. See Cell-mediated immunity Cellular immunology, 34–37, 153 development of, 39–41 Cellular interstitial pneumonia, 552 Cellular oncogene. See Protooncogene CentiMorgan (cM), 186 Central lymphoid organs, 140 Central MHC, 190 Central tolerance, 440 Centriole antibodies, 485 Centroblasts, 142 Centrocytes, 143 Centromere autoantibodies, 487 Cerebrospinal fluid (CSF) immunoglobulins, 242 Cetuximab, 790 CFA. See Complete Freund’s adjuvant CFU. See Colony-forming unit CFU-GEMM, 375 CFU-S, 99 CGD. See Chronic granulomatous disease CGMP. See Acyclic guanosine monophosphate CH, 239 CH1, 239 CH2, 240 CH3, 240 CH4, 240

K10141_IDX.indd 905

905 CH50 unit, 833 CH50 unit, 402 Chachexia, 716 CHAD. See Cold hemagglutinin disease Chagas’ disease, 763 Challenge, 175, 769 Challenge stock, 770 Chancre immunity, 732 Chaperones, 94, 266 Charcot-Leyden crystals, 422, 550 Chediak-Higashi syndrome, 633 Chemical “splenectomy,” 653 Chemical adjuvants, 180 Chemiluminescence, 851 Chemoattractant, 93 Chemokine autoantibodies, 491 Chemokine β receptor-like 1, 347 Chemokine receptor, 347 Chemokines, 347 Chemokinesis, 93 Chemotactic assays, 631, 851 Chemotactic deactivation, 93 Chemotactic disorders, 631 Chemotactic factors, 92 Chemotactic peptide, 93 Chemotactic receptors, 92–93 Chemotaxis, 92, 723–724 defective, 629 macrophage/monocyte, 117 Chemotherapy, 796 Chicken pox (varicella), 748 Chido (Ch) antigens, 519 Chief cell autoantibodies, 473 Chimera, 686 Chimeric antibodies, 239 Chimeric protein, 284 Chimerism, 686 Chlamydia immunity, 755 Chlorambucil (4-bis(2-chloroethyl)amino-phenylbutyric acid), 659, 795 Chlorodinitrobenzene (1-chlor-2,4-dinitrobenzene), 429 Cholera toxin, 732, 744, 778–779 Cholera vaccine, 779 Cholinergic urticaria, 421 CHOP therapy, 796 Chorea, 552 Choriocarcinoma, 699 Chromatin remodeling, 156 Chromatin remodeling complexes, 156 Chromatography, 816 Chromium release assay, 862 Chromogenic substrate, 840 Chromogranin monoclonal antibody, 888 Chromosomal translocation, 528 Chronic active hepatitis, autoimmune, 458, 556–557, 751 Chronic disease, 717 Chronic fatigue syndrome (CFS), 590 Chronic graft rejection, 693 Chronic graft vasculopathy, 693 Chronic graft-vs.-host disease (GVHD), 696–698 Chronic granulomatous disease (CGD), 630 Chronic lymphocytic leukemia (CLL), 529–530 Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 600–601, 620 immunophenotype, 601 Chronic lymphocytic thyroiditis, 545 Chronic mucocutaneous candidiasis, 624 Chronic myelogenous leukemia (CML), 532 immunophenotype, 591 Chronic myeloid leukemia, 533 Chronic neutropenia, 631 Chronic progressive vaccinia, 774

3/25/10 12:57:07 PM

906 Chronic rejection, 693 Chronic xenograft rejection, 682 Chrysotherapy. See Gold therapy Churg-Strauss syndrome (allergic granulomatosis), 407, 550 CIA. See Collagen-induced arthritis CIC. See Circulating immune complexes Cicatrical ocular pemphigoid, 569–570 Ciclosporin, 653–654 CIE, 824 CIIV. See Class II vesicle Ciliary neurotrophic factor (CNTF), 357 Cimetidine, 795 Circulating anticoagulant, 481 Circulating dendritic cell, 121, 206 Circulating immune complexes (CIC), 426 Circulating lupus anticoagulant syndrome (CLAS), 481 Circulating lymphocytes, 105 Circulatory system infections, 732 Cisterna chyll. See Thoracic duct CL, 239 Clade, 639 Cladosporium species, 419 Claman, Henry, 36 Class I antigen, 187 Class I MHC molecules, 187 Class I region, 186 Class IB genes, 186 Class II antigens, 190 Class II region, 189 Class II transactivator (CIITA). See MHC class II transactivator Class II vesicle (CIIV), 210 Class III molecules, 190 Class III region, 190 Class switching (isotype switching), 279–280 Classic pathway of complement, 383 Classical C5 convertase. See C5 convertase Classical Hodgkin lymphoma (CHL), immunophenotype, 615 Classical pathway. See Classic pathway of complement Clathrin, 98 Cleveland procedure, 842 Clinical trials, 797 CLIP, 210 Clonal, 267 Clonal anergy, 443 Clonal balance, 443 Clonal deletion, 442, 663 Clonal exhaustion, 443 Clonal expansion, 443 Clonal ignorance, 440 Clonal restriction, 437 Clonal selection, 267 Clonal selection theory, 267 Clone, 111 Cloned DNA, 864 Cloned T cell line, 334 Clonotypic, 223 Clonotypic, 330 Closed enzyme donor immunoassay, 823 Clostridium immunity, 739 Clotting system, 94 Cluster of differentiation (CD), 79, 318 antigens, 79 molecules, 79 Clusterin (serum protein SP-40,40), 393 Clustering, 224 CMI. See Cell-mediated immunity CML. See Chronic myelogenous leukemia CNS prophylaxis, 796 Coagglutination, 730 Coagulation system, 94 Coated pit, 98

K10141_IDX.indd 906

Index Coated vesicles, 98 Cobra venom factor (CVF), 390 Coca, Arthur Fernandez, 31 Cocapping, 224 Coccidiodes immunity, 761–762 Coccidiodin, 744, 853 Coding joint, 228, 327 Codominant, 507 Codominantly expressed, 186 Codon, 159 Coelomate, 802 Coelomocyte, 803 Cogan’s syndrome, 569 Cognate antigen, 163 Cognate interaction, 213 Cognate recognition, 213 Cohn fraction II, 234 Coisogenic, 812 Coisogenic strains, 813 Cold agglutination, 520 Cold agglutinin syndrome, 520 Cold antibodies, 520 Cold chain (vaccination), 770 Cold ethanol fractionation, 820 Cold hemagglutinin disease, 520 Cold hypersensitivity, 421 Cold target inhibition, 820 Cold urticaria, 421 Cold-antibody autoimmune hemolytic anemia, 453 Cold-reacting autoantibodies, 453–454 Collagen, 88 type IV autoantibodies, 481 types I, II and III autoantibodies, 481 Collagen disease, arthritis panel and, 481 Collagen disease/lupus erythematosus diagnostic panel, 842 Collagen type IV (CIV22), 870 Collagen vascular disease, 481 Collagen-induced arthritis (CIA), 576 Collateral injury, 343 Collectin receptor, 387 Collectins, 387 Colocalization, 224 Colon antibodies, 554 Colon autoantibodies, 456, 554 Colon-ovary tumor antigen (COTA), 895 Colony-forming unit (CFU), 99, 375 Colony-forming units, spleen (CFU-S), 99 Colony-stimulating factors (CSFs), 373–374 Colostrum, 502 Colsogenic, 191–192 Combinatorial diversity, 228, 327 Combinatorial joining, 228 Combined immunodeficiency, 624 Combining site. See Antigen-binding site Commensal mice, 813 Common acute lymphoblastic leukemia antigen (CALLA/CD10), 527–528, 880 Common gamma chain (γc chain), 368 Common leukocyte antigen (LCA), 883 Common lymphoid progenitors, 100 Common mucosal immune system, 495 Common myeloid progenitor, 123 Common variable antibody deficiency. See Common variable immunodeficiency Common variable immunodeficiency (CVID), 622 Competitive binding assays, 820 Competitive inhibition assay, 820 Complement (C), 383 Complement activation, 385 Complement control protein (CCP) modules, 389 Complement deficiency conditions, 404, 634

3/25/10 12:57:08 PM

907

Index Complement deviation (Neisser-Wechsberg phenomenon), 402 Complement fixation assay, 402, 833 Complement fixation inhibition test, 402 Complement fixation reaction, 401, 832 Complement fixing antibody, 401 Complement inhibitors, 399 Complement membrane attack complex. See Membrane attack complex Complement multimer, 392 Complement receptor 1(CR1), 399 Complement receptor 2 (CR2), 400 Complement receptor 3 (CR3), 400 Complement receptor 4 (CR4), 400 Complement receptor 5 (CR5), 400 Complement receptors (CR), 399 Complement system, 24–28, 385 Complement-dependent cytotoxicity test, 403 Complementarity, 158 Complementarity-determining region (CDR), 240 Complementation, 111 Complete carcinogen, 699 Complete clinical response of hematopoietic neoplasms, 525 Complete Freund’s adjuvant, 182 Complex allotype, 259 Complex release activity, 426 Complotype, 190–191, 388 Concanavalin A (con A), 335 Concatamer integration, 864 Concomitant immunity, 714, 730 Conditional knockout mouse, 860 Confocal fluorescent microscopy, 835 Conformational determinant, 167 Conformational epitopes, 168 Congenic, 812 Congenic mice. See Congenic strains Congenic strains, 813 Congenital agammaglobulinemia, 619 Congenital immunodeficiencies, 62, 617–618 Congenital neutropenia, 631 Conglutinating complement absorption test, 406, 820 Conglutination, 406, 820 Conglutinin, 406 Conglutinin solid phase assay, 406, 820 Conjugate, 171 Conjugate vaccine, 783 Conjugated antigen. See Conjugate Connective tissue disease, 481–482 Connective tissue-activating peptide-III (CTAP-III), 349 Consensus sequence, 159 Constant domain, 240 Constant exon, 229 Constant region, 240 Constitutive defense system, 152, 730 Consumption test, 820 Contact dermatitis, 434 allergic, 434, 538 Contact hypersensitivity reaction, 433 Contact sensitivity (CS), 433–434 Contact system, 150 Continuous epitopes, 168 Contrasuppression, 326, 437 Contrasuppressor cell, 437 Control tolerance, 440, 820 Convalescent serum, 730 “Conventional (holoxenic) animals,” 813 Conventional mouse, 813 Convertase, 385 Cooke, Robert Anderson, 31 Coombs’ test, 511–512 Coombs, Robin R.A., 41–42 Coons, Albert Hewett, 64 Cooperation, 213

K10141_IDX.indd 907

Cooperative determinant, 168 Cooperativity, 213 Copolymer, 180 Copper deficiency, 632 immunity and, 631–632 Coprecipitation, 296, 820 Coproantibody, 502 Coral immunity, 801 Cords of Billroth, 144 Core, 641 Coreceptor, 208 Corneal response, 430 Corneal test. See Corneal response Corneal transplants, 687–688 Coronavirus immunity, 756–757 Cortex, 140 Cortical thymic epithelial cells (cTECs). See Thymic epithelial cells Corticosteroids, 651–653 Corticotropin receptor autoantibodies (CRA), 464 Corynebacterium diphtheriae immunity, 732 Costimulator, 207 Costimulatory blockade, 207 Costimulatory molecules, 207 Costimulatory signal, 207 Coulombic forces, 286 Counter electrophoresis, 824 Counter migration electrophoresis, 825 Countercurrent electrophoresis, 824 Counterimmunoelectrophoresis (CIE), 825 Coverage (vaccine), 769 Cowden syndrome, 701 Cowpox, 773 Coxsackie, 757 CpG nucleotides, 772 CR1. See Complement receptor 1 CR2. See Complement receptor 2 CR2, Type II complement receptor, 400 CR3 deficiency syndrome. See Leukocyte adhesion deficiency CREGs, 675 CREST complex, 580–581 CREST syndrome, 487 Creutzfeldt-Jakob syndrome, 755 Crithidia assay, 478, 820 Crithidia luciliae, 478, 820 CRM 197, 779 Crohn’s disease, 553–554 Cromolyn, 414 Cromolyn sodium, 412 Cross-absorption, 304 Cross-linking, 81 Cross-match testing, 676 Cross-presentation, 208 Cross-priming, 207–208 Cross-reacting antibody, 237, 304 Cross-reacting antigen, 164, 304 Cross-reaction, 303–304 Cross-reactivity, 304 Cross-sensitivity, 438 Cross-tolerance, 438 Crossed immunoelectrophoresis, 825 Crotalidae polyvalent immune Fab (ovine) (injection), 793 Crow-Fucase syndrome. See POEMS syndrome CRP. See C-reactive protein Cryofibrinogenemia, 587 Cryoglobulin, 587 Cryoglobulinemia, 587 Cryopreservation, 863 Cryostat, 835 Cryptantigens, 520 Cryptic epitopes, 168

3/25/10 12:57:08 PM

908 Cryptococcus neoformans immunity, 762 Cryptodeterminant. See Hidden determinant Cryptosporidium immunity, 762 Crystallographic antibodies, 578 CSF, 374 CSIF. See Interleukin-10 CSMAC, 204 CTL. See Cytotoxic T lymphocytes CTLA-4, 208–209 CTLA4-Ig, 209 Cκ, 239 Cλ, 239 Cu-18, 895 Cunningham plaque technique, 846 Cutaneous anaphylaxis, 422 Cutaneous basophil hypersensitivity, 435 Cutaneous immune system, 503 Cutaneous lymphocyte antigen, 503 Cutaneous sensitization, 430, 503 Cutaneous T cell lymphoma, 530 CXCL8, 347 CXCR-4, 348 Cyanogen bromide, 240 Cycle-specific drugs, 659 Cyclic neutropenia, 631 Cyclin D1 (polyclonal), rabbit, 885 Cyclooxygenase pathway, 410 Cyclophilins, 655 Cyclophosphamide (N,N-bis-[2-choroethyl]-tetrahydro-2H-1,3, 2-oxazaphosphorine-2-amine-2-oxide), 658 Cyclosporin A, 653–654 Cyclosporine, 653–654 Cyclostomes, 804 CYNAP antibodies, 675 CYNAP phenomenon. See CYNAP antibodies Cytochalasins, 762 Cytochrome b deficiency, 631 Cytochrome c, 135 Cytokeratin (34betaE12), mouse, 870–871 Cytokeratin 7 (K72), mouse, 871 Cytokeratin 8, 18, 872 Cytokine assays, 346 Cytokine autoantibodies, 346, 448 Cytokine inhibitors, 346 Cytokine receptor classes, 343 Cytokine receptor families, 343–344 Cytokine receptors, 343 Cytokine synthesis inhibitory factor. See Interleukin-10 Cytokine upregulation of HIV coreceptors, 640 Cytokine-specific subunit, 345 Cytolysin, 325 Cytolytic, 325, 402 Cytolytic reaction, 325, 403 Cytolytic T lymphocytes (CTLs), 325 Cytomegalovirus (CMV), 645, 747–748 immunity, 748 Cytomegalovirus immune globulin intravenous (human—injection), 786 Cytopathic effect (of viruses), 757 Cytophilic antibody, 237 Cytoplasmic antigens, 490 Cytosine arabinoside, 795 Cytoskeletal antibodies, 449 Cytoskeletal autoantibodies, 448–449 Cytoskeleton, 97 Cytosolicaspartate-specific proteases (CASPases), 134 Cytotoxic, 322 Cytotoxic agents, 658–659 Cytotoxic antibody, 238 Cytotoxic CD8 T cells, 324 Cytotoxic cytokines, 370 Cytotoxic drugs, 658–659

K10141_IDX.indd 908

Index Cytotoxic T cells, 323 Cytotoxic T lymphocyte precursor (CTLp), 322 Cytotoxic T lymphocytes (CTLs), 322–323, 695–696 Cytotoxicity, 238, 322, 403 natural, 113 perforin/granzyme-mediated, 112 tests, 238, 821 Cytotoxicity assays, 324, 820–821 Cytotoxicity tests, 324 Cytotoxins, 325 Cytotrophic antibodies, 238 Cytotropic anaphylaxis, 413

D δ chain, 252 D exon, 228, 282 D gene, 227 D gene region, 227 D gene segment, 227 D region, 227 D-amino acid polymers, 166 D3TX mice, female, 466 Daclizumab, 660–661 DAF. See Decay-accelerating factor Dale, Henry Hallett, 29 Dalen-Fuchs nodule, 474 Dameshek, William, 41 Dander antigen, 409 DANE particle, 750 Danger signals, 137 Danysz effect, 299–300 Danysz phenomenon, 299–300 Dapsone, 732 Dark zone, 143 DAT, 512 Dausset, Jean Baptiste Gabriel, 57 DdC (dideoxycytidine), 647 DdI (2’-,3’dideoxyinosine), 647 DDS syndrome, 732 De novo pathway of nucleic acid synthesis, 156 Dead vaccine. See Inactivated vaccine Dean and Webb titration, 295 Death by neglect, 314 Death domains, 136–137 Death receptor, 137 Decay-accelerating factor (DAF), 400–401 Decomplementation, 402 Decorate, 867 Defective endogenous retroviruses, 757 Defensins, 128 Degranulation, 414 Delayed xenograft rejection, 682 Delayed-type hypersensitivity (DTH), 430 Deletional joining, 226 Delta agent (hepatitis D virus [HDV]), 558, 751 Denaturation, 169–170 Dendritic cells (DC), 120–121 circulating, 206 follicular, 141–142 immunotherapy, 792 mature, 206 Dendritic epidermal cells, 121 Dendritic epidermal T cells (DETC), 108 Dengue, 757 Dense-deposit disease, 564 Density gradient centrifugation, 827 Deoxyguanosine. See Purine nucleotide phosphorylase Deoxyribonuclease, 157 Deoxyribonuclease I, 157 Deoxyribonuclease II, 157

3/25/10 12:57:08 PM

909

Index Deoxyribonucleoprotein antibodies, 492–493 Depot-forming adjuvants, 180 Dermatitis herpetiformis (DH), 539 Dermatitis venenata. See Contact dermatitis Dermatographism, 415 Dermatomyositis, 489, 543 Dermatopathic lymphadenitis, 141 Dermatophagoides, 419 Dermatophagoides pteronyssinus, 419 Dermatophytid reaction. See Id reaction Dermis, 152 Desensitization, 413 Desetope, 210 Designer antibody, 273 Designer lymphocytes, 708 Desmin, 878 Desmin (D33), mouse, 878 Desmoglein, 473 Desmosomes, 152 Despecification, 792 DETC. See Dendritic epidermal T cells Determinant groups (or epitopes), 169 Determinant selection model, 202–203 Determinant spreading, 449 Deuterostomes, 803 Dextrans, 167, 511 Dhobi itch, 431 Diabetes insipidus, 470 Diabetes mellitus, 558 insulin dependent (Type 1), 45–46 Diacylglycerol (DAG), 96 Dialysis, 816 Diapedesis, 126 Diathelic immunication, 773 Diazo salt, 172 Diazotization, 171–172 DIC. See Disseminated intravascular coagulation Dick test, 852 Differential RNA processing, 156 Differential signaling hypothesis, 315 Differentiation, 99 Differentiation antigen, 165 Differentiation factors, 354 Diffuse large B cell lymphoma (DLB), NOS, 606–607 immunophenotype, 607–608 subgrouping of by immunophenotyping, 608 Diffusion coefficient, 302 DiGeorge syndrome, 617, 622–623 Digestive system, immunological diseases and immunopathology of, 553–556 Dilution end point, 304 Dim, 851 Dinitrochlorobenzene (DNCB), 170 Dinitrofluorobenzene (2,4-dinitro-1-fluorobenzene) (DNFB), 171 Diphtheria and tetanus toxoids (absorbedinjection), 780 and acellular pertussis vaccine (adsorbed DTaP—injection), 780 Diphtheria antitoxin, 785 Diphtheria immunization, 779 Diphtheria toxin, 737, 779 Diphtheria toxoid, 779 Diphtheria vaccine, 779 Diploid, 158 Direct agglutination, 508 Direct allorecognition, 688 Direct amplicon analysis, 675 Direct antigen presentation, 203 Direct antiglobulin test, 513 Direct Coombs’ test, 512 Direct fluorescence antibody method, 836 Direct immunofluorescence, 836 Direct reaction, 684

K10141_IDX.indd 909

Direct staining, 836 Direct tag assays, 821 Directional selection, 199 Discoid lupus erythematosus, 575 Discontinuous epitopes. See Conformational epitopes Disease, autoimmune manifestations of, 41–52 Disodium cromoglycate, 412 Disseminated intravascular coagulation (DIC), 428 Dissociation constant, 294 Distemper vaccine, 778 Distribution ratio, 240 Disulfide bonds, 244 Diversity, 104 Diversity (D) gene segments, 228 Diversity (D) segments, 327 Dixon, Frank James, 32 Dl-β-(3,4-dihydroxyphenyl)-α-isopropylaminoethanol, 423 Dl-β-(3,5-dihydroxyphenyl)-α-isopropylaminoethanol, 423 DM, 189 DN thymocytes, 314 DNA binding motif, 156 DNA fingerprinting, 160, 844 DNA laddering, 160 DNA library, 156–157 DNA ligase, 157 DNA microarray, 844 DNA nucleotidylexotransferase, 157, 315 DNA polymerase, 157 DNA polymerase I, 157 DNA polymerase II, 157 DNA polymerase III, 157 DNA repair, 158 DNA vaccination, 772 DNA vaccine, 771 DNA-dependent RNA polymerase, 157 DNBS (2,4-dinitrobenzene sulfonate), 170 DNCB. See Dinitrochlorobenzene DNFB. See Dinitrofluorobenzene DNP. See 2,4-dinitrophenyl (DNP) group DO, 189 Doctrine of original antigenic sin, 172, 269–270 Doherty, Peter, 59 Domain, 244 Dome, 498 Dominant negative transgene, 859 Dominant phenotype, 158 Donath-Landsteiner antibody, 515 Donor, 683 Donor cell infusion, 687 Dopamine neuron autoantibodies, 470 Dot blot, 844 Dot DAT, 512 Double diffusion test, 823 Double immunodiffusion, 816 Double-emulsion adjuvant, 181 Double-layer fluorescent antibody technique, 836 Double-negative (DN) cell, 314 Double-negative thymocytes, 314 Double-positive (DP) cell, 314 Double-positive thymocytes, 314 Double-stranded DNA autoantibodies, 478–479 Doubling dilution, 817 Doughnut structure, 392 DP pause, 314 DP thymocytes, 314 DPT vaccine, 780 Draining lymph node, 141 Drakeol 6VR, 180 DRiP pathway, 202 Drug allergy, 419 Drug-induced autoimmunity, 449

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910 Drug-induced immune hemolytic anemia, 452 Drug-induced lupus (DIL), 572–573 Drug-induced lupus erythematosus, 482 DTaP vaccine, 780 DTH. See Delayed-type hypersensitivity DTH T cell, 431 Dual-tropic HIV, 639 Duffy antigen/chemokine receptor (DARC), 348 Duffy blood group, 518 Duncan’s syndrome, 619 Dye exclusion test, 861 Dye test, 861 Dysferlin, 891 Dysgammaglobulinemia, 587 Dystrophin (C-terminus)2, 891 Dystrophin (N-terminus)3, 891

E E allotype, 809 E antigen, 751 E rosette, 318 E rosette-forming cell, 318 E-cadherin, 91–92 E-selectin (CD62E), 90 E2A, 228 E32, 231 E5, 790 EAC, 403, 821 EAE. See Experimental allergic encephalomyelitis EAMG (experimental autoimmune myasthenia gravis), 467, 545 Early B cell factor (EBF), 230 Early induced responses, 152 Early phase reaction, 408 EAT, 546 EAU. See Experimental autoimmune uveitis EBF (early B cell factor), 230 EBI1, 348 EBNA (Epstein–Barr virus nuclear antigen), 757 EBV. See Epstein–Barr virus ECF-A (eosinophil chemotactic factor of anaphylaxis), 411 Echinococcus immunity, 763–764 Echinoderm leukocytes, 804 Echinoderms, 804 ECHO virus, 748 Eclipsed antigen, 165 Ecotopic, 95 ECRF3, 348 Ecto-5’-nucleotidase deficiency, 620 Eczema, 419, 629 Eczema vaccinatum, 773 Eczematoid skin reaction, 419 ED50, 821 Edelman, Gerald Maurice, 24 Edema, 150 Edible vaccine, 771 Efalizumab, 791 Effector B cells, 109 Effector function, 240 Effector lymphocytes, 103 Effector mechanism, 730 Effector phase, 730 Effector response, 730 Effector site, 498 Effector stage (hypersensitivity). See Elicitation Effector T cells. See Effector lymphocytes Efferent lymphatic vessel, 139 Ehrlich side-chain theory (historical), 18, 263 EIA, 410 Eicosanoid, 416 Eisen, Herman Nathaniel, 24

K10141_IDX.indd 910

Index ELAM-1, 87 Electroimmunodiffusion, 827 Electrophoresis, 826 Electrophoretic mobility, 826 Electroporation, 864 Elek plate, 825 Elephantiasis, 764 Elicitation, 407 ELISA, 841 ELISPOT assay, 842 Embryo fibroblast protein-1. See EMF-1 Embryonic antigens, 165, 703–704 Embryonic stem cells, 99 Emerging infectious disease, 717 Emerin, 891 EMF-1, 93 EMIT, 841 Emperipoiesis, 104 ENA antibodies, 478 ENA autoantibodies, 479 ENA-78, 93 Enactin, 561 Encapsulated bacteria, 732 Encapsulation, 734, 804 Encephalitogenic factors, 470 End cell, 111 End piece, 385 End point, 304 End-binders, 164, 732 End-point immunoassay, 304, 823 End-stage renal disease (ESRD), 564 Enders, John F., 60 Endocrine, 95 Endocytic pathway, 202 Endocytic vesicle, 118 Endocytosis, 118 Endogenous, 118 Endogenous antigen, 163 Endogenous antigen processing and presentation, 202 Endogenous pyrogen, 351 Endometrial antibodies, 466 Endometrial autoantibodies, 466 Endomysial autoantibodies, 456–457 Endophthalmitis phacoanaphylactica, 474 Endoplasmic reticulum, 97, 285–286 autoantibodies, 459 Endorinopathies, autoimmune, 46 Endosome, 98 Endothelial cell autoantibodies (ECA), 490 Endothelial leukocyte adhesion molecule-1 (ELAM-1), 86–87 Endothelin, 87 Endotoxin shock, 728–729 Endotoxins, 727–728 Engraftment, 688 Enhancement, 680 Enhanceosome, 189 Enhancer, 227 Enhancing antibodies, 680 Entactin/nidogen autoantibodies, 462 Entamoeba histolytica antibody, 764 Enteric cytopathogenic human orphan virus, 748 Enteric neuronal autoantibodies, 470 Enterocytes, 146 Enterotoxin, 729 Envelope, 642 Envelope glycoprotein (env), 643–644 Enzyme immunoassay (EIA), 841 Enzyme labeling, 841 Enzyme replacement therapy, 786 Enzyme-linked immunosorbent assay (ELISA), 841 Enzyme-multiplied immunoassay technique, 841

3/25/10 12:57:08 PM

911

Index Eosinophil cationic protein (ECP), 128–129 Eosinophil chemotactic activities, 129 Eosinophil chemotactic factors, 128 Eosinophil differentiation factor, 128, 356 Eosinophil granule major basic protein (EGMBP), 129 Eosinophilia, 454 Eosinophilic granuloma, 530 Eosinophilic myalgia syndrome (EMA), 590 Eosinophils, 128 Eotaxin, 347 Eotaxin-1, 347 Eotaxin-2, 347 eph receptors, 107 Ephrins, 107 Epibody, 263 Epidermal growth factor receptor (EGFR), 893 Epidermis, 152 Epithelial cell adhesion molecule (EpCAM), 894 Epithelial derived neutrophil attractant-78. See ENA-78 Epithelial membrane antigen (EMA), 872 Epithelial reticular cells, 307 Epithelial thymic-activating factor (ETAF), 314 Epitheliod cell, 116 Epitope spreading, 168 Epitopes, 167–168 Epitype, 168 Epivir, 647 Epstein–Barr immunodeficiency syndrome, 619 Epstein–Barr nuclear antigen, 537 Epstein–Barr virus (EBV), 537, 753, 877 Equilibrium dialysis, 292–293 Ergotype, 470 Erp57, 204 Erythema marginatum, 552 Erythema multiforme, 539–540 Erythema nodosum, 555 Erythroblastosis fetalis, 514 Erythrocyte agglutination test. See Hemagglutination test Erythrocyte autoantibodies, 508 Erythrocytes, 100 Erythroid progenitor, 99 Erythropoiesis, 99 Erythropoietin, 99 Escherichia coli immunity, 739 Essential mixed cryoglobulinemia, 588 Estradiol, 873 Estrogen/progesterone receptor protein, 872 Etanercept (injection), 662 Euglobulin, 234 Eukaryote, 156 Everolimus, 655 EVI antibodies, 474 Exchange transfusion, 523 Excitation filter, 834 Exercise, immunity and, 154 Exercise-induced asthma, 423 Exoantigen, 165 Exocytosis, 130 Exogenous, 130 Exogenous antigen, 163 Exogenous antigen processing and presentation, 202 Exon, 157 Exotoxin, 744 Experimental allergic encephalomyelitis, 566–567 Experimental allergic neuritis, 567 Experimental allergic orchitis, 590 Experimental allergic thyroiditis, 546 Experimental autoimmune encephalomyelitis, 568 Experimental autoimmune myasthenia gravis (EAMG), 545 Experimental autoimmune myocarditis (EAM), 590 Experimental autoimmune neuritis (EAN), 568

K10141_IDX.indd 911

Experimental autoimmune oophoritis, 590 Experimental autoimmune sialoadenitis (EAS), 579 Experimental autoimmune thyroiditis (EAT), 546 Experimental autoimmune uveitis (EAU), 569 Experimental tolerance, 438 Exposure of humans to xenobiotics, 491–492 Extended haplotype, 670 Extracellular antigen. See Exogenous antigen Extracellular bacteria, specific immune response to, 731 Extracellular pathogens, 733 Extramedullary plasmacytoma, 534 Extramedullary tissues, 144 Extranodal lymphoma, 535 Extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT lymphoma). See MALT lymphoma Extrathymic T cell development, 313 Extravasation, 150 Extrinsic allergic alveolitis, 549 Extrinsic apoptotic pathway. See Apoptosis Extrinsic asthma, 423 Exudate, 150 Exudation, 150 Eye, immunological diseases and immunopathology of, 568–570

F F allotype, 810 F protein, 813 F(ab′)2 fragment, 257 F-actin, 95 F-Met peptides, 93 F-Met-Leu-Phe. See Formyl-methionyl-leucyl-phenylalinine F1 hybrid resistance, 681 Fab (fragment, antigen-binding) fragment, 254–255 Fab′ fragment, 257 Fabc fragment, 257 Fab≤ fragment. See F(ab′)2 fragment Facb fragment, 257 Facilitation immunologique, 710 FACS, 850 Factor B, 397 Factor D, 397 Factor D deficiency, 397 Factor H, 397 Factor H deficiency, 398 Factor H receptor (fH-R), 397 Factor I, 398 Factor P (properdin), 395, 397 Factor VIII, 878 Factor XIIIa, 893 Facultative phagocytes, 118 FAE. See Follicle-associated epithelium Faenia rectivirgula, 548 Fagraeus-Wallbom, Astrid Elsa, 36 Familial Mediterranean fever (FMF), 624 FANA, 479 Fare technique, 828 Farmer’s lung, 548 Fas (APO-1/CD95), 137 Fas ligand, 137 Fas pathway, 137 Fascin (55k-2), mouse, 888 Fasciola immunity, 768 FasL/Fas toxicity, 137 Fatty acids, immunity and, 150 Fb fragment, 257 Fc fragment (Fc piece), 256 Fc receptor, 275–276 on human T cells, 334 Fc region. See Fc fragment (Fc piece) Fc′ fragment, 257

3/25/10 12:57:08 PM

912

Fcε receptor (FcεR), 277 Fcε receptor (FcεRI), 277 Fcγ receptors (FcγR), 276 FcγRI, 276 FcγRII, 276 FcγRIII, 276 Fd fragment (Fd piece), 255 Fd′ fragment, 257 FDCs. See Follicular dendritic cells Feline immunity, 810 Felton phenomenon, 446 Female D3TX mice, 466 Fernandez reaction, 732 Ferritin, 836–837 Ferritin labeling, 837 Fertilizin, 804 Fetal antigen, 703 Fetal thymic organ culture (FTOC), 340 Fetal-material tolerance, 439–440 Fetus allograft, 680–681 Feulgen reaction, 864 Fever, 717 Fibrin, 87–88 Fibrinogen, 87 Fibrinoid necrosis, 427 Fibrinopeptides, 87 Fibronectin, 87, 720 Fibrosis, 150 FICA (fluoroimmunocytoadherence), 816 Ficin, 513 Ficoll, 863 Ficoll-Hypaque, 863 FIGE, 827 Filarial immunity, 764 Filbrillarin autoantibodies, 485–486 Filovirus immunity, 757 Final serum diluation, 304 First-set rejection, 689 First-use syndrome, 415 FISH (fluorescence in situ hybridization), 843 Fish immunity, 805 Fixed drug eruption, 436 FK506, 654 FKBP (FK-binding proteins), 655 Flagellar antigens, 727, 728 Flagellin, 728 Flame cells, 585 Flavivirus immunity, 757 FLIP/FLAM, 135 Flocculation, 299 Flow cytometry, 676, 849–850 FLP ratio, 833 Flt3 ligand, 111 FLU/v, 775 Fludarabine, 796 Fluid mosaic model, 97 FluMist, 775 Fluorescein, 833 Fluorescein isothiocyanate (FITC), 833 Fluorescein-labeled antibody, 833 Fluorescence, 834 Fluorescence enhancement, 835 Fluorescence microscopy, 834 Fluorescence quenching, 294, 834 Fluorescence treponema antibody absorption. See FTA-ABS Fluorescence-activated cell sorter (FACS), 850–851 Fluorescent antibody, 833 Fluorescent antibody technique, 833 Fluorescent protein tracing, 835 Fluorochrome, 833

K10141_IDX.indd 912

Index Fluorodinitrobenzene. See Dinitrofluorobenzene Fluorography, 827 Fog fever, 549 Follicle-associated epithelium (FAE), 140 Follicles, 140 Follicular B cells, 143 Follicular center cells, 140 Follicular dendritic cells, 141–142 Follicular hyperplasia, 140 Follicular lymphoma, 530 Follicular lymphoma (FL), immunophenotype, 606 Food allergy, 420 Food and drug additive reactions, 421 Footprinting, 864 Footprints, 863 Forbidden clone theory, 283 Foreign gene, 859 Formol toxoid, 779 Formyl-methionyl-leucyl-phenylalinine (F-Met-Leu-Phe), 93 Forssman antibody, 175 Forssman antigen, 174–175 Forward genetics approach, 859 Foscarnet, 648 FoxP3, 441 Fractional catabolic rate, 271 Fragment crystallizable, 256 Fragmentins, 112 Framework regions (FR), 283 Francisella immunity, 739 Freemartin, 438 Frei test, 740 Freund’s adjuvant, 181 Freund’s complete adjuvant. See Freund’s adjuvant Freund’s incomplete adjuvant. See Freund’s adjuvant Freund, Jules, 64 Front typing, 509 Frustrated phagocytosis, 118 FTA-ABS, 732–733 FTY720, 660 Full chimerism, 687 Functional affinity, 272 Functional antigen. See Protective antigen Functional immunity. See Protective immunity Fungal immunity, 761 Fungi, 761 Fusin, 640 Fusobacterium immunity, 740 Fv fragment, 257 Fv region, 255 Fyn, 326–327

G γ globulin, 234 γ globulin fraction, 234 γ heavy-chain disease, 587–588 γ interferon. See Interferon γ γ macroglobulin. See Immunoglobulin M γδ, 329–330 γδ T cell receptor (TCR), 329 γδ T cells, 331 G protein-coupled receptor family, 150 G proteins, 150 G-CSF, 375 GAD-65, 465 Gag, 644 Gammapathies, 581–590 Gammopathy, 586 Gancyclovir (9-[2-hydroxy-1(hydroxymethyl) ethoxymethyl] guanine), 648 Ganglioside autoantibodies, 470–471

3/25/10 12:57:09 PM

913

Index Gas gangrene antitoxin, 786 Gastric cell cAMP stimulating autoantibodies, 457 Gastric parietal cells, autoantibodies to, 456 Gastrin receptor antibodies, 487 Gastrin-producing cell autoantibodies (GPCA), 457 GATA-2 gene, 159 Gatekeeper effect, 87 Gay bowel syndrome, 641 GCDFP-15 (23A3), mouse, 868 GEFs. See Guanine nucleotide exchange factors Gel diffusion, 816 Gel filtration chromatography, 815 Gene bank. See DNA library Gene cloning, 159, 279 Gene conversion, 157–158, 808 hypothesis, 186, 280 Gene diversity, 279 Gene knockout, 860 Gene mapping, 158 Gene rearrangement, 227, 280, 327 Gene segments, 227, 327 Gene therapy, 636 Generalized anaphylaxis, 414 Generalized vaccinia, 774 Generative lymphoid organ, 140 Genetic code, 158, 278 Genetic immunization, 773 Genetic knockout, 860 Genetic polymorphism, 159 Genetic switch hypothesis, 280 Genome, 278 Genomic DNA, 156, 278 Genomics, 156 Genotype, 158, 283 Germ line, 156 Germ-free animal, 860 Germ-line configuration, 226 Germ-line organization, 281 Germinal centers, 142 Germinal follicle. See Germinal centers Gershon, Richard K., 36 Ghon complex, 742–743 Giant cell arteritis, 490 Glatiramer acetate (injection), 791 Gld gene, 483, 813 Gld mouse, 483 Gleevec, 791 Gliadin autoantibodies, 457 Glialfibrillary acidic protein (GFAP), 889 Glick, Bruce, 36 Globulins, 234 Glomerular basement membrane autoantibodies, 462 Glomerulonephritis (GN), 560 Glucocorticoids (GCs), 652 Glucose-6-phosphate dehydrogenase deficiency, 632 Glutamic acid decarboxylase autoantibodies, 471 Gluten-sensitive enteropathy, 554 GlyCAM-1, 89 Glycocalyx, 499 Glycosylphosphatidylinositol-(GPI)-linked membrane antigens, 166 Gm allotype, 257, 261 Gm marker. See Gm allotype GM1 autoantibodies, 493 Gnotobiotic, 628 Goblet cells, 499 Gold compounds, 792 Gold therapy, 483 Golgi apparatus, 97 Golgi complex, 97 Gonads, autoimmunity and, 46

K10141_IDX.indd 913

Gonococcal complement fixation test, 832–833 Good, Robert Alan, 36 Goodpasture antigen, 463, 565 Goodpasture syndrome, 463, 565 GOR autoantibodies, 459 Gorer, Peter Alfred, 55 Gowans, James, 36 Gp160, 648 Gp41, 642 GPLA, 191, 809 Graft, 678 Graft arteriosclerosis, 693 Graft facilitation, 679–680 Graft rejection, 688–689 Graft-vs.-host disease (GVHD), 694–695 Graft-vs.-host reaction (GVHR), 693–694 Graft-vs.-leukemia (GVL), 695 Granular cells, 802 Granule exocytosis pathway, 323 Granulocyte antibodies, 525 Granulocyte autoantibodies, 454 Granulocyte chemotactic protein-2 (GCP-2), 347 Granulocyte colony-stimulating factor (G-CSF), 375 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 375 Granulocyte-monocyte colony-stimulating factor, 375 Granulocyte-specific antinuclear autoantibodies (GS-ANA), 483 Granulocytes, 123 Granulocytopenia, 454 Granuloma, 211 Granulomatous hepatitis, 557 Granulopoietin, 374 Granulysin, 325 Granzymes, 112 Graves’ disease (hyperthyroidism), 546–547 Gravity, immunity and, 153–154 Gross cystic disease fluid protein 15 (GCDFP-15) antigen, 869 Group agglutination, 744 Growth factors, 345 Guanine nucleotide exchange factors (GEFs), 150 Guillain-Barré syndrome, 471 Gut-associated lymphoid tissue (GALT), 495 GVH disease. See Graft-vs.-host disease

H H antigens, 507, 727, 733 H chain (heavy chain), 243 H substance, 507 H-2, 192 H-2 complex, 192 H-2 locus, 192 H-2 restriction, 192 H-2D, 192 H-2I region, 192 H-2K, 192 H-CAM, 82 H-chain disease, 585 H-Y, 666 H-Y antigen, 666 H-Y system, 666 H1 receptors, 411 H1, H2 blocking agents. See Antihistamine H2 receptors, 411 H65-RTA, 662 H7, 337 HA-1A, 790 HAART, 647 Haemolin, 801 Haemophilus immunity, 740 Hageman factor (HF), 94

3/25/10 12:57:09 PM

914 Hagfish, 804 Hairplin loop, 157 Hairy cell leukemia, 530 immunophenotype, 601 Half-life (T1/2), 270–271 Halogenation, 733 Halothane antigens, 166 HALV (human AIDS-lymphotropic virus) (historical), 640 HAM test. See Paroxysmal noctural hemoglobinuria HAM-1, 211 HAM-2 (histocombatibility antigen modifier), 211 HAMA, 787 Hanganitziu-Deicher antigen, 895 Haploid, 158 Haploididentical transplate, 675 Haplotype, 199, 283, 675 ancestral, 199, 675 MHC, 675 shared, 675 Hapten, 170–171 Hapten conjugate response, 171 Hapten conjugates, 171 Hapten inhibition test, 171, 821 Hapten X. See CD15 Hapten-carrier conjugate, 171 HAR. See Hyperacute graft rejection Harderian gland, 808 Hasek, Milan, 36 Hashimoto disease (Hashimoto thyroiditis), 545–546 Hassall’s corpuscles, 310 HAV, 748 Hay fever, 419 HbcAg, 750 HbeAg, 750 HBIG (hepatitis B immunoglobulin), 787 HBLV, 747 HbsAg, 750 HBV, 750 HBx, 749–750 HCC-1, 347–348 HCG, 96 HD50, 402 HDN. See Hemolytic disease of the newborn Heaf test, 854 Heart-lung transplantation, 678 Heat inactivation, 403 Heat shock protein antibodies, 96 Heat shock proteins (HSPs), 96 Heat-aggregated protein antigen, 166 Heat-labile antibody, 283 Heavy chain, 243 class, 243 subclass, 243 Heavy chain class (isotype) switching, 280 Heavy-chain disease, 585–587 Heidelberger, Michael, 22–23 Helicobacter pylori immunity, 500 Helminth, 764 Helper CD4+ T cells, 319 Helper T cells, 320 Helper/suppressor ratio, 334 Hemadsorption inhibition test, 821 Hemagglutination, 508, 828 Hemagglutination test, 508 Hemagglutinin, 508 Hematocytes, 802 Hematocytoblast, 100 Hematogones, 215 Hematopoiesis, 99 Hematopoietic cell, 100 transplant, 686

K10141_IDX.indd 914

Index Hematopoietic chimerism, 686–687 Hematopoietic lineage, 99 Hematopoietic neoplasms, complete clinical response of, 525 Hematopoietic stem cell, 100, 686 transplants, 686 Hematopoietic system, 100 Hematopoietic tumors, 525 Hematopoietic-inducing microenvironment (HIM), 99 Hematopoietic/lymphoid, 880 Hematoxylin bodies, 479 Hematuria, 562 Hemocyanin, 166 Hemolymph, 801 Hemolysin, 402–403 Hemolysis, 306 Hemolytic anemia, 525 alloimmune, 526 Hemolytic anemia of the newborn. See Hemolytic disease of the newborn Hemolytic disease of the newborn (HDN), 513 Hemolytic system, 403 Hemophilia, 95 Hemophilus influenzae type b, 733, 783 conjugate vaccine, 783 vaccine (HB), 783 Hemostatic plug, 82 Henoch-Schoenlein purpura, 543 HEP, 774 Heparan sulfate, 83 Heparin, 83 Hepatitis, 458–459, 556–558 lupoid, 460 vaccine, 777 Hepatitis A, 749 vaccine (inactivated—injection), 777 Hepatitis A inactivated and hepatitis B recombinant vaccine (injection), 777–778 Hepatitis B, 557, 749 immune globulin (human—injection), 787 vaccine, 777 vaccine (recombinant—injection), 777 virus immunity, 750 Hepatitis B surface antigen (HbsAg) antibody, 750 Hepatitis B virus protein X. See HBx Hepatitis B virus surface antigen, 876 Hepatitis C virus immunity, 750–751 Hepatitis E virus (HEV), 751 Hepatitis E virus immunity, 751 Hepatitis immunopathology panel, 557, 751 Hepatitis, non-A, non-B (C) (NAN BH), 558, 750 Hepatitis serology, 558, 749 Hepatocyte specific antigen, 893 Hepatocyte-stimulating factor, 357 Hepatosplenomegaly, 537 Her2/Neu(c-erb-B-2), 873 Herbimycin A, 337 Herceptin, 790 Herd immunity, 152 Hereditary angiodema (HAE), 404, 634 Hereditary ataxia telangiectasia. See Ataxia telangiectasia Hereditary complement deficiencies, 405 Herpes gestationis (HG) autoantibodies, 473 Herpes simplex virus 1 and 2, 876 Herpes simplex virus 1 and 2 polyclonal antibody, 747 Herpes simplex virus immunity, 747 Herpes zoster, 747 Herpesvirus, 746 Herpesvirus-6 immunity, 747 Herpesvirus-8 immunity, 747 Herxheimer reaction, 426 Heteroantibody, 491 Heteroantigen, 166

3/25/10 12:57:09 PM

915

Index Heteroclitic antibody, 233 Heterocliticity, 285 Heteroconjugate, 709 Heteroconjugate antibodies, 709 Heterocytotropic antibody, 233, 284 Heterodimer, 330 Heterogeneic. See Xenogeneic Heterogeneous nuclear ribonucleoprotein (RA-33) autoantibodies, 483 Heterogenetic antibody. See Heterophile antibody Heterograft. See Xenograft Heterokaryon, 863 Heterologous antigen, 166 Heterologous vaccine, 771 Heterophile antibody, 233 Heterotopic, 678 Heterotopic graft, 678 Heterotypic vaccine, 771 Heterozygosity, 159 Heterozygous, 159 Hetrophile antigen, 174 Heymann antigen, 562 Heymann glomerulonephritis, 563 HGG. See Human γ globulin HGP-30, 778 HHV, 746 HHV-8, 877 Hib, 733, 783 Hidden determinant, 168 HIG. See Human immune globulin High endothelial postcapillary venules, 143 High endothelial venules (HEV), 143 High-dose tolerance, 440 High-titer low-avidity antibodies (HTLA), 233 High-zone tolerance, 440 Higher vertebrates, 803 Highly active antiretroviral therapy (HAART), 647 Highly polymorphic, 159 Hinge region, 240 Histaminase, 415 Histamine, 414–415 Histamine release assay, 856 Histamine-releasing factors (HRF), 415 Histidyl t-RNA synthetase (anti-HRS), antibodies to, 489 Histiocyte, 117 Histiocytosis X, 530 Histocompatibility, 183, 665 Histocompatibility antigen, 184, 665 Histocompatibility locus, 183–184, 665 Histocompatibility testing, 666 Histocytic lymphoma, 530 Histone (H2A-H2B)-DNA complex autoantibodies (IgG), 479 Histone antibodies, 479 Histone autoantibodies (non-[H2A-H2B]-DNA), 479 Histoplasma immunity, 762 Histoplasmin, 853 Histoplasmin test, 853 HIV. See Human immunodeficiency virus HIV infection, 640 HIV-1 genes, 643 HIV-1 virus structure, 641 HIV-2, 644 HIV-2V, 644 Hives, 411 HLA, 192–193, 667 HLA allelic variation, 193, 671 HLA class I molecules, 454 HLA class II molecules, 454 HLA class III. See Class III molecules; MHC genes HLA complex. See Human leukocyte antigen (HLA) complex HLA disease association, 193–195, 670 HLA locus, 193, 667

K10141_IDX.indd 915

HLA nonclassical class I genes, 187, 196, 670 HLA oligotyping, 671 HLA tissue typing, 671 HLA type, 187, 667 HLA-A, 193, 454, 667 HLA-B, 454, 667–668 HLA-B27-related arthropathies, 484 HLA-C, 454, 668 HLA-D region, 193, 668 HLA-DM, 195, 669–670 HLA-DO, 195, 670 HLA-DP subregion, 195, 669 HLA-DQ subregion, 195, 669 HLA-DR antigenic specificities, 195, 669 HLA-DR subregion, 195, 669 HLA-E, 197, 670 HLA-F, 197, 670 HLA-G, 196, 670 HLA-H, 188, 670 Hm-1, 808 Hodgkin disease, 534–535 Hof, 111 Hole in the repertoire model, 441 Homing-cell adhesion molecule. See H-CAM Homing receptors, 81 Homobody, 263 Homocytotrophic antibody, 233–234 Homodimer, 330 Homograft, 681 Homograft reaction, 682 Homograft rejection, 682 Homokaryon, 117 Homologous, 681 Homologous antigen, 166 Homologous chromosomes, 681 Homologous disease, 688 Homologous recombination, 158 Homologous restriction factor (HRF), 401 Homologous vaccine, 771 Homology region, 241 Homology unit, 240 Homopolymer, 163 Homotransplantation, 682 Homozygote, 733 Homozygous, 676 Homozygous typing cell (HTC) technique, 676 Homozygous typing cells (HTCs), 193, 676 Hook effect, 857 Hookworm immunity, 764–765 Hookworm vaccine, 784 Hormone immunoassays, 823 Hormones, 95 Horror autotoxicus (historical), 447 Horse serum sensitivity, 420 Host-vs.-graft disease (HVGD), 691 Hot antigen suicide, 166 Hot spot, 240 House dust allergy, 420 HPV, 746 HR. See hypersensitive response HSA, 164 HSC. See Hematopoietic stem cell HSV, 747 HTLV, 528 HTLV-IV, 640 Humagglutination inhibition reaction, 508 Humagglutination inhibition test, 508 Human B lymphotropic virus (HBLV), 747 Human choriogonadotrophic hormone. See hCG Human diploid cell rabies vaccine (HDCV), 775 Human immune globulin (HIG), 636, 786

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916 Human immune globulin (intravenous—IVEEGAMEN), 786 Human immunodeficiency virus (HIV), 638–639, 641 anti-retroviral drugs for, 787 carriage, 639 Human leukocyte antigen (HLA). See HLA Human leukocyte antigen (HLA) complex, 187, 667 Human macrophage marker (anti-CD68), 884 Human MHC class II region, 668 Human milk-fat globulin (HMFG), 895 Human γ globulin (HGG), 786 Human papillomavirus recombinant vaccine (quadrivalent—injection), 778 Human SCID (hu-SCID) mouse, 625 Human T lymphocyte, 89 Humanization, 240–241 Humanized antibody, 241 Humoral, 109–110 Humoral immune response, 153 Humoral immunity, 236 Humps, 563 HUT 78, 641 HV regions. See Hypervariable regions HY, 666 Hybrid antibody, 272 Hybrid cell, 863 Hybrid hapten, 171 Hybrid resistance, 714 Hydrogen bonds, 287 Hydrophilic, 287 Hydrophobic, 287 Hydrophobic bonding, 286–287 Hydrops fetalis, 514 Hydroxychloroquine (2-[[4-[7-chloro-4-quinolyl]amino]ethyl-amino] ethanol sulfate), 659, 795 Hydroxychloroquine sulfate (oral), 795 Hyemann’s nephritis. See Heymann glomerulonephritis Hyper-IgD with periodic fever syndrome, 624 Hyper-IgM syndrome (HIGM), 633 Hyperacute graft rejection (HAR), 692 Hyperacute rejection, 691–692 Hypercosinophilia, 411 Hypergammaglobulinemia, 586 Hypergammaglobulinemic purpura, 543 Hyperimmune, 773 Hyperimmunization, 773 Hyperimmunized individual, 689 Hyperimmunoglobulin E syndrome (HIE), 633 Hyperimmunoglobulin M syndrome (HIGM), 633 Hyperplasia, 140 Hypersensitive response (HR), 407 Hypersensitivity, 407 Hypersensitivity angiitis, 420, 553 Hypersensitivity diseases, 407 Hypersensitivity pneumonitis, 408, 420–421, 550–551 Hypersensitivity vasculitis, 421, 553 Hyperthyroidism (Graves’ disease), 546–547 Hypervariable regions, 245–246 Hypocomplementemia, 385 Hypocomplementemic glomerulonephritis, 562 Hypocomplementemic vasculitis, 541 Hypogammaglobulinemia, 618 Hyposensitization, 413 Hypothyroidism, 546

I I region, 188 I-exons, 281 I-J, 188 I-K, 491 I-Selectin, 498

K10141_IDX.indd 916

Index Ia antigens (immune-associated antigen), 188 IBD. See Inflammatory bowel disease Ibuprofen, 484 IC3b-Neo, 396–397 IC4b, 399 ICA512 (IA-2), 465 ICAM-1, 84–85 ICAM-2. See Intracellular adhesion molecule-2 ICAM-3. See Intracellular adhesion molecule-3 Iccosomes (immune complex-coated antibodies), 177 ICIRs. See Killer cell immunoglobulin-like receptors ICOS, 337 Id reaction, 434 IDDM. See insulin-dependent (Type 1) diabetes mellitus Identity testing, 862 Idiopathic thrombocytopenic purpura, 456, 525 Idiotope, 262 Idiotype, 261–262 Idiotype network, 262 Idiotype network theory. See Network theory Idiotype suppression, 262 Idiotype vaccine, 772 Idiotypic determinant. See Epitope Idiotypic specificity, 262 IE, 824 IEF. See Isoelectric focusing IEP, 824 IFE, 824 IFN. See Interferons IFN-α. See Interferon(s) α IFN-β. See Interferon β IFN-γ. See Interferon γ Ig, 242 Ig domain. See Immunoglobulin domain Ig myeloma subclasses, 584 IgA. See Immunoglobulin A IgA deficiency. See Immunoglobulin A deficiency IgA nephropathy (Berger’s disease), 563–564 IgA paraproteinemia, 585 IgD. See Immunoglobulin D IgE. See Immunoglobulin E IgG. See Immunoglobulin G (IgG) IgG index, 248 IgG subclass deficiency, 621 IgG-induced autoimmune hemolysis, 493 IgM. See Immunoglobulin M IgM deficiency syndrome, 621 IgM paraproteinemia, 585 Igα, 219 IgαIgβ (CD79a/CD79b), 219 Igαβ complex, 219 Igβ, 219 IgR, 808 IgW, 806 IgX, 807 IgY, 807 Ii. See Invariant (Ii) chain Ii antigens, 519 IIEL, 499 Iinterleukin-17, 322 Ikaros, 137 IL. See Interleukins IL-1. See Interleukin-1 IL-10. See Interleukin-10 IL-11. See Interleukin-11 IL-12. See Interleukin-12 IL-13. See Interleukin-13 IL-14. See Interleukin-14 IL-17, 322 IL-18. See Interleukin-18 IL-19. See Interleukin-19

3/25/10 12:57:09 PM

Index IL-2. See Interleukin-2 IL-2 receptor (CD25). See Interleukin-2 receptor (IL-2R) IL-2/LAK cells, 714 IL-20. See Interleukin-20 IL-21. See Interleukin-21 IL-22. See Interleukin-22 IL-23. See Interleukin-23 IL-24. See Interleukin-24 IL-25. See Interleukin-25 IL-26. See Interleukin-26 IL-27. See Interleukin-27 IL-28. See Interleukin-28 IL-29. See Interleukin-29 IL-3. See Interleukin-3 IL-30. See Interleukin-30 IL-31. See Interleukin-31 IL-32. See Interleukin-32 IL-33. See Interleukin-33 IL-34. See Interleukin-34 IL-4. See Interleukin-4 IL-5. See Interleukin-5 IL-6. See Interleukin-6 IL-6 receptor, 357 IL-7, 359 IL-9, 361 IL15. See Interleukin-15 IL16. See Interleukin-16 Imbritumomab tiuxetan, 791 ImD50, 175 IMiDs, 659 Immature B lymphocyte, 217 Immature dendritic cells, 121 Immediate hypersensitivity, 408 Immediate-spin cross-match, 523, 678 Immobilization test, 831 Immune, 77 Immune adherence, 399–400 Immune adherence receptor. See Complement receptor 1 Immune antibody, 507 Immune cell cryopreservation, 863 Immune cell motility, 77 Immune clearance. See Immune elimination Immune complex disease (ICD), 559–560 Immune complex glomerulonephritis, 561 Immune complex pneumonitis, 560 Immune complex reactions, 423 Immune complex-coated antibodies (iccosomes), 177 Immune complexes, tissue injury and, 42 Immune costimualtory molecules, 209 Immune cytolysis, 393 Immune deviation, 444–445 Immune elimination, 295, 859 Immune exclusion, 499 Immune inflammation, 150 Immune interferon. See Interferon γ Immune network hypothesis of Jerne, 263 Immune neutropenia, 454 Immune privilege. See Immunologically privileged sites Immune response, 175–176 Immune response (Ir) genes, 224 Immune serum, 242, 786 Immune serum globulin, 242 Immune suppression, 651 Immune system, 77, 443 anatomy, 77–78 Immune tolerance, 438 Immune-neuroendocrine axis, 443–444 Immunity, 79 aging and, 474–475 antioxidants and, 785 arginine and, 785–797

K10141_IDX.indd 917

917 copper and, 631–632 exercise and, 154 fatty acids and, 150 gravity and, 153–154 in prokaryotes, 801 invertebrate, 801 iron and, 156 mercury and, 463 natural anti-viral, 118 nutrition and, 154 radiation and, 662–663 sex hormones and, 450 stress and, 444 vitamin A and, 154–155 vitamin B and, 155 vitamin C and, 155 vitamin D and, 155 vitamin E and, 155 zinc and, 155–156 Immunization, 175, 773 Immunize, 175 Immunizing dose (ImD50), 769 Immunoablation, 794 Immunoabsorbent, 815 Immunoassay, 821 Immunoaugmentive therapy (IAT), 794 Immunobeads, 849 Immunobiology, 34–37 development of, 39–41 Immunoblast, 79 Immunoblastic lymphadenopathy. See Angioimmunoblastic lymphadenopathy Immunoblastic sarcoma, 534 Immunoblot (Western blot), 842 Immunoblotting, 842 Immunochemistry, 20–24 Immunocompetent, 79 Immunoconglutination, 406 Immunoconglutinin, 406 Immunoconjugate, 711 Immunocyte, 79, 540 Immunocytoadherence, 863 Immunocytochemistry, 79 Immunocytokine, 711 Immunodeficiencies, congenital, 62 Immunodeficiency, 617 associated with hereditary defective response to Epstein–Barr virus, 636 from hypercatabolism of immunoglobulin, 636 from severe loss of immunoglobulins and lymphocytes, 636 with partial albinism, 629 with thrombocytopenia and eczema, 629 with thymoma, 620 Immunodeficiency animal models, 625 Immunodeficiency disorders, 617 Immunodeficiency with T cell neoplasms, 623–624 Immunodiagnosis, 867 Immunodiffusion, 816 Immunodominance, 168 Immunodrug Platform, 792 Immunodysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome. See IPEX Immunoelectroadsorption, 821 Immunoelectron microscopy, 837 Immunoelectroosmophoresis, 824 Immunoelectrophoresis (IEP), 824 Immunoenhancement, 177 Immunoferritin method, 837 Immunofluorescence, 835 Immunofluorescent “staining” of C4d, 689 Immunogen, 167

3/25/10 12:57:10 PM

918 Immunogenetics, 52–60 Immunogenic, 167 Immunogenic carbohydrates, 165 Immunogenicity, 167 Immunoglobulin, 228, 241, 886 Immunoglobulin A (IgA), 251, 886 Immunoglobulin A deficiency, 620 Immunoglobulin alpha (α) chain, 252 Immunoglobulin class, 247 Immunoglobulin class switching, 252. See also Isotype switching; Switch; Switch cells; Switch region; Switch site Immunoglobulin D (IgD), 252 Immunoglobulin deficiency with elevated IgM, 622 Immunoglobulin domain, 244 Immunoglobulin E (IgE), 252 elevated, 629 Immunoglobulin epsilon (ε) chain, 252–253 Immunoglobulin evolution, 806 Immunoglobulin fold, 245 Immunoglobulin fragment, 253–254 Immunoglobulin function, 242 Immunoglobulin G (IgG), 248, 886 Immunoglobulin gamma (γ) chain, 249–250 Immunoglobulin gene superfamily, 225 Immunoglobulin genes, 224, 278–279 Immunoglobulin heavy chain, 242–243 Immunoglobulin heavy chain-binding protein (BiP), 246 Immunoglobulin light chain, 246 Immunoglobulin M (IgM), 250, 886 Immunoglobulin monomer, 241 Immunoglobulin mu (µ) chain, 250–251 Immunoglobulin δ chain, 252 Immunoglobulin structure. See Immunoglobulin Immunoglobulin subclass, 248 Immunoglobulin superfamily, 234 Immunoglobulin κ chain, 246 Immunoglobulin λ chain, 246 Immunoglobulin-like domain, 245 Immunogold labeling, 837 Immunogold silver staining (IGSS), 838 Immunohematology, 505 Immunohistochemistry, 837, 867 Immunoincompetence, 617 Immunoinformatics, 815 Immunoinhibitory genes, 671 Immunoisolation, 681 Immunologic (or immune) paralysis, 445 Immunologic adjuvant, 179 Immunologic barrier, 679 Immunologic colitis, 554–555 Immunologic competence, 437 Immunologic competency, 182 Immunologic enhancement, 446, 680, 710 Immunologic facilitation, 680, 710 Immunologic tolerance, 438 Immunologic(al), 80 Immunological contraception, 772 Immunological deficiency state, 617 Immunological escape, 710 Immunological ignorance, 445 Immunological inertia, 437 Immunological infertility, 466 Immunological memory, 730. See also Anamnesis Immunological reaction, 80 Immunological rejection, 689 Immunological suicide, 324, 446 Immunological synapse, 204 Immunological unresponsiveness, 437 Immunologically activated cell, 80 Immunologically competent cell, 80 Immunologically privileged sites, 679

K10141_IDX.indd 918

Index Immunologist, 80 Immunology, 80 advent of classical, 6–20 landmarks in the history of, 71–76 transplantation, 665 Immunolymphoscintigraphy, 711 Immunomagnetic technique, 861 Immunomodulation, 793 Immunomodulator, 439 Immunoosmoelectrophoresis, 824 Immunoparasitology, 525 Immunopathic damage, 343, 525 Immunopathology, 525 Immunoperoxidase, 838 Immunoperoxidase method, 867 Immunophelometry, 861 Immunophenotyping, 851 Immunophilin ligands, 653–656 Immunophilins, 655 Immunophysiology, 80 Immunopotency, 165 Immunopotentiation, 177 Immunoprecipitation, 822 Immunoproliferative small intestinal disease (IPSID), 537–538 Immunoprophylaxis, 773 Immunoproteasome. See Proteasome Immunoprotein, 794 Immunoradioisotope, 711 Immunoradiometric assay (IRMA), 857 Immunoradiometry, 857 Immunoreactant, 295 Immunoreaction, 295 Immunoreceptor tyrosine-based activation motif. See ITAM Immunoreceptor tyrosine-based inhibition motif. See ITIM Immunoregulation, 437 Immunoscintigraphy, 711 Immunoselection, 710 Immunosenescence, 662 Immunosome, 329 Immunostimulants, 663 Immunosuppression, 651 specific, 662–663 Immunosuppressive agents, 651–660 Immunosuppressive cytokines, 651 Immunosuppressive drugs, 651 Immunosurveillance, 709–710 Immunotactoid glomerulopathy, 561 Immunotherapy, 710, 785 Immunotoxin, 685, 711–712 Immunotoxin-induced apoptosis, 135 Immunotyping. See Immunophenotyping Immunuabsorption, 815 In situ transcription, 864 In vitro, 79 In vivo, 79 In-situ hybridization, 840, 843 Inaccessible antigens. See Hidden determinant Inactivated C3b, 396 Inactivated poliovirus vaccine, 775 Inactivated vaccine, 770 Inactivated virus vaccine. See Killed virus vaccines Inactivation, 403 Inbred mouse strain, 812 Inbred strain, 812 Incompatibility, 523, 681 Incomplete antibody, 513 Incomplete Freund’s adjuvant (IFA), 180–181, 182 Incubation time, 717 Indicible nitric oxide synthase (iNOS), 116 Indirect agglutination (passive agglutination), 830 Indirect allorecognition, 690

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919

Index Indirect antigen presentation, 203–204, 689–690 Indirect antiglobulin test, 513 Indirect complement fixation test, 402 Indirect Coombs’ test, 512 Indirect fluorescence antibody technique, 834, 836 Indirect immunofluorescence, 836 Indirect tag assays, 821 Indirect template theory (historical), 18, 264 Indolent, 700 Indomethacin, 484 Induced fit, 172 Inducer determinant, 168, 374 Inducer T lymphocyte, 322 Inductive phase, 175 Inductive sites, 499 Infantile agammaglobulinemia, 619 Infantile sex-linked hypogammaglobulinemia, 618 Infection, 717 Infection allergy, 431, 732 Infection hypersensitivity, 431 Infectious agents, 876 Infectious diseases, immunication against, 60–61 Infectious mononucleosis, 757–758 syndromes, 758 Infectious tolerance, 445 Inflammation, 147–149 Inflammatory bowel disease (IBD), 555 Inflammatory CD4 T cells, 319 Inflammatory cells, 150 Inflammatory macrophage, 150 Inflammatory mediator, 150 Inflammatory myopathy, 543 Infliximab, 661–662 Infliximab, 790 Influenza, 753 virus vaccine, 775 Influenza hemagglutinin, 755 Influenza virus immunity, 754–755 Influenza viruses, 753–754 Inhibin, alpha (R1), mouse, 889 Inhibition test, 821 Inhibition zone. See Prozone Initiation, 699 Innate defense system, 152, 730 Innate immunity, 152, 726 against intracellular bacteria, 729 mechanisms against parasites, 729 Innocent bystander, 80 Innocent bystander hemolysis, 454 Inoculation, 175 Inosiplex, 794 Inositol 1, 4, 5-triphosphate (IP3), 230, 317 Insect resistance (immunity), 803 Instructional model, 285 Instructive theory (of antibody formation), 285 Insulin receptor autoantibodies, 465 Insulin resistance, 465 Insulin-dependent (Type 1) diabetes mellitus (IDDM), 45–46, 465, 558–559 Insulin-like growth factor-II (IGF-II), 96 Insulin-like growth factors (IGFs), 96, 377 Insulitis, 465 Intal, 412 Integrin family of leukocyte adhesive proteins, 83 Integrins, 82 HGF/SF activation of, 83 Interallelic conversion, 671 Intercalated cell autoantibodies, 462 Intercrine cytokines, 350 Interdigitating reticular cells. See Dendritic cells (DC) Interfacial test, 821

K10141_IDX.indd 919

Interferon, 62 Interferon α (IFN-α), 710–711 Interferon α-2a, recombinant (injection), 664, 793 Interferon α-2b, recombinant (injection), 664, 793 Interferon α-n3 (injection), 664, 793–794 Interferon β (IFN-β), 369 Interferon γ (IFN-γ), 369 Interferon γ (IFN-γ) inducible protein-10 (IP-10), 369 Interferon γ receptor, 370 Interferon β-1a, 369 Interferon γ-1b (injection), 664, 794 Interferon regulatory factors (IRF), 368 Interferon response, 368 Interferon λ, 369–370 Interferon(s) α (IFN-α), 369 Interferon-producing cells (IPCs), 368 Interferons (IFNs), 368 Interfollicular region, 140 Interleukin-1 (IL-1), 350–351 Interleukin-1 receptor, 351 deficiency, 352 Interleukin-1 receptor antagonist (IL-1ra), 351 Interleukin-1 receptor antagonist protein (IRAP), 352 Interleukin-1 receptor deficiency, 623 Interleukin-10 (IL-10), 362–363 Interleukin-11 (IL-11), 363 Interleukin-12 (IL-12), 363 Interleukin-13 (IL-13), 363–364 Interleukin-14 (IL-14), 364 Interleukin-15 (IL-15), 364 Interleukin-16 (IL-16), 364–365 Interleukin-17 (IL-17), 365 Interleukin-18 (IL-18), 365 Interleukin-19 (IL-19), 365 Interleukin-2 (IL-2), 352–353 Interleukin-2 receptor (IL-2R), 353 Interleukin-2 receptor α subunit (IL-2Rα), 354 Interleukin-2 receptor αγβ subunit (IL-α2Rβγ), 354 Interleukin-2 receptor β subunit (IL-2Rβ), 354 Interleukin-2 receptor γ subunit (IL-2Rγ), 354 Interleukin-2 receptor βγ subunit (IL-2Rβγ), 354 Interleukin-20 (IL-20), 365 Interleukin-21 (IL-21), 365–366 Interleukin-22 (IL-22), 366 Interleukin-23 (IL-23), 366 Interleukin-24 (IL-24), 366 Interleukin-25 (IL-25), 366 Interleukin-26 (IL-26), 366 Interleukin-27 (IL-27), 366–367 Interleukin-28 (IL-28), 367 Interleukin-29 (IL-29), 367 Interleukin-3 (IL-3), 354 Interleukin-3 receptor (IL-3R), 354 Interleukin-30 (IL-30), 367 Interleukin-31 (IL-31), 367 Interleukin-32 (IL-32), 367 Interleukin-33 (IL-33), 367 Interleukin-34 (IL-34), 367–368 Interleukin-4 (IL-4), 354–355 Interleukin-4 receptor (IL-4R), 355–356 Interleukin-5 (IL-5), 356 Interleukin-5 receptor complex, 357 Interleukin-6 (IL-6), 357 Interleukin-6 receptor, 358–359 Interleukin-7 (IL-7), 359 Interleukin-8 (IL-8), 359 Interleukin-8 receptor type A (IL-8RA) (alpha), 360 type B (IL-8RB) (beta), 360 Interleukin-9 (IL-9), 361 Interleukins (IL), 350

3/25/10 12:57:10 PM

920 Intermediate filaments, 895 Intermolecular epitope spreading, 168 Internal image, 263 International Unit of Immunological Activity, 769 Interstitial dendritic cells, 121 Interstitial fluid, 146 Interstitial nephritis, 562 Interstitial pneumonitis, usual, 547 Intervening sequence. See Intron Intestinal cryopatches, 145 Intestinal follicles, 145 Intestinal lymphangiectasia, 635 Intimin, 733 Intolerance, 797 Intrabody, 237 Intracellular adhesion molecule-1 (ICAM-1), 84 Intracellular adhesion molecule-2 (ICAM-2), 85 Intracellular adhesion molecule-3 (ICAM-3), 85 Intracellular adhesion molecules (ICAMs), 84 Intracellular antigen. See Endogenous antigen Intracellular cytokine staining, 346, 840 Intracellular immunization, 641 Intracellular pathogens, 733 Intracellular signaling pathway, 81 Intraepidermal lymphocytes, 503 Intraepithelial pocket, 140 Intraepithelial T lymphocytes, 330 Intramolecular epitope spreading, 168 Intravenous immune globulin (IVIG), 786 Intrinsic affinity, 294 Intrinsic association constant, 294 Intrinsic asthma, 550 Intrinsic factor, 457 Intrinsic factor autoantibodies, 457 Intron, 157 Inulin, 395 Inv, 244 allotypes, 244 Invariant (Ii) chain, 209 Invasin, 742 Invasive pathogens, 733 Invasive tumor, 700 Inversional joining, 225 Invertebrate coagulation, 803 Invertebrate cytokine-like molecules, 804 Invertebrate immunity, 801 Invertebrates, 804 Inverted repeat, 157 Ion exchange chromatography, 816 Ionic bonds, 286 Ionic forces, 286 IPEX, 629 IPV-injection, 775–776 Ir genes, 186 Iridovirus immunity, 803–804 Iron, immunity and, 156 Irradiation chimera, 687 Ischemia, 95 ISCOMs, 180 Ishizaka, Kimishige, 29 Ishizaka, Terako, 29 Islet cell autoantibodies (ICA), 465 Islet cell transplantation, 684 Islets of Langerhans, 684 Isoagglutinin, 258 Isoallergens, 419 Isoallotypic determinant, 258 Isoantibody, 683 Isoelectric focusing (IEF), 861 Isoelectric point (pI), 861 Isoforms, 95

K10141_IDX.indd 920

Index Isogeneic (isogenic), 683 Isograft, 683 Isoimmunization, 523 Isoleukoagglutinins, 683 Isologous, 683 Isophile antibody, 523 Isophile antigen, 523 Isoprinosine, 794 Isoproterenol, 423 Isoschizomer, 160 Isotope, 258 Isotopic labeling, 818 Isotype, 258 Isotype switching, 279 Isotypic determinant, 258 Isotypic exclusion, 281 Isotypic specificities, 258 Isotypic variation, 258 ITAM, 82 ITIM, 83 ITIM/ITAM immunoreceptors, 82 ITP. See Idiopathic thrombocytopenic purpura IVIG. See Intravenous immune globulin

J J chain, 246–247 J exon, 225 J gene segment, 225, 327 J region, 225, 327–328 JAK-STAT signaling pathway, 345–346 JAK3-SCID, 626 Janus kinases (Jaks), 345 Jarisch-Herxheimer reaction, 733 Jawless fishes, 804 JC/70A, 882 Jenner, Edward, 3–4 Jerne network theory, 262 Jerne plaque assay, 846 Jerne, Niels Kaj, 16–17 Jo-1 autoantibodies, 489 Jo-1 syndrome, 489 Job’s syndrome, 632 Johnin, 853 Jones criteria, 552 Jones-Mote hypersensitivity, 435 Jones-Mote reaction, 435 Jugular bodies, 807 Junctional diversity, 225, 282 Juvenile onset diabets, 465 Juvenile rheumatoid arthritis (JRA), 485

K κ chain, 243 κ light-chain deficiency, 621–622 K (killer) cells, 114–115 K antigens, 733 K cells (killer cells), 726 K region, 189 K562 cells, 114 Kabat, Elvin Abraham, 23 Kabat-Wu plot. See Wu-Kabat plot Kallidin. See Kinins Kallikrein, 412 Kallikrein inhibitors, 412 Kallikrein-kinin system, 411 Kaposi’s sarcoma, 641 Kappa (κ), 243 Kappa-lambda exclusion, 243 Karatinocytes, 152

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921

Index Karyotype, 158 Kawasaki’s disease, 553 Kell blood group system, 517–518 Keratin layer, 152 Keratinocyte growth factor (KGF), 381 Keratoconjunctivitis sicca, 474 Kernicterus, 514 Ketokonazole, 762 Keyhole limpet hemocyanin (KLH), 166 Ki autoantibodies, 479 Ki-1, 882 Ki-67, 873 Ki-780, 873 Kidd blood group system, 518 Killed vaccine, 770 Killed virus vaccines, 770 Killer (K) cells, 725 Killer activatory receptors (KARs), 113 Killer cell immunoglobulin-like receptors, 113, 191 Killer cells, 114–115 Killer inhibitory receptors (KIRs), 191 Killer T cell, 323 Kilobase (kb), 157 Kinetochore autoantibodies, 487 Kininases, 412 Kininogens, 412 Kinins, 412–413 Kitasato, Shibasaburo, 11 Klebsiella immunity, 738 KLH. See Keyhole limpet hemocyanin Km allotypes, 244 Knock-in transgene, 859 Knockout gene, 860 Knockout mouse, 860 Koch phenomenon, 33–34, 431 Koch, Robert, 8 Köhler, Georges J.F., 65–67 Ku, 479 antibodies, 479 autoantibodies, 468 Kunkel, Henry George, 44 Kupffer cell, 118 Kuru, 758 Kveim reaction (historical), 550

L λ cloning vector, 845 λ5, 216, 217 λ5 B cell development, 216 L cell conditioned medium, 376 L chain, 244 L+ dose (historical), 305–306 L-phenylalamine mustard, 797 L-plastin (LPL), 101 L-selectin (CD62L), 91 L3T4, 322 L3T4+ T lymphocytes, 322 La/SS-B, 488 Lactalbumin, 895 Lactoferrin, 153 Lactoperoxidase, 153 LAF (lymphocyte-activating factor). See Interleukin-1 LAG-3, 113 LAK cells. See Lymphokine-activated killer (LAK) cells LAM-1, 89 Lambda (λ), 243 Lambda (λ) chain, 244 Lamin, autoantibodies against, 459 Lamina propria, 496 Laminin, 88–89

K10141_IDX.indd 921

receptor, 89 LAMP 1, 98, 103 LAMP 2, 98, 103 Lampreys, 804 Lancefield precipitation test, 825 Landsteiner’s rule (historical), 52, 506 Landsteiner, Karl, 51–52, 285–286 Lane, 826–827 Langerhans cells, 121–122, 503 Lapinized vaccine, 771 LAR. See Local acquired resistance Large granular lymphocytes (LGL). See Natural killer (NK) cells Large lymphocyte, 103 Large pre-B cells, 215 Large pyroninophilic blast cells, 140 LAT. See Linker of activation in T cells Late-onset immunodeficiency, 635 Late-phase reaction, 149 Latency, 745 Latent allotype, 259 Latent infection, 717 Latex allergy, 412 Latex fixation test, 829 Latex particles, 830 LATS. See Long-acting thyroid stimulator LATS protector, 452 Lattice theory, 297–298 Laurell crossed immunoelectrophoresis, 827 Laurell rocket test, 827 LAV, 642 Lawrence, Henry Sherwood, 33 Lazy leukocyte syndrome, 632 LCA. See Leukocyte common antigen LCAM, 80 Lck, 326–327 LCM. See Lymphocytic choriomeningitis LD50, 769 LDCF (lymphocyte-derived chemotactic factor), 360 LE cell, 479, 574 “prep,” 480 test, 480 LE factor, 480 Leader sequence, 266 Leading front technique, 821 Lectin pathway of complement activation, 385 Lectins, 95 Lederberg, Joshua, 18 Leflunomide, 659, 795 Legionella immunity, 740 Leishmania, 765 Leishmaniasis, 765 Lenalidomide (oral), 796 Lens-induced uveitis, 474 Lentiviruses, 642 LEP (low egg passage), 775 Lepra cells, 740 Lepromatous leprosy, 740 Leptin, 688 Leptospira immunity, 740 Lesch-Nyhan syndrome, 622 LESTR, 350 Lethal dose, 865 Lethal hit, 324 Letterer-Siwe disease, 530 Leu M1, 881. See also CD15 Leu-CAM, 101 Leukapheresis, 865 Leukemia, 526. See also specific types of leukemia Leukemia inhibitory factor (LIF), 378–379 Leukemia viruses, 526 Leukoagglutinin, 683

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922 Leukocidin, 129 Leukocyte activation, 100, 726 Leukocyte adhesion deficiency (LAD), 632 Leukocyte adhesion molecule-1 (LAM-1), 89, 101 Leukocyte adhesion molecules, 100 Leukocyte adhesion proteins, 101 Leukocyte adhesive proteins, integrin family of, 83 Leukocyte cell adhesion molecule, 80 Leukocyte chemotaxis inhibitors, 101 Leukocyte common antigen (LCA), 100 Leukocyte common antigen (LCA, CD45), 332 Leukocyte culture, 863 Leukocyte functional antigens (LFAs), 101 Leukocyte groups, 187 Leukocyte inhibitory factor (LIF), 378 Leukocyte integrins, 101 Leukocyte migration inhibitory factor. See Leukocyte inhibitory factor Leukocyte sialoglycoprotein. See LGSP Leukocyte transfer. See Adoptive transfer Leukocytes, 100 echinoderm, 804 neutrophil, 123–124 Leukocytoclasis (nuclear dust), 541 Leukocytoclastic vasculitis, 540–541 Leukocytosis, 100 Leukopenia, 100 Leukophysin, 323 Leukotaxis, 93 Leukotrienes, 409–410, 416–417 Levamisole, 663, 795 Levine, Phillip, 52 Lewis blood group system, 516–517 Lewisx/Sialyl-Lewisx CD15/CD15S, 517 Lf dose (historical), 306 Lf flocculating unit (historical), 306 LFA-1. See Leukocyte functional antigens LFA-1 deficiency, 630 LFA-2, 84 LFA-3, 84 LGL (large granular lymphcyte or null cell), 112 LGSP (leukocyte sialoglycoprotein), 310 Li-Fraumeni syndrome, 701 Liacopoulos phenomenon (nonspecific tolerance), 445 Liberated CR1, 400 Ligand, 81 Light chain subtype, 243 Light chain type, 243 Light scatter, 851 Light zone, 143 Light-chain deficiencies, 621 Light-chain disease, 586 Limiting dilution, 852 Lineage infidelity, 532 Linear determinants, 168–169 Linear epitope, 169 Linear staining, 565 Linkage disequilibrium, 670 Linked recognition, 317 Linked suppression, 440 Linker of activation in T cells (LAT), 337 Lipid raft, 205 Lipocortins, 94 Lipopolysaccharide (LPS). See Endotoxin; LPS Liposomes, 179 Lipoxygenase pathway, 410 Liquefactive degeneration, 573 Lissamine rhodamine (RB200), 833 Listeria, 744 Listeria immunity, 744 Listeria monocytogenes, 744 Live attenuated measles (rubeola) virus vaccine, 776

K10141_IDX.indd 922

Index Live attenuated vaccine, 770 Live measles and mumps virus vaccine, 777 Live measles and rubella virus vaccine, 777 Live measles virus vaccine, 776 Live measles, mumps, and rubella virus vaccine (live—injection), 777 Live oral poliovirus vaccine, 776 Live rubella virus vaccine, 776 Live vaccine, 770 Liver autoimmunity and, 47 immunological diseases and immunopathology of, 556–558 Liver cytosol autoantibodies, 460 Liver membrane antibodies, 460 Liver membrane autoantibodies, 460 Liver-kidney microsomal antibodies, 461 Liver-kidney microsome 1 (LKM-1) autoantibodies, 461 Liver-kidney microsome 2 (LKM-2) autoantibodies, 461 Liver-kidney microsome 3 (LKM-3) autoantibodies, 461 LM autoantibodies, 460 LMP genes, 205 LMP-2, 205 LMP-7, 205 L o dose (historical), 306 Local acquired resistance (LAR), 799 Local anaphylaxis, 414 Local immunity, 495 Locus (pl.: loci), 186 Locus accessibility, 280 London forces (van der Waals forces), 288 Long homologous repeat, 398 Long terminal repeat (LTR), 644 Long-acting thyroid stimulator (LATS), 423, 452, 547 Long-lived lymphocyte, 103 Long-term nonprogressors (LTNPs), 645 Low responder mice, 813 Low-dose tolerance, 440 Low-zone tolerance, 440 Lower invertebrates, 801 LPAM-1, 91 LPR. See Late-phase reaction LPS, 230, 727–728 LPS-binding protein (LBP), 230 L r dose (historical), 306 LT. See Lymphotoxin LTα, 323 Lung autoantibodies, 463 Lungs, immunological diseases and immunopathology of, 547–552 Lupoid hepatitis, 460 Lupus anticoagulant, 482, 575 Lupus erythematosus, 482 pregnancy and, 482 Lupus nephritis, 482 Lutheran blood group, 519 Lw antibody, 511 Ly antigen, 230, 333 Ly1 B cell, 230 Ly6, 230, 334 Lyb, 230 Lyb-3 antigen, 230 Lyme disease, 733 Lymph, 139 Lymph gland. See Lymph node Lymph node, 138–139 draining, 141 Lymphadenitis, 141 Lymphadenoid goiter. See Hashimoto disease Lymphadenopathy, 141 Lymphatic system, 139 Lymphatic vessels, 139 Lymphatics, 139 Lymphoblast, 101

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923

Index Lymphocyte activation, 105, 175 three signal model of, 317–318 threshold, 105 Lymphocyte activation factor (LAF). See Interleukin-1 Lymphocyte activation threshold, 175 Lymphocyte anergy, 442 Lymphocyte antigen receptor complex, 223 Lymphocyte antigen stimulation test, 848–849 Lymphocyte chemokine (BLC), 348 Lymphocyte chemotaxis, 106 Lymphocyte defined (LD) antigens, 670 Lymphocyte determinants, 676 Lymphocyte function-associated antigen-1 (LFA-1), 84 Lymphocyte function-associated antigen-2. See CD2 Lymphocyte function-associated antigen-3 (LFA-3), 84 Lymphocyte immune globulin (injection), 690 Lymphocyte maturation, 216, 313 Lymphocyte mitogen stimulation test, 849 Lymphocyte precursors, 101 Lymphocyte receptor repertoire, 104 Lymphocyte recirculation, 104 Lymphocyte specificity, 102 Lymphocyte tolerization, 441 Lymphocyte toxicity assay, 849 Lymphocyte trafficking, 104 Lymphocyte transfer reaction. See Normal lymphocyte transfer reaction Lymphocyte transformation, 847–848 Lymphocytes, 101 activated, 103, 337 circulating, 105 effector, 103 intraepidermal, 503 large, 103 long-lived, 103 memory, 178 naïve, 104 primed, 176 recirculation of, 105 resting, 103–104, 336 short-lived, 104 small, 103 Lymphocytic choriomeningitis (LCM), 813–814 Lymphocytic interstitial pneumonia (LIP), 548 Lymphocytopenic center. See Germinal centers Lymphocytosis, 104 Lymphocytotoxic autoantibodies, 454 Lymphocytotoxin. See Lymphotoxin Lymphocytotrophic, 104 Lymphogranduloma venereum (LGV), 755–756 Lymphoid, 140 Lymphoid cell, 102 series, 102 Lymphoid enhancer factor-1 (LEF-1), 330 Lymphoid follicle, 140 Lymphoid lineage, 103 Lymphoid nodules, 140 Lymphoid organs, 140 secondary, 141 Lymphoid patches, 140 Lymphoid progenitor cell, 101 Lymphoid system, 133 Lymphoid tissues, 133 secondary, 141 tertiary, 146 Lymphokine, 346 Lymphokine-activated killer (LAK) cells, 713–714 Lymphoma, 535 Lymphoma belt, 537 Lymphomatoid granulomatosis, 553 Lymphomatosis, 536 Lymphopenia, 102

K10141_IDX.indd 923

Lymphopoiesis, 102 Lymphoreticular, 102 Lymphorrhages, 102 Lymphotactin (Ltn), 348 Lymphotoxin (LT), 373 Lysins, 239 Lysis, 306 Lysogeny, 745 Lysosomes, 98 primary, 98 secondary, 128 Lysozyme, 719 Lyt 1,2,3, 326 Lyt antigens, 326 Lytic granules, 323

M M antigen, 734 M cells, 498–499 M component, 586 M macroglobulin, 589 M protein, 584 Mab. See Monoclonal antibodies MAC, 742. See also Membrane attack complex MAC-1, 89, 236 Macroglobulin, 589 Macroglobulinemia, 589 Macrophage (HAM-56), 885 Macrophage chemotactic and activating factor (MCAF), 119, 348 Macrophage chemotactic factor (MCF), 119, 370 Macrophage colony-stimulating factor (M-CSF), 375 Macrophage cytophilic antibody, 120 Macrophage functional assays, 120, 849 Macrophage immunity, 117 Macrophage inflammatory peptide-2 (MIP-2), 119 Macrophage inflammatory protein-1-α (MIP-1α), 119, 379 Macrophage inflammatory protein-1-β (MIP-1β), 379–380 Macrophage inflammatory protein-2 (MIP-2), 380 Macrophage migration inhibitory factor, 347 Macrophage migration test, 849 Macrophage-activating factor (MAF), 119, 370 Macrophage-monocyte inhibitory factor (MIF), 346–347, 849 Macrophage/monocyte chemotaxis, 117 Macrophages, 115, 706–708 activated, 115–116 alveolar, 120 angry, 116 armed, 118 resident, 116 stimulated, 116 suppressor, 128 tingible body, 117 tissue-fixed, 116 Macropinocytosis, 202 Macropinosome, 202 MadCAM-1, 91, 498 MAF. See Macrophage-activating factor Magic bullet, 712 MAIDS, 649 MAIS complex, 742 Major basic protein (MBP), 422 Major histocompatibility complex (MHC), 183 class II deficiency, 626 molecule, 187 Major histocompatibility complex restriction. See MHC restriction Major histocompatibility system. See Major histocompatibility complex Malaria, 765 vaccine, 784 Malignant, 700 Malignant conversion, 699

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924 Malignant transformation, 699 Malignolipin (historical), 701 MALT lymphoma, 604 immunophenotype, 605 Mammals, 808 Mancini technique, 818 Mancini test, 819 Mannan-binding protein, 385 Mannose receptor, 117 Mannose-binding lectin (MBL), 117 Mannose-binding pathway. See Lectin pathway of complement activation Mantle cell lymphoma (MCL), immunophenotype, 606 Mantle zone, 141, 143 lymphoma, 530, 620 Mantoux test, 742 MAPK (mitogen-activated protein kinase). See Ras/MAPK signaling pathway Marek’s disease, 471 Marginal zone, 145 Marginal zone B cells, 145 Margination, 126 Marrack, John Richardson, 24 Marsupial immunity, 812 MART-1 (M2-7C10), mouse, 893 Mass vaccination, 769 Mast cell activation, 410 Mast cell growth factor-1. See Interleukin-3 Mast cell growth factor-2. See Interleukin-4 Mast cell tryptase, 410–411 Mast cell-eosinophil axis, 411 Mast cells, 129–130 armed, 410 Masugi nephritis, 562 Maternal antibodies, 270 Maternal immunity, 502 Maternal immunoglobulins, 270 Maternal imprinting, 159 Mature B cell, 217 Mature dendritic cells, 206 Mature T cells, 335 MBP, 471 McCleod phenotype, 518 Mcg isotypic determinant, 244 MCP-1 in atherosclerosis, 350 MCTD. See Mixed-connective tissue disease MDP. See Muramyl dipeptide Measles vaccine, 776 Measles virus vaccine (live—injection), 776 Mechanical barriers, skin and mucous membrane as, 718 Mechnikov, Elia (Illya). See Metchnikoff, Elie (Illya) Medawar, Peter Brian, 55 Medulla, 140 Medullary cord, 140 Medullary sinuses, 141 Medullary thymic epithelial cells (mTECs). See Thymic epithelial cells Megakaryocytes, 132 MEL-14, 79 antibody, 79–80 Melanocytes, 705 Melanoma antigen-1 gene (MAGE-1), 705 Melanoma growth stimulatory activity (MGSA), 349 Melanoma-associated antigens (MAA), 705–706 Melanomas, 705 Melanosome, 705 Melphalan (1-phenylalanine mustard), 659 Membrane attack complex (MAC), 392–393 Membrane attack unit, 385 Membrane cofactor of proteolysis (MCP or CD46), 398 Membrane cofactor protein (MCP), 398 Membrane complement receptors, 399 Membrane immumoglobulin, 229

K10141_IDX.indd 924

Index Membrane immunofluorescence, 835 Membrane nibbling, 121 Membrane-bound immunoglobulin (mIg), 219 Membranoproliferative glomerulonephritis (MPGN), 561 Membranous glomerulonephritis, 560 Memory, 178 Memory B cells, 217 Memory cells, 178 Memory lymphocytes, 178 Memory response. See Anamnesis Memory T cells, 178 Meningococcal vaccine, 781–782 Mercury, immunity and, 463 Merosin, 891 Metal hypersensitivity, cellular and humoral, 431 Metaproterenol, 423 Metastases, 700 Metastasis, 700 Metatype autoantibodies, 454 Metchnikoff, Elie (Illya), 10 Methotrexate (N-[p-[[2,4-diamino-6-pteridinyl-methyl]methylamino] benzoyl] glutamic acid), 658 Methyl green pyronin stain, 864 Metronidazole, 765 MGUS. See Monoclonal gammopathy of undertermined significance MHC. See Major histocompatibility complex MHC class 1 molecules, 187 MHC class I deficiency, 626 MHC class I proteins, 187 MHC class Ib and IIb molecules, 189 MHC class II compartment (MIIC), 189 MHC class II molecules, 189–190 MHC class II proteins, 190 MHC class II region, 189 MHC class II transactivator (CIITA), 189 MHC class III proteins, 190 MHC congenic mice, 191 MHC disease associations. See HLA disease association MHC functions, 186 MHC genes, 184–186 MHC haplotype, 675 MHC II deficiency, 626 MHC molecules, 186 MHC mutant mice, 191 MHC peptide tetramers, 186 MHC peptide-binding specificity, 187 MHC recombinant mice, 186 MHC restriction, 197, 208 MHC-I antigen presentation, 211 MHC-like proteins, 190 MIC molecules, 499 MICA, 191 MICB, 191 Microchimerism, 687 Microenvironment, 80 Microfilaments, 80 Microfold cells. See M cells Microglial cell, 118 Microlymphocytotoxicity, 672, 846 Microorganisms, phagocytosis of, 117–118 Microtiter method. See Takatsy method Microtubules, 98 Mid-piece, 385 MIG, 349 MIg, 219, 241 MiHA. See Minor histocompatability antigens MIIC, 210 Mikulicz’s syndrome, 579 Miller, J.F.A.P, 36 Mini pigs, 682 Minimal hemagglutinating dose (MHD), 829

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Index Minimal hemolytic dose (MHD), 402 Minimum lethal dose (MLD), 865 Minisatellite, 864 Minor blood group antigens, 516 Minor H peptides, 665 Minor histocompatability antigens (MiHA), 665 Minor histocompatability locus, 665 Minor histocompatability peptides, 666 Minor lymphocyte stimulatory (MIs) loci, 666 Minor lymphocyte-stimulating (MIs) determinants, 666 Minor lymphocyte-stimulating genes, 666 Minor transplantation antigens. See Minor histocompatability antigens Miotic spindle apparatus autoantibodies, 484 MIP-1. See Macrophage inflammatory protein-1-α MIP-1α receptor, 379 MIP-1β, 379 MIRL (membrane inhibitor of reactive lysis), 393 MIs genes, 666 “Missing self” hypothesis, 105, 708 Mitochondria, 98 Mitochondrial antibodies, 460 Mitochondrial autoantibodies (MA), 460 Mitogen, 729 Mitsuda reaction, 742 Mixed agglutination, 821, 830 Mixed chimerism, 687 Mixed hemadsorption, 822 Mixed leukocyte reaction (MLR), 676 Mixed lymphocyte reaction (MLR), 847 Mixed vaccine, 770 Mixed-antiglobulin reaction, 821 Mixed-connective tissue disease, 573 Mixed-lymphocyte culture (MLC), 676 Mixed-lymphocyte reaction (MLR), 676 MK-571, 423 MLC. See Mixed-lymphocyte culture MLH1, 873 MMR vaccine, 777 MNSs blood group system, 515 Modulation, 706 Moesin, 82 Moferumomab, 790–791 Mol, 396 Molecular (DNA) typing, sequence-specific priming (SSP), 673–675 Molecular hybridization probe, 843 Molecular mimicry, 492 Molluscs, 803 Moloney test, 737 Monoclonal, 268 Monoclonal antibodies (MAb), 267–268, 660–662 therapy, 787 Monoclonal antiinsulin antibody, 889 Monoclonal gammopathy, 586 Monoclonal gammopathy of undertermined significance (MGUS), 586, 602–603 immunophenotype, 603 Monoclonal immunoglobulin, 588 Monoclonal immunoglobulin deposition disease (MIDD), 582 Monoclonal protein, 588 Monocyte chemoattractant protein (MCP-1), 349 in atherosclerosis, 350 Monocyte chemoattractant protein-2 (MCP-2), 349 Monocyte chemoattractant protein-3 (MCP-3), 349 Monocyte colony-stimulating factor (MCSF), 375–376 Monocyte-derived neutrophil chemotactic factor. See Interleukin-8 Monocyte-phagocyte system, 132 Monocytes, 130–131 Monogamous bivalency, 275 Monogamous multivalency, 275 Monokine, 346 Monomorphic, 159

K10141_IDX.indd 925

925 Monomorphic population, 159 Mononuclear cells, 101 peripheral blood, 104 Mononuclear phagocyte, 721 Mononuclear phagocyte system, 122 Montenegro test, 854 Mooren’s ulcer, 569 Moreschi, Carlo Alberto, 51 Moro test, 740 Moth-eaten mouse, 483 Motor inhibitors, 653–656 MOTT (mycobacteria other than Mycobacterium tuberculosis), 741 MOTT cell, 765 Mouse hepatitis virus (MHV), 558 Mouse immunoglobulin antibodies, 690 MRL-lpr/lpr mouse, 482 MS. See Multiple sclerosis MSH2, 873 Mucins, 79 Mucocutaneous candidiasis, 762–763 Mucocutaneous lymph node syndrome (MLNS). See Kawasaki’s disease Mucosa, 495 as mechanical barrier, 718 Mucosa homing, 498 Mucosa-associated lymphoid tissue (MALT), 495 Mucosal immune system, 495 Mucosal lymphoid follicles, 498 Mucosal tolerance, 502 Mucous adhesive, 499 Mucous membrane. See Mucosa Multicatalytic proteinase autoantibodies, 469 Multilocus probes, 677, 844 Multiple autoimmune disorders (MAD), 493 Multiple myeloma, 583–584 Multiple sclerosis (MS), 565–566 Multiple-emission adjuvant, 181 Multiplicity, 159 Multisystem immunological diseases and immunopathology, 552–553 Multivalent, 285 Multivalent antiserum, 285 Multivalent vaccine. See Polyvalent vaccine Mumps vaccine, 776 Mumps virus vaccine (live—injection), 776 Muramyl dipeptide (MDP), 182 Murine growth factor P40. See Interleukin-9 Muromonab-CD3 (injection), 791 Murray, Joseph E., 59 Muscle immunological diseases and immunopathology, 543–544 Mutagen, 158 Mutant, 228 Mutation, 228 Myasthenia gravis (MG), 48, 544 experimental autoimmune, 545 Mycardial autoantibodies (MyA), 469 Mycobacteria immunity, 741 Mycobacterial adjuvants, 181 Mycobacterial peptidoglycolipid, 181 Mycobacterium, 740–741 Mycophenolate mofetil, 656–657 Mycoplasma immunity, 733 Mycoplasma-AIDS link, 649 Mycoses, 761 Mycosis fungoides (MF), 530 immunophenotype, 610 Myelin autoantibodies, 471 Myelin basic protein (MBP), 471, 566 antibodies, 471 Myelin-associated glycoprotein (MAG) autoantibodies, 471 Myeloablative conditioning, 687 Myeloblast, 100 Myelodysplastic syndromes (MDS), 532

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926 Myeloid antigen, 531 Myeloid cell series, 123 Myeloid cells, 100 Myeloid lineage, 100 Myeloid progenitors, 100 Myeloma, 584–585 IgD, 585 protein, 585 Myelomatosis, 585 Myeloperoxidase, 125, 633 Myeloperoxidase (MPO) deficiency, 633 Myelopoiesis, 100 Myeloproliferative diseases (MPD), 532 Myogenin (F5D), mouse, 879 Myoglobin, 879 Myoglobin antibody, 879 Myoid cells, 310 Myositis-associated autoantibodies, 489 Myositis-specific autoantibodies, 489

N N region, 228 N-addition, 228 N-formyl peptide receptor (FPR), 348 N-formylmethionine, 150 N-linked oligosaccharide, 96 N-nucleotides, 228 N-Region diversification, 228 N-terminus, 241 Naïve B cell, 217 Naïve lymphocyte, 104 Naïve T cell, 334 Naked DNA vaccine, 771 NALT. See Nasopharyngeal-associated lymphoreticular tissue NAP, 125 NAP-1. See Interleukin-8 NAP-2 (neutrophil activating protein-2), 125 Naprosyn, 796 Naproxen (2-naphthaleneacetic acid, 6-methoxy-α-methyl), 796 Nasopharyngeal-associated lymphoreticular tissue (NALT), 139, 495 Native immunity, 152 Natural anti-viral immunity, 118 Natural autoantibodies, 237, 449 Natural cytotoxicity, 113 Natural cytotoxicity receptors (NCRs), 113 Natural fluorescence, 834 Natural immunity, 152, 717 against viruses, 729 Natural interferon-producing cells (NIPCs). See Interferons Natural killer (NK) cells, 111–112, 708, 724–725 Natural killer gene complex, 113 Natural killer T (NKT) cells, 112 Naturally acquired immunity, 731 NCAM-L1, 80 NCR. See Natural cytotoxicity receptors Necrosis, 136, 137 Nef, 642 Negative induction apoptosis, 136 Negative phase, 176 Negative selection, 315, 442, 663 Neisser-Wechsberg phenomenon, 402 Neisseria immunity, 743 Neonatal Fc receptor (FcRn), 238 Neonatal immunity, 237 Neonatal thymectomy syndrome. See Wasting disease Neonatal tolerance, 438 Neoplasm, 699 Neopterin, 641 Nephelometry, 300, 822

K10141_IDX.indd 926

Index Nephritic factor, 462 autoantibodies, 462–463 Nephritic syndrome, 562 Nephrotic syndrome, 562 Nervous system, immunological diseases and immunopathology of, 565–568 Network hypothesis, 263 Network theory, 262–263 Neural cell adhesion molecule-L1. See NCAM-L1 Neuraminidase, 745 Neuroendocrine system, 443 Neurofilament, 889 Neurofilament (2F11), mouse antibody, 889–890 Neurologic disorders, autoimmune, 47–48 Neurological autoimmune diseases, 471 Neuromuscular immunological diseases and immunopathology, 544–545 Neuromuscular junction autoimmunity, 467 Neuron-specific enolase (NSE), 841, 890 Neuron-specific enolase (NSE) antibody, 889 Neuronal autoantibodies, 471 Neuropeptides, 147 Neuropilin, 82, 96 Neutralization, 746 Neutralization test, 862 Neutralizing antibody, 745 Neutropenia, 125 Neutrophil activating protein-1 (NAP-1). See Interleukin-8 Neutrophil activating protein-2 (NAP-2), 125 Neutrophil alkaline phosphatase (NAP), 125 Neutrophil chemotactic activities, 129 Neutrophil cytoplasmic antibodies, 454–455 Neutrophil leukocyte, 123–124, 454 Neutrophil microbicidal assay, 124, 850 Neutrophil nicotinamide adenine dinucleotide phophate oxidase. See Chronic granulomatous disease Neutrophil-activating factor. See Interleukin-8 Neutrophil-activating peptide 2, 360–361 Neutrophil-activating protein 2 (NAP-2), 349 Neutrophil-attracting peptide 2 (NAP-2), 361 Neutrophilia, 125 Neutrophils, 124 Neutrophils chemotaxis. See Chemotactic factors; Chemotaxis New Zealand black (NZB) mice, 455 New Zealand white (NZW) mice, 483 Newcastle disease, 758 vaccines, 778 Nezelof’s syndrome, 623 NF-AT, 337 NF-κB, 108–109 NFc (nephritic factor of the classical pathway), 397 NFid3 nuclear factor KB, 109 NFt (C3bBb-P stabilizing factor), 397 NHEJ pathway, 158 Nick translation, 864 Nidogen, 561 Nijmegen breakage syndrome (NBS), 624 NIP (4-hydroxy,5-iodo,3-nitrophenylacetyl), 171 NIPC. See Interferon-producing cells Nitric oxide (NO), 116 synthetase, 117 Nitroblue tetrazolium (NBT) test, 849 NK 1.1, 113 NK activatory receptors, 113 NK cell lectin-like receptors, 113 NK cells. See Natural killer (NK) cells NK inhibitory receptors, 113 NK tolerance, 442 NK-T cells, 113 NK/T precursor, 111 NK1-T, 113

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927

Index NK1.1, 113 NKT cells. See Natural killer T (NKT) cells Nocardia immunity, 763 NOD (nonobese diabetic) mouse, 465 NOD proteins, 127 Nodal marginal zone lymphoma (NMZL), 605 immunophenotype, 606 Non-Hodgkin lymphomas (NHL), 535 Non-homologous end-joining (NHEJ) pathway, 158 Non-myeloablative conditioning, 687 Nonadherent cell, 103 Noncovalent forces, 286 Noncytopathic virus, 745 Nonimmunologic classic pathway activators, 386 Nonprecipitating antibodies, 238 Nonproductive rearrangement, 158 Nonresponder, 176 Nonsecretor, 509 Nonsequential epitopes, 169 Nonspecific esterase (α naphthyl acetate esterase), 122 Nonspecific fluorescence, 835 Nonspecific immunity, 154 Nonspecific suppression, 651 Nonspecific T cell suppressor factor, 325, 651 Nonspecific T lymphocyte helper factor, 104, 321 Nonspecific tolerance, 445 Nonsquamous keratin (NSK), 895 Nonsterile immunity, 153 Nonsteroidal antiinflammatory drugs (NSAIDS), 417, 796 Nontissue-specific antigen, 166 Nontropical sprue, 554 Normal body constituents, 492 Normal cellular antigen (class of TAA), 702 Normal lymphocyte transfer reaction, 684 Northern blotting, 843 Norvir, 647 Nossal, Gustav Joseph Victor, 17–18 Notch 1, 101, 314 NP (4-hydroxy,3-nitrophenylacetyl), 171 Nuclear dust (leukocytoclasis), 541 Nuclear matrix proteins (NMPs), 158 Nucleic acid synthesis de novo pathway of, 156 salvage pathway of, 628 Nucleolar autoantibodies, 486 Nucleoside phosphorylase, 628 Nucleotide excision pathway (NER), 158 Nude mouse, 339–340 Null cell, 111 Null phenotype, 507 Nurse cells. See Thymic nurse cells Nutrition, immunity and, 154 Nylon wool, 846 NZB/NZW F1 hybrid mice, 483

O O antigen, 507, 727 O blood group, 507 O phage antibody library, 238 O125 (ovarian celomic), 895 Oakley-Fulthorpre test, 825 Oct-2, 231 OKT monoclonal antibodies, 238 OKT3, 660, 690, 698 OKT4. See CD4 OKT8. See CD8 Old tuberculin (OT), 854 Oligoclonal bands, 566 Oligoclonal response, 566

K10141_IDX.indd 927

Oligomorphic, 159 Oligosaccharide determinant, 169 Omenn’s syndrome, 626 Onchocerciasis volvulus immunity, 765–766 Oncofetal antigen, 703 Oncogene theory, 701 Oncogenes, 701 Oncogenesis, 701 Oncogenic virus, 705 Oncomouse, 701 One gene, one enzyme theory (historical), 227 One-hit theory, 403 One-turn recombination signal sequences, 227 Open reading frame (ORF), 158 Opisthorchiasis-clonorchiasis immunity, 765 Opsonin, 127 Opsonins, 719–720 Opsonization, 127 Opsonophagocytosis, 127 Oral immunology, 501 Oral tolerance, 444, 501, 502 Oral unresponsiveness, 501 Organ bank, 683 Organ brokerage, 683 Organ-specific antigen, 450 Organ-specific autoimmune diseases, 450 Organism-specific antibody index (OSAI), 733 Orthoclone OKT3, 660, 690, 698 Orthotopic, 678 Orthotopic graft, 678 Oseltamivir, 787 Osteocalcin antibody, 874 Osteoclast, 145 Osteoclast-activating factor (OAF), 381 OT (historical), 854 Ouchterlony test, 825 Ouchterlony, Orjan Thomas Gunnersson, 64 Oudin test, 825 Oudin, Jacques, 64 Outbreeding, 861 Ovalbumin (OA), 164 Ovary antibodies (OA), 466 Ovary autoantibodies, 466 Ovine immune system, 811 Owen, Ray David, 55 Oxazolone (4-ethoxymethylene-2-phenyloxazol-5-one), 433 Oxidized low-density lipoprotein autoantibodies, 493 Oxygen-dependent killing, 125, 724 Oxygen-independent killing, 125–126 Oz isotypic determinant, 258

P P. See Properdin P antigen, 515 P-80, 822 P-addition, 227 P-K reaction. See Prausnitz-Küstner reaction P-nucleotides, 225 P-selectin (CD62P), 90 P1 kinase, 758 P1A1 antibodies, 521 P24 antigen, 642 P53, 701 P63 (ap53 homolog at 3q27-29) Ab-4 (cocktail) mouse monoclonal antibody, 874 PAF. See Platelet-activating factor Palindrome, 157 Palivizumab (injection), 789 PAMP. See Pathogen-associated molecular pattern

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928 Pan keratin antibodies, 840 Pan-T cell markers, 318 Panagglutination, 509 Pancreas, immunological diseases and immunopathology of, 558–559 Pancreatic islet cell hormones, 840 Pancreatic transplantation, 684 Pandemic, 717 Panel reactive antibody (PRA) test, 673 Paneth cells, 720 Panning, 849 Pannus, 576 PAP (peroxidase-antiperoxidase) technique, 839–840 Papain, 253 Papain hydrolysis, 253 Paper radioimmunosorbent test (PRIST), 857 Papillomavirus immunity, 758 Papovaviruses, 746 Para-Bombay phenotype, 507 Parabiotic intoxication, 695 Paracortex, 103 Paracrine, 345 Paracrine factor, 345 Paradoxical reaction (historical), 411 Paraendocrine syndromes, 590 Paraimmunoglobulins, 590 Parainfluenza virus (PIV) immunity, 758 Paralysis, 445 Paralyzed TCRs, 166 Paramyxovirus immunity, 758 Paraneoplastic autoantibodies, 472 Paraneoplastic autoimmune syndromes, 491 Paraneoplastic pemphigus, 472 Paraprotein, 585 Paraproteinemias, 589–590 Parasite immunity, 763 Parasites, 763 Parathyroid hormone autoantibodies, 451 Parathyroid, autoimmunity and, 46 Paratope, 237 Parenteral, 175 Parietal cell antibodies, 457, 556 Parietal cell autoantibodies, 457–458 Paroxysmal cold hemoglobinuria (PCH), 455 Paroxysmal nocturnal hemoglobinuria (PNH), 401, 526 Partial identity. See Reaction of partial identity Parvovirus, 751 immunity, 751–752 Parvovirus B19, 878 PAS, 841 Passive agglutination, 830 Passive agglutination test, 831 Passive anaphylaxis, 413 Passive Arthus reaction, 427 Passive cutaneous anaphylaxis (PCA), 422, 855 Passive hemagglutination, 303 Passive hemolysis, 303 Passive immunity, 153, 731 Passive immunization, 273, 773 Passive sensitization, 407 Passive systemic anaphylaxis, 414 Passive transfer, 407 Pasteur, Louis, 8–9 Pasteurella immunity, 743 Patch test, 430, 852 Patching, 223 Paternity testing, 862 Pathogen-associated molecular pattern (PAMP), 733 Pathogenesis-related (PR) proteins, 799 Pathogenicity, 717 Pathogens, 733 attenuated, 769

K10141_IDX.indd 928

Index Pathologic autoantibodies, 449 Pattern recognition molecules (PRMs), 117 Pattern recognition receptors (PRRs), 734 Paul-Bunnell test, 829 PAX-5, 887 Pax-5 gene, 227 Payne, Rose, 57 PBC. See Primary biliary cirrhosis PCA. See Passive cutaneous anaphylaxis PCH. See Paroxysmal cold hemoglobinuria PCP, 641 PCR. See Polymerase chain reaction PECAM (CD31), 86 Pediatric AIDS, 648 Pegademase bovine, 786 Peginterferon α-2a, 794 Pemphigoid, 472 Pemphigus erythematosus (Senear-Usher syndrome), 472 Pemphigus foliaceus, 473 Pemphigus vulgaris, 473, 539 Penicillin hypersensitivity, 435–436 Pentadecacatechol, 435 Pentamidine isoethionate, 647 Pentraxins, 151 Pepsin digestion, 253 Pepsinogen, autoantibodies against, 458 Peptide interception, 208 Peptide regurgitation, 208 Peptide T, 642 Peptide vaccine, 771 Peptide-binding cleft, 202 Peptide-binding motif, 199 Percoll, 828 Perforin, 324–325 Perforin/granzyme-mediated cytotoxicity, 112 Periarteriolar lymphoid sheath (PALS), 145 Periarteritis nodosa, 543 Perinuclear antibodies, 578 Perinuclear factor (profillagrin) autoantibodies, 484 Peripheral blood mononuclear cells, 104 Peripheral lymphoid organs, 137 Peripheral lymphoid tissues. See Secondary lymphoid tissues Peripheral T cell lymphoma, NOS, 611 immunophenotype, 612 Peripheral tolerance, 440 Permeability factors. See Vascular permeability factors Permeability-increasing factor, 345 Pernicious anemia (PA), 455, 555–556 Peroxidase-antiperoxidase (PAP) technique, 838–839, 867 Persistent generalized lymphadenopathy (PGL), 641 Persistent infection, 717 Pertussis adjuvant, 181 Pertussis vaccines, 780 Peyer’s patches, 145 PFC (plaque-forming cell), 846 PFc′ fragment, 253 Pfeiffer phenomenon (historical), 306 PFU, 846 PHA, 335 Phacoanaphylactic endophthalmitis, 474 Phacoanaphylaxis, 421 Phage antibody library, 227 Phage display, 238–239 library, 239 Phagocyte disorders, 630 Phagocytes, 126 Phagocytic cell function deficiencies, 630 Phagocytic cells, 720–721 Phagocytic dysfunction, 631 Phagocytic index, 631 Phagocytosis, 117, 721–723, 800

3/25/10 12:57:12 PM

Index frustrated, 118 of microorganisms, 117–118 Phagolysosome, 118, 127 Phagosome, 118 Pharyngeal pouch, 307 Pharyngeal pouch syndrome, 307 Pharyngeal tonsils, 141 Phenotype, 675 Phenylbutazone, 578, 796 Philadelphia chromosome, 532–533 Phoma species, 408 Phorbol ester(s), 107, 166–167, 335 Phosphatase, 95 Phosphatidylinositol bisphosphate (PIP2), 107 Phosphocholine antibodies, 734–735 Phospholipid autoantibodies, 480 Phosphotyrosine kinases in T cells, 326–327 Photoallergy, 416 Photoimmunology, 503 Phycoerythrin, 851 Phylogeny, 814 Phytoalexins, 799 Phytogenetic-associated residues, 806 Phytohemagglutinin (PHA), 799 Phytoimmunity, 799 Phytomitogens, 799 Phytonicides, 799 PI. See Primary immunodeficiency Picornavirus, 748, 759 immunity, 759 Picryl chloride (1-chloro-2,4,6-trinitrobenzene), 433 Piecemeal necrosis, 557 Pigeon breeder’s lung, 408 Pili, 735 Pinocytosis, 118 Pituitary autoantibodies, 472 autoimmunity and, 46 PK test. See Prausnitz-Küstner reaction Plague vaccine, 779 Plant immunity, 799 Plantibodies, 800 Plaque neutralization assay, 862 Plaque technique, 862 Plaque-forming cell (PFC) assay, 845, 861 Plaque-forming cells, 861 Plasma, 505 Plasma cell antigen, 222 Plasma cell dyscrasias, 586 Plasma cell myeloma, immunophenotype, 603 Plasma cell neoplasms, 601–602 Plasma cells, 110–111, 221 Plasma half-life (T1/2), 283 Plasma histamine, 415 Plasma pool, 284 Plasmablast, 221 Plasmacyte, 221 Plasmacytoid dendritic cells (PDCs), 121 Plasmacytoma, 534, 586 Plasmapheresis, 863–864 Plasmid, 864 Plasmin, 254 Plasminogen, 119–120 Platelet, 132 Platelet antigens, 520–521 Platelet autoantibodies, 456 Platelet endothelial cell adhesion molecule-1 (PECAM-1)(CD31), 86 Platelet factor 4 (PF4), 380 Platelet transfusion, 521 Platelet-activating factor (PAF), 410 Platelet-activating factor receptor (PAFR), 380

K10141_IDX.indd 929

929 Platelet-associated immunoglobulin (PAIgG), 685 Platelet-derived growth factor (PDGF), 132 Platelet-derived growth factor receptor (PDGF-R), 132 Pleitropic, 368 Pleuropneumonia-like organisms. See PPLO Pluripotency, 99 Pluripotent stem cell, 99 PM-Scl autoantibodies, 486 PMN. See Polymorphonuclear leukocytes Pneumococcal 7 valent conjugate vaccine, 783 Pneumococcal 7 valent conjugate vaccine (diphtheria CRM197 protein) (injection), 783 Pneumococcal polysaccharide, 164 vaccine, 782 Pneumococcal vaccine polyvalent (injection), 783 Pneumocystis carnii (formerly Pneumocystis jiroveci) (PCP), 645 PNH. See Paroxysmal noctural hemoglobinuria PNH cells, 526 POEMS syndrome, 589 Poison ivy, 434 hypersensitivity, 434 Pol, 644 Poliomyelitis vaccines, 776 Poliovirus, 752 Pollen hypersensitivity, 420 Poly-Ig receptor, 278 Polyagglutination, 508 Polyarteritis nodosa, 542–543 Polyarthritis, 552 Polyclonal, 111 Polyclonal activators, 166 Polyclonal antibodies, 269, 660 Polyclonal antiserum, 235 Polyclonal hypergammaglobulinemia, 587 Polyclonal rabbit anti-calretinin, 892 Polyclone proteins, 284 Polyendocrine autoimmunity, 493 Polyendocrine deficiency syndrome, 464 Polyethylene glycol assay for CIC, 822 Polygenic, 159 Polygenic inheritance, 176 Polyglandular autommune syndrome, 464 Polyimmunoglobulin receptor, 278, 496 Polymerase chain reaction (PCR), 843–844 Polymeric immunoglobulins (Ig), 278 Polymers, 253 Polymorphism, 511, 667 Polymorphonuclear leukocytes (PMNs), 123, 720 Polymyositis (PM), 544 Polynucleotide, 180 Polyomavirus immunity, 663 Polyspecific antihuman globulin (AHG), 512 Polyspecificity, 285 Polyvalent antiserum, 239, 285 Polyvalent pneumococcal vaccine, 783 Polyvalent vaccine, 770 Popcorn cells, 536 Porcine immunity, 811 Porter, Rodney Robert, 24 Portier, Paul Jules, 30 Positive induction apoptosis, 136 Post-GC, 530 Post-transplant lymphoproliferative disorder (PTLD), 698 Postcapillary venules, 141 Postcardiotomy syndrome, 469 Postinfectious encephalomyelitis, 472, 568 Postinfectious iridocyclitis, 474 Postrabies vaccination encephalomyelitis, 775 Poststreptococcal glomerulonephritis, 563 Posttransfusion graft-vs.-host disease, 694 Postvaccinal encephalomyelitis, 774

3/25/10 12:57:12 PM

930 Postzone, 305 Poxvirus immunity, 758–759 PPD. See Purified protein derivative PPLO (pleuropneumonia-like organisms), 520 PRA, 673 Prausnitz-Giles, Carl, 31 Prausnitz-Küstner (PK) reaction (historical), 421–422 Pre-B cell receptor, 216 Pre-B cells, 215 Pre-GC, 530 Pre-T cell alpha (pTα), 314 Pre-T cell receptor (Pre-TCR), 314 Pre-T cells, 314 Pre-T lymphocyte, 315 Pre-TCR activation, 315 Pre-vertebrates, 803 Preactivation, 745 Precipitating antibody, 237 Precipitation, 295 Precipitation reaction, 295 Precipitin, 237, 295 Precipitin curve, 296 Precipitin reaction, 295 Precipitin test, 295 Preclinical trials, 797 Precursor B lymphoblastic leukemia/lymphoma, NOS, immunophenotype, 598–599 Precursor T lymphoblastic leukemia/lymphoma, immunophenotype, 599–600 Prednisolone (1,4-Pregnadiene-11β,17α,21-Triol-3,20-Dione), 653 Prednisone (1,4-Pregnadiene-17α,21-Diol-3,11,20-Trione), 653 Preemptive immunity, 153 Pregnancy, lupus erythematosus and, 482 Preintegration complex, 642 Prekallikrein, 95 Premalignant clone, 710 Preneoplastic clone, 710 Preprogenitor cells, 217 Present, 201 Presentosome, 205 Prick test, 412, 852 Primary agammaglobulinemia, 618 Primary allergen, 420 Primary biliary cirrhosis (PBC), 557 Primary follicle, 141–142 Primary granule, 125 Primary immune response, 176 Primary immunodeficiency (PI), 617 Primary interaction, 285 Primary lymphoid follicles, 109 Primary lymphoid organs, 139 Primary lymphoid tissues, 139 Primary lysosome, 98 Primary nodule. See Primary follicle Primary reaction, 285 Primary response, 176 Primary sclerosing cholangitis (PSC), 460 Primary structure, 169 Primary tumor, 700 Primate (nonhuman) immune system, 814 Prime-boost strategy, 771 Primed, 176 Primed lymphocyte, 176 typing, 669 Primed lymphocyte test (PLT), 668–669 Priming, 176 Prion, 752 Private antigen, 174, 677 Private idiotypic determinant, 262 Private specificity, 174 Privileged sites, 678–679

K10141_IDX.indd 930

Index Pro-C3, 389 Pro-C4, 391 Pro-C5, 391 Pro-drug, 792 Pro-T cell, 313 Probe hybridization (tissue typing), 675 Procomplementary factors, 385 Productive infection, 717 Productive rearrangement, 104 Productivity testing, 226, 328 Proenzyme, 395 Professional antigen-presenting cells, 206 Progenitor cell, 101 Progerssive systemic sclerosis (scleroderma), 580 Programmed cell death, 134 Progressive multifocal leukoencephalopathy, 641 Progressive transformation of germinal centers (PTGC), 536 Progressive vaccinia, 773 Prokaryotes, immunity in, 801 Proleukin (aldesleukin), 664 Promiscuous binding, 81 Promoter, 701 Properdin (factor P), 395 Properdin deficiency, 396 Properdin pathway, 395 Properdin system, 395 Prophylactic immunization, 773 ProPO system, 803 Propylthiouracil, 796 Prostaglandins (PG), 417 Prostate-specific antigen (PSA), 702–703, 875 Prostatic acid phosphatase (PAP)/prostatic epithelial antigen, 875 Proteasome, 203 Proteasome genes, 205 Protectin (CD59), 394 Protective antigens, 153 Protective epitopes, 153 Protective immunity, 153, 718 Protein A, 229 Protein AA, 583 Protein B, 822 Protein blotting. See Immunoblotting Protein kinase C (PKC), 95, 336 Protein M, 734 Protein P, 583 Protein S, 95 Protein SAA, 583 Protein separation techniques, 842 Protein tyrosine kinases, 107 Proteinuria, 562 Proteolipid protein autoantibodies, 472 Proteomics, 156 Proteus immunity, 743 Prothombin antibodies, 455–456 Prothymocyte, 315 Protooncogene, 701 Protoplast, 734 Protoplast fusion, 864 Protostomes, 803 Protozoa, 800 Protozoans, 763 Proviral DNA. See Provirus Provirus, 638, 759 Provocation poliomyelitis, 776 Prozone, 304 PRP antigen, 734 Pruritis, 558 PSA. See Prostate-specific antigen Pseudoalleles, 159 Pseudoallergic reaction, 415–416 Pseudoallergy, 416

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931

Index Pseudogene, 158 Pseudolymphoma, 536 Pseudolymphomatous lymphadenitis, 585 Pseudomonas aeruginosa immunity, 743 Pseudoparaproteinemia, 590 Pseudopodia, 127 PSMAC, 204 Psoriasis vulgaris, 540 Psychoneuroimmunology, 443 PTAP, 779 Public antigen, 164, 174, 513, 677 Public idiotypic determinant (IdX or CRI), 262 Public specificity, 186 Pulmonary vasculitis, 550 Pulsed-field gel electrophoresis, 827 Pulsing, 203 Purified protein derivative (PPD), 430 Purine nucleotide phosphorylase (PNP), 627 deficiency, 627–628 Purpura, 543 Purpura hyperglobulinemia, 588 Pyogenic bacteria, 734 Pyogenic infection, 734 Pyogenic microorganisms, 734 Pyrogen, 351, 734 Pyroglobulins, 234 Pyroninophilic cells, 222

Q Q fever, 755 Qa antigens, 189 Qa locus, 188–189 Qa region. See Tla complex Qa-2 antigen, 189 Quantitative gel diffusion test, 296 Quantitative precipitin reaction, 296, 818 Quarternary syphilis, 641 Quaternary structure, 169 Quellung phenomenon, 735 Quellung reaction, 735 Quenching, 835–836 Quni-2, 836 Qunidine (6'-methoxycinchonan-9-ol), 796

R R5 viruses, 639 RA. See Rheumatoid arthritis RA cell. See Rheumatoid arthritis cell RA-33, 578 Rabbit immunity, 809 Rabbit immunoglobulin allotypes, 809 Rabies, 752 vaccine, 774 Rabies vaccination, 775 Rac, 337 Radiation bone marrow chimeras, 687 Radiation chimera. See Irradiation chimera Radiation therapy, 796 Radiation, immunity and, 662–663 Radical immunodiffusion, 302 Radioallergosorbent test (RAST), 856 Radioimmunodiffusion, 819 Radioimmunodiffusion test. See Single radial immunodiffusion Radioimmunoelectrophoresis, 824 Radioimmunoprecipitation assay (RIPA), 822 Radioimmunoscintigraphy, 711 Radioimmunosorbent test (RIST), 856–857 Radioimmunotherapy, 796 Radiolabeling, 818

K10141_IDX.indd 931

Radiomimetic drug, 662 Radionuclide labeling, 818 RAG blastocyst complementation, 861 RAG recombinases, 226 RAG-1, 225 RAG-2, 225 Ragg, 578 Ragweed, 421 Raji cell assay, 831–832 Ramon test (historical), 865 Ramon, Gaston, 63 RANA (rheumatoid arthritis-associated nuclear antigen), 577 RANA autoantibodies, 485 Random breeding, 812 RANK Ligand, 372 RANTES, 349 Rapamycin, 654–655 Ras, 159, 701 Ras/MAPK signaling pathway, 159, 346 Raynaud’s phenomenon, 573 RB200. See Lissamine rhodamine RCA locus (regulator of complement activation), 385 Reaction of identity, 302 Reaction of nonidentity, 301, 302 Reaction of partial identity, 301, 302 Reactive arthritis, 553 Reactive lysis, 403 Reactive nitrogen intermediates, 127 Reactive oxygen intermediates (ROIs), 83 Reactive oxygen species (ROS), 116 Reagin (historical), 241 Reaginic antibodies. See Reagin (historical) Reassortant vaccine, 771 Rebuck skin window, 856 Receptor blockade, 442 Receptor editing, 227 Receptor-associated tyrosine kinases, 105 Receptor-mediated endocytosis, 224 Receptors, 109 adrenergic, 109 antigen, 110 Recirculating pool, 105 Recirculation of lymphocytes, 105 Recognition phase, 141 Recognition unit, 384 facilitation of binding by C1q, 387 Recombinant cytokines, 664 Recombinant DNA, 159 technology, 864 Recombinant DNA technology, 159 Recombinant inbred strains, 813 Recombinant vaccine, 772 Recombinant vector vaccine, 772 Recombination activating gene 1 (RAG-1), 226 Recombination activating gene 2 (RAG-2), 226 Recombination activating genes (RAG-1 and RAG-2), 283 Recombination recognition sequences, 226 Recombination signal sequences (RSSs), 284 Recombinatorial germ-line theory, 286 Recticulum cell. See Reticular cells Recurrent infection, 629 Red cell-linkied antigen antiglobulin test, 829 Red marrow. See Bone marrow Red pulp, 145 Reed-Sternberg cells, 535 Refractory cancer, 699 Regional enteritis, 555 Regocyte, 578 Regulation of complement activation (RCA) cluster, 389 Regulators of complement activity (RCA), 389 Regulatory CD4+ cells, 441

3/25/10 12:57:13 PM

932 Regulatory peptides, 799 Regulatory T cells, 441 Reiter complement fixation test (historical), 833 Rejection, 689 Relapse, 525 Relapsing polychondritis, 570 Relative risk (RR), 191 Relative sibling risk, 683 Released antigen, 768 REMICADE (infliximab), 790 Remission, 699–700 Renal cell carcinoma, 894 Renal immunological diseases and immunopathology, 559–565 ReoPro (abciximab), 790 Reovirus immunity, 759 Replicative senescence, 136 Reptile immunity, 807 Reptiles, 807–808 Repulsion, 288 Rescue graft, 678 Reservoir, 717 Resident macrophage, 116 Respiratory burst, 125 Responder animals, 176 Resting lymphocytes, 103–104, 212, 336 Restriction endonucleases, 159–160 Restriction fragment length polymorphism (RFLP), 160, 279, 844 Restriction map, 160 Reticular cells, 141 Reticular dysgenesis, 624 Reticulin autoantibodies, 458 Reticuloendothelial blockade, 664 Reticuloendothelial system (RES), 122 Reticulosis. See Lymphoma Reticulum cell sarcoma, 530 Retina autoantibodies, 474 Retrovir, 647 Retrovirus, 638 Retrovirus immunity, 641 Rev protein, 642 Reverse anaphylaxis, 427 Reverse genetics approach, 859 Reverse immunology, 714 Reverse Mancini technique, 819 Reverse passive Arthus reaction, 427 Reverse plaque method, 846 Reverse radioimmunodiffusion, 819 Reverse transcriptase, 642 Reverse transcriptase polymerase chain reaction (RT-PCR), 844 Reverse vaccinology, 771 RF. See Rheumatoid factor RFLP (restriction fragment length polymorphism), 160, 845 Rh disease. See Erythroblastosis fetalis Rh factor, 510 Rhabdovirus immunity, 759–760 Rhesus antibody, 511 Rhesus antigen, 510 Rhesus blood group system, 510 Rhesus incompatibility, 511 Rheumatic, 553 Rheumatic fever (RF), 552 Rheumatoid arthritis (RA), 43–44, 484, 575 juvenile, 485 Rheumatoid arthritis cell (RA cell), 484, 577 Rheumatoid arthritis-associated nuclear antigen, 577 Rheumatoid factor (RF), 43–44, 484–485, 576–577 Rheumatoid nodule, 485, 577 Rheumatoid pneumonitis, 485 Rhinovirus immunity, 760 RhLA locus, 191 Rhnull, 510

K10141_IDX.indd 932

Index RhoD immune globulin, 510–511 Rhodamine isothiocyanate, 836 RhoGAM, 511 RIA, 822 Ribavarin (1-8-5-3 ribofuranosyl-1,2,4-trizole-3-carboxamide), 648 Ribosomal P protein autoantibodies (RPP), 478 Ribosome, 98 Ribozyme, 786 Richet, Charles Robert, 30 Ricin, 712 Ricinus communis, 712 Rickettsia immunity, 755 Rieckenberg reaction, 822 Rinderpest vaccines, 772 Ring precipitation test, 296 Ring test, 296 Rituxan (rituximab), 790 Rituximab, 790 RNA polymerase, 486 RNA polymerases I, II, and III autoantibodies, 486 RNA splicing, 865 RNA-directed DNA polymerase (reverse transcriptase), 642 RNAse protection assay, 864 Ro/SS-A, 488 Rocket electrophoresis, 826 Rodgers (Rg) antigens, 519–520 Romer reaction (historical), 854 Roquinimex, 794 ROS. See Reactive oxygen species (ROS) Rose, Noel Richard, 43–44 Rose-Waaler test, 829 Rosette, 108, 318 Rotavirus, 752 Round cells, 104 Rous sarcoma virus (RSV), 701 RPR (rapid plasma reagin) test, 830 RS61443 (Mycophenolate mofetil), 657 Rubella vaccine, 776 Runt disease, 340 Runting syndrome, 340 Russell body, 111

S S, 828 S antibody, 245 S protein, 393 S region, 190 S value (Svedberg unit), 828 S-100, 879 S-100 protein, 879 S-100 protein antibody, 879 Sabin vaccine, 776 Sabin-Feldman dye test, 766 Saccharated iron oxide, 123 Sacculus rotundus, 500, 809 Sago spleen, 582 SAIDS (simian acquired immunodeficiency syndrome), 648 Saline agglutinin, 508 Salk vaccine, 775 Salmonella immunity, 743 SALT. See Skin-associated lymphoid tissue Salt precipitation, 828 Salting out, 828 Salvage pathway of nucleic acid synthesis, 628 Salvage therapy, 699 SAM. See Substrate adhesion molecules Sanarelli-Shwartzmann reaction, 428 Sandoglobulin, 660 Sandwich ELISA, 835 Sandwich immunoassay, 834

3/25/10 12:57:13 PM

Index Sandwich method, 834 Sandwich technique, 834 SAPK/JNK signaling pathway, 346 Saponin, 772 SAR. See Systemic acquired resistance Sarcoidosis, 549–550 Sarcomas, 699 SCAB, 241 Scarlet fever, 735 Scatchard analysis, 288 Scatchard equation, 288 Scatchard plot, 288 Scavenger receptors, 123 ScFv, 242 Schick test, 853 Schick, Bela, 63 Schistosoma immunity, 766 Schistosomiasis, 768 vaccines, 784 Schlepper, 171 Schultz-Dale test (historical), 411 SCID. See Severe combined immunodeficiency syndrome SCID (severe combined immundeficiency) human mouse, 625 SCID mouse, 625 Scl-70 (topoisomerase I) autoantibody, 486–487 Scl-70 antibody, 580 Scleroderma, 486 SCR1 (soluble complement receptor type 1), 400 Scratch test, 411 Scurfy mouse, 441 SDS-PAGE, 827 SE. See Staphylococcal enterotoxins Second messengers (IP3 and DAG), 330 Second signals, 106 Second-set rejection, 689 Second-set response, 689 Secondary allergen, 420 Secondary antibody, 280 response, 177 Secondary disease, 694 Secondary follicle, 143 Secondary granules, 125, 720 deficiency of, 631 Secondary immune response, 176–177 Secondary immunodeficiency, 635 Secondary lymphoid follicle, 218 Secondary lymphoid organs, 141 Secondary lymphoid tissues, 141 Secondary lysosome, 128 Secondary reactions, 285 Secondary response, 177 Secondary structure, 169 Secreted immunoglobulin (sIg), 251 Secretor, 509 Secretory antibodies, 499 Secretory component (T piece), 500 Secretory component deficiency, 500 Secretory IgA, 499 Secretory immune system, 499 Secretory immunoglobulin A (SIgA), 718 Secretory piece, 251–252, 500 Sedimentation coefficient, 828 Sedimentation pattern, 828 Sedormid purpura (historical), 493 Segmental exchange, 158 Selectins, 79 Selective IgA deficiency, 620–621, 621–622 Selective IgG deficiency, 621 Selective IgM deficiency, 621–622 Selective immunoglobulin deficiency, 620 Selective theory, 285

K10141_IDX.indd 933

933 Self-marker hypothesis (historical), 18, 264 Self-MHC, 187 Self-MHC restriction, 330 Self-peptides, 439 Self-renewal, 136 Self-restriction. See Self-MHC restriction Self-tolerance, 439 Semisyngeneic graft, 679 Senear-Usher syndrome, 472 Senescent cell antigen, 522 Sensitization, 407 Sensitized lymphocyte, 337–338 Sensitized vaccine, 771 Sensitizing agent, 407 Sephadex, 816 Sepharose, 827 Septic shock, 735 Septicemia, 717 Sequence-specific priming (SSP), 845 Sequential determinant, 169 Sequestered antigen, 449–450 Serial dilution, 817 Serial passage, 866 Serial TC triggering model, 313 Seroconversion, 735 Serological determinants, 676 Serological epitopes, 168 Serologically defined (SD) antigens, 676 Serology, 285 Serotherapy, 785 Serotonin (5-hydroxytryptamine) [5-HT], 416, 890 Serotype, 735 Serpins, 647 Serum albumin, 165, 513 Serum amyloid A component (SAA), 582 Serum antitoxins, 235 Serum hepatitis (hepatitis B), 558 Serum sickness, 426 Serum virus vaccination, 772 Severe combined immunodeficiency (Swiss-type agammaglobulinemia), 617 Severe combined immunodeficiency syndrome, 624–625 X-linked, 626 Sex hormones, immunity and, 450 Sex-limited protein, 389 Sézary syndrome, 537 Sézary syndrome (SS), immunophenotype, 611 SH-2 domain, 105 Shared haplotype, 675 Sheep red blood cell agglutination test, 829 Sheep red blood cells (SRBC), 318 Shift assay, 865 Shigella immunity, 735 Shingles (herpes zoster), 751 SHIV (simian-human immunodeficiency virus), 649 Shock organ, 412 Shocking dose, 412 Short-lived lymphocytes, 104 Shwartzman reaction, 427 Shwartzman, Gregory, 31 Shwartzman-Sanarelli reaction, 427 Sialophorin (CD43), 310 Sicca complex, 488–489 Sicca symptoms, 579 Side chain theory, 263–264 Side effect, 493 Signal hypothesis, 286 Signal joint, 226, 328 Signal peptide, 222 Signal recognition particle autoantibodies against SRP, 489–490 Signal sequence. See Signal hypothesis

3/25/10 12:57:13 PM

934 Signal transducers and activators of transcription (STATs), 345. See also Janus kinases Signal transduction, 105 Silencer sequence, 331 Silencers, 331 Silica adjuvants, 181 Silicate autoantibodies, 490–491 Silicosis, 551–552 Simian immunodeficiency virus (SIV), 648 Simonsen phenomenon, 695 Simple allotype, 259 Simulect (basiliximab), 790 Sin test (historical), 822 Single chain antigen-binding proteins, 241 Single cysteine motif-1 (SCM-1), 380 Single diffusion test, 819 Single domain antibodies, 258 Single hit theory, 386 Single immunodiffusion, 818 Single locus probes (SLPs), 678, 845 Single nucleotide polymorphism (SNP), 158 Single radial immunodiffusion, 819 Single-chain Fv fragment, 258 Single-positive thymocytes, 311 Sips distribution, 294 Sips plot, 294 Sirolimus, 655 Site-directed mutagenesis, 865 SIV (simian immunodeficiency virus), 648 SIVmac, 649 Sjögren’s syndrome, 45, 487–488, 578–579 Skin as mechanical barrier, 718 autoantibodies, 473–474 autoimmune reactions of, 48 immunological diseases and immunopathology of, 538–540 Skin graft, 684 Skin immunity, 502–503 Skin test, 852 Skin window, 856 Skin-associated lymphoid tissue (SALT), 502 Skin-fixing antibody, 239 Skin-reactive factor (SRF), 345 Skin-sensitizing antibody, 408 Skin-specific histocompatability antigen, 684 Slide agglutination test, 830 Slide flocculation test. See Slide agglutination test Slot blot analysis, 864 Slow viruses, 752 Slow-reacting substance of analphylaxis (SRS-A), 417 Slp. See Sex-limited protein Sm, 477 SMAA, 771 SMAC, 204 Small “blues,” 675 Small G proteins, 95 Small lymphocyte, 103 Small molecules, 660 Small pre-B cells, 215 Smallpox, 1–6, 760, 773 vaccination, 774 vaccine, 774 Smooth muscle antibodies, 557 Smooth muscle autoantibodies, 459 SNagg, 485 Sneaking through, 699 Snell, George Davis, 56 Snell-Bagg mice, 341 SOD. See Superoxide dismutase SODD (silencer of death domains), 371

K10141_IDX.indd 934

Index Solid organ transplant, 686 Solid-phase radioimmunoassay, 823 Solitary plasmacytoma of bone, 534 Solubilized water-in-oil adjuvant, 181 Soluble antigen, 163 Soluble complex, 305 Soluble cytokine receptors, 345 Soluble liver antigen antibodies, 557 Somatic antigen, 727, 728 Somatic cell hybrid, 111 Somatic gene conversion, 806 Somatic gene therapy, 627 Somatic hybrid selection, 111 Somatic hypermutation, 286 Somatic mutation, 286 Somatic recombination, 226, 313–314 Southern blotting, 842 Southwestern blot, 842 SP thymocytes, 312 SP-40,40, 394 Spar, 302 Species specificity, 814 Specific granule, 125 Specific immunity, 153 Specificity, 219, 328 Speckled pattern, 490 Spectratyping, 865 Spectrin, 891 Spectrotype, 861 Sperm antibodies, 466 Sperm autoantibodies, 467 Spermatozoa, immunological diseases and immunopathology of, 570 Spheroplast, 735 Spherulin, 763 Spleen, 143–144 Splenic cords, 144 Spliceosomal snRNP autoantibodies, 478 Split thickness graft, 684 Split tolerance, 444 Splits, 676–677 Sponges, 801 Spongiform encephalopathies, 752 Spontaneous autoimmune thyroiditis (SAT), 451 Spontaneous cancer, 699 Spontaneous remission, 700 Sporadic cancer, 699 Spot ELISA, 841 Sprue. See Gluten-sensitive enteropathy Squalene, 181 SRBC (sheep red blood cells), 318 Src homology-2 (SH-2) domain, 105 Src homology-3 (SH-3) domain, 106 SRS-A, 410 SRV-1, 649 SRY, 865 Ss protein, 389 SS-A, 579 SS-A Ro, 488 SS-A/Ro antibodies, 488 SS-B, 580 SS-B La, 488 SS-B/La antibodies, 489 SSPE, 579 SSS III, 164 St. Vitus dance (chorea), 552 Staphylococcal enterotoxins (SEs), 735 Staphylococcal protein A, 231 Staphylococcus immunity, 735–736 STAT transcription factors, 345 Status asthmaticus, 423

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935

Index Status thymolymphaticus (historical), 310 Stem cells, 98, 685–686 pluripotent, 99 Stem-cell factor (SCF), 99 Steroid cell antibodies, 464 Stiff man syndrome (SMS), 467 Stimulated macrophage, 116 Stochastic models, 866 Stormont test, 853 Strain, 859 Street virus, 755 Streptavidin, 867 Streptobacillus immunity, 736 Streptococcal M protein, 736 Streptococcus immunity, 736 Streptolysin O test. See ASO Stress proteins, 95 Stress, immunity and, 444 Striational antibodies, 467 Striational autoantibodies (StrAb), 467–468 Stroma/sarcoma, 878 Stromal cell-derived factor-1 (SDF-1), 380–381 Stromal cells, 133, 307, 310 Strongyloides hyperinfection, 766 Strongyloides immunity, 766 STS, 822 Sub-exon, 280 Subacute sclerosing panencephalitis, 755 Subset, 334 Substance P, 410 Substrate adhesion molecules (SAM), 82 Subunit vaccine, 771 Sugercane worker’s lung. See Bagassosis; Farmer’s lung Sulfite sensitivity, 411 Sulzberger-Chase phenomenon, 444, 502 Superantigen, 163, 210–211 Superinfection “immunity,” 736 Superoxide anion, 125 Superoxide dismutase, 116 Suppressin, 325 Suppression, 651 Suppressor CD4+ cells, 441 Suppressor cell, 325 Suppressor macrophage, 128 Suppressor T cell factor (TsF), 325 Suppressor T cells (Ts cells), 325 Suppressor/inducer T lymphocytes, 325 Supratypic antigen, 163–164, 513, 667, 677. See also Public antigen Suramin, 394, 766 Surface antigens, 166 Surface immunoglobulin, 229 Surface phagocytosis, 127 Surface plasmon resonance (SRP), 293 Surrogate light chains, 229 Sustiva, 647 SV40 (simian virus 40), 704–705 Svedberg unit, 828 Sweet’s syndrome (acute febrile neutrophilic dermatosis), 540 Swiss agammaglobulinemia, 627 Swiss type immunodeficiency. See Swiss-type agammaglobulinemia Swiss-type agammaglobulinemia, 617, 620 Switch, 286 Switch cells, 286 Switch defect disease, 266 Switch recombination, 226 Switch region, 266–267 Switch site, 267 SXY-CIITA regulatory system, 189 Syk PTK, 218 Sympathetic nervous system autoantibodies, 473

K10141_IDX.indd 935

Sympathetic ophthalmia, 474, 568 Synaptophysin, 890 Syncytia formation, 642 Synergism, 345 Syngeneic, 683 Syngeneic preference, 681 Syngraft, 683 Synthetic antigen, 161 Synthetic polypeptide antigens, 167 Synthetic vaccines, 772 Systemic acquired resistance (SAR), 799 Systemic anaphylaxis, 413–414 Systemic autoimmune diseases, 475, 570–581 Systemic autoimmunity, 475 Systemic immunoblastic proliferation, 533–534 Systemic inflammatory response syndrome (SIRS), 730 Systemic lupus erythematosus (SLE), 42–43, 475–476, 570–572, 579–580 animal models of, 482 Systemic sclerosis, 486

T θ antigen. See Thy 1 antigen T activation, 522 T agglutinin, 522 T antigens, 521–522 T cell antigen receptors, 328 T cell antigen-specific suppressor factor, 325 T cell areas, 317 T cell clonal expansion, 334 T cell domains, 317 T cell growth factor (TCGF). See Interleukin-2 T cell growth factor 1. See Interleukin-2 T cell growth factor 2. See Interleukin-4 T cell growth factor III. See Interleukin-9 T cell help, 317 T cell hybridoma, 334, 863 T cell immunodeficiency syndromes (TCIS), 623 T cell large granular lymphocytic leukemia (T-LGL), immunophenotype, 610 T cell leukemia, 530 T cell leukemia viruses, 530 T cell lymphoma (TCL), 530 T cell maturation. See Thymus cell differentiation T cell nonantigen-specific helper factor, 321 T cell prolymphocytic leukemia (T-PLL), 609 immunophenotype, 610 T cell receptor (TCR), 328 T cell receptor complex, 329 T cell receptor genes, 328 T cell receptor α chain (TCRα), 328 T cell receptor β chain (TCRβ), 328 T cell replacing factor (TRF), 334–335 T cell rosette. See E rosette T cell specificity. See MHC restriction T cell system, 312–313, 805 T cell tolerance, 441 T cell vaccination (TCV), 491, 772 T cell-independent (TI) antigen, 317 T cells, 107, 333–334. See also T lymphocytes alpha-beta, 108 CD8, 206 dendritic epidermal, 108 development of, 313 DTH, 431 helper, 320 migration, 313 phosphotyrosine kinases in, 326–327 regulatory, 108 T cells, memory, 178

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936 T globulin, 812 T lymphocyte (T cell), 332–333 T lymphocyte clone, 335 T lymphocyte helper factor, nonspecific, 104 T lymphocyte hybridoma, 335 T lymphocyte receptor, 329 T lymphocyte subpopulation, 335 T lymphocyte-B lymphocyte cooperation, 211 T lymphocyte-conditioned medium, 345 T lymphocyte-T lymphocyte cooperation, 284 T lymphocytes, armed cytotoxic, 108 T piece. See Secretory piece T-200, 332 T-dependent antigen. See Thymus-dependent antigen T-independent antigen. See Thymus-independent antigen T1 antigen. See CD5 T1DM. See Type 1 diabetes mellitus T2DM. See Type 2 diabetes mellitus T3 antigen. See CD3 T4 antigen. See CD4 T4 count, 640 T8 antigen. See CD8 TAA. See Tumor-associated antigens TAB vaccine, 778 Tac, 337 Tac antigen. See CD25 TACI (transmembrane activator and CAML-interactor), 371 Tacrolimus, 654 Taenia solium immunity, 766–767 TAF. See Toxoid-antitoxin floccules Takatsy method, 831 Takayasu’s arteritis, 553 Take, 688 Tall peptide, 242 Talmage, David Wilson, 15–16 Tamiflu, 787 Tamm-Horsfall glycoprotein (uromodulin), 588–589 Tandem immunoelectrophoresis, 825 Tanned red cell test, 829 Tanned red cells, 829 TAP, 205 TAP 2 gene, 205 TAP1 gene, 205 TAPA-1, 220 Tapasin, 205 Tapioca adjuvant (historical), 180 Taq polymerase, 844 Target cell, 403 Tat, 642 Tat gene, 642 TATA, 706 TATA box, 157 Tau antibody, 890 TB, 741 Tc cells, 323 Tc lymphocyte. See Cytotoxic T lymphocytes TCGF (T cell growth factor). See Interleukin-2 TCR complex, 331 TCR tickling, 313 TCRαβ transgenic mouse (TCR tg), 341 TD antigen. See Thymus-dependent antigen TdT, 157, 315–316, 887 Tec kinase, 218, 337 Telencephalin, 242 Teleosts, 805 Template theory (historical), 18, 264 Tenascin, 88 Terasaki, Paul, 58 Terminal complement complex (TCC), 394 Terminal complement complex deficiency, 394

K10141_IDX.indd 936

Index Terminal complement components, 394 Terminal deoxynucleotidyl-transferase (TdT), 157, 216, 315–316 Terminal transferase. See DNA nucleotidylexotransferase Termination of tolerance, 439 Tertiary granule, 125 Tertiary immune response, 178 Tertiary immunization, 178 Tertiary lymphoid tissues, 146 Tertiary reactions, 285 Tertiary response, 178 Tertiary structure, 169 Test dosing, 855 Testicular autoimmunity, 466 Tetanus, 736 Tetanus antitoxin, 779, 785 Tetanus toxin, 736 Tetanus toxoid, 164, 780 Tetanus vaccine, 779 Tetraparental chimera, 439 Tetraparental mouse, 439 Texas red, 851 TFA antigens, 164, 809 TGF. See Transforming growth factor(s) TGF-β. See Transforming growth factor-βs TH cells, 320 TH0 cells, 321 TH1 cells, 321 TH1/TH2 differentiation, 321 TH17 cells, 321 TH17 hypothesis, 322 TH2 cells, 321 TH3 cells, 441 Thalidomide, 659 Theiler’s virus myelitis, 760 Theiler, Max, 61 Theileria immunity, 767 Theliolymphocytes, 104 Theophylline (1,3,dimethylxanthine), 423 Therapeutic antisera, 785 Therapeutic vaccination, 769 Thermoactinomyces species, 549 Thomas, E. Donnall, 59 Thoracic duct, 137 drainage, 138 Thorium dioxide, 123 Thorotrast (thorium dioxide 33THOT), 123 Three signal model of lymphocyte activation, 317–318 Threonyl-transfer RNA synthetase antibodies, 490 Threonyl-transfer RNA synthetase autoantibodies, 469–470 Thrombocyte, 132 Thrombocytopenia, 521 Thrombocytosis, 521 Thromboxanes, 417 Thy (θ), 326 Thy 1 antigen, 326 Thy-1, 326 Thy-1+ dendritic cells, 326, 503 Thymectomy, 340 Thymic alymphoplasia, 629 Thymic anlage, 307 Thymic epithelial cells (TECs), 310 Thymic hormones, 338 Thymic hormones and peptides, 338 Thymic humoral factor(s) (THFs), 338 Thymic hypoplasia (DiGeorge syndrome), 617, 623 Thymic involution, 339 Thymic leukemia antigen (TL), 312 Thymic medullary hyperplasia, 545 Thymic nurse cells, 310 Thymic stromal-derived lymphopoietin (TSLP), 326, 357

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937

Index Thymicleukemia antigen (TL), 702 Thymin, 339 Thymocytes, 310–311 Thymoma, 310 Thymopentin (TP-5), 339 Thymopoietin, 338–339 Thymosin α-1 (thymopoietin), 338 Thymosine, 338 Thymulin, 326 Thymus, 133–134, 307 Thymus cell differentiation, 316 Thymus cell education. See Thymus cell differentiation Thymus-dependent (TD) antigen, 173, 316–317 Thymus-dependent antigen, 317 Thymus-dependent areas, 141, 317 Thymus-independent (TI) antigen, 173, 317 Thymus-replacing factor (TRF). See Interleukin-5 Thyroglobulin, 451 autoantibodies, 451 Thyroid gland, 545 immunological diseases and immunopathology of, 545–547 Thyroid antibodies, 451 Thyroid autoantibodies, 472, 546 Thyroid autoimmunity animal models, 451 Thyroiditis, autoimmune, 45, 451 Thyrotoxicosis, 451, 547 Thyrotropin, 451 Thyrotropin receptor authoantibodies, 452 Thyroxine, 545 Tie-2, 146 Tight junction, 380 Tight skin-1 mouse (Tsk1), 580 Tight skin-2 mouse (Tsk2), 580 TIL. See Tumor-infiltrating lymphocytes Tingible body, 117 Tinglible body macrophages, 117 Tiselius, Arne W.K., 23–24 Tissue injury, immune complexes and, 42 Tissue transglutinase autoantibodies, 458 Tissue typing, 672 Tissue-fixed macrophage, 116 Tissue-plasminogen activator (TPA), 119 Tissue-specific antigen, 451 Titer, 237, 303 TL (thymic-leukemia antigen), 188 Tla antigen, 188 Tla complex, 188 TLR1-10. See Toll-like receptors TNF. See Tumor necrosis factor TNF receptor-associated factors (TRAFs), 371–372 TNF receptor-associated period syndrome (TRAPS), 624 TNF-α. See Tumor necrosis factor α TNF-β. See Tumor necrosis factor β TNF-related activation-induced cytokine (TRANCE), 372 TNFR1 pathway, 372 TNP. See Trinitrophenyl (picryl) group Togavirus immunity, 760 Tolerance, 437–438 Tolerization, 438 Tolerogen, 439 Tolerogenic, 438 Tolerosome, 439 Toll-like receptors, 127 Tolmetin, 796 Tonegawa, Susumu, 18–19 Tonsils, 146 Topoisomerase I, 486 TORCH panel, 760 Tositumomab, 791

K10141_IDX.indd 937

Total body irradiation (TBI), 687 Total lymphoid irradiation (TLI), 662 Totipotent, 99 Toxic complexes, 425–426 Toxic epidermal necrolysis, 695 Toxic shock syndrome, 736 Toxin neutralization (by antitoxin), 305 Toxin-1 (TSST-1), 736 Toxins, 163 Toxocara canis immunity, 767 Toxoid, 779–780 Toxoid-antitoxin floccules, immunizing preparation containing, 779 Toxoplasma gondii, 878 Toxoplasma gondii immunity, 767 Toxoplasmosis, 767 Tp44 (CD28), 207, 331 TPA (tissue-plasminogen activator), 119 TPHA. See Treponema pallidum hemagglutination assay TPI. See Treponema pallidum immobilization test TR1. See Regulatory T cells TR1 cells, 441 Trace labeling, 819 Traffic area. See Thymus-dependent areas TRAFs, 371–372 TRAIL (TNF-related apoptosis-inducing ligand), 372 TRALI (transfusion-related acute lung injury), 523–524 Transcobalamin II deficiency, 622 with hypogammaglobulinemia, 622 Transcription factors, 156, 865 Transcriptomics, 156 Transcytosis, 500 Transduction, 865 Transfection, 865 Transfectoma, 266 Transfer factor (TF), 376–377 Transferrin, 736 Transferrin receptor (T9), 337 Transformation, 175 Transforming growth factor(s), 377–378 Transforming growth factor-α (TGF-α), 377 Transforming growth factor-βs (TGF-βs), 377–378 Transfusion, 522–523 Transfusion effect, 669 Transfusion reactions, 523 Transfusion-associated graft-vs.-host disease (TAGVHD), 523 Transfusion-related acute lung injury. See TRALI Transgenes, 859 Transgenic(s), 860 Transgenic line, 860 Transgenic mice, 860 Transgenic organisms, 860 Transient hypogammaglobulinemia of infancy, 618 Transmissible spongiform encephalopathy (TSE) immunity, 568 Transplantation, 665 Transplantation antigens, 665 Transplantation immunology, 665 Transplantation rejection, 690 Transport piece. See Secretory piece Transporter associated with antigen processing (TAP), 204 Transudation, 146 Trastuzumab, 790 Treponema immunity, 736 Treponema pallidum hemagglutination assay, 829 Treponema pallidum immobilization test, 822 TRF, 357 Trichuris trichiura immunity, 767 Trinitrophenyl (picryl) group, 170 Triple response of Lewis, 408 Triple vaccine, 780 Triton X-100, 180

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938 Trophoblast, 132, 664 Tropical eosinophilia, 767 Trypan blue, 675 Trypan blue dye exclusion test, 675 Trypanosome adhesion test, 822 Trypanosome immunity, 767–768 Tryptic peptides, 284 Ts, 325 Ts cells, 441 Ts1 lymphocytes, 325 Ts3 lymphocytes, 325 TsF. See Suppressor T cell factor TTF-1 (8G7G3/a), mouse, 876 Tuberculid, 432 Tuberculin, 432 Tuberculin hypersensitivity, 432 Tuberculin reaction, 432 Tuberculin test, 432, 853 Tuberculin-type reaction, 432 Tuberculosis immunization, 432, 781 Tubular basement membrane, autoantibodies, 463 Tuftsin, 118 Tuftsin deficiency, 632 Tumor, 700 Tumor antigens, 702 Tumor enhancement, 710 Tumor hypoxia, 700 Tumor imaging, 702 Tumor immunity, 37–38 Tumor immunotherapy, 792 Tumor necrosis factor (TNF), 370 Tumor necrosis factor (TNF) family, 370 Tumor necrosis factor α (TNF-α), 370–371, 715–716 Tumor necrosis factor β (TNF-β), 372, 716 Tumor necrosis factor receptor, 372, 716 Tumor promoter. See Phorbol ester(s) Tumor regression, 700 Tumor rejection antigen, 706 Tumor-associated antigens, 702 Tumor-infiltrating lymphocytes (TIL), 714 Tumor-specific antigen (TSA), 706 Tumor-suppression genes, 701 TUNEL assay (TdT-dependent dUTP-biotin nick end labeling), 864 TUNEL-based assays, 864–865 Tunicates, 804 Turbidimetry, 822 Tween, 80, 180 Twelve/twenty-three (12/23) rule, 225 Two-dimensional gel electrophoresis, 824 Two-signal hypothesis, 175 Type 1 diabetes mellitus (T1DM), 465 Type 2 diabetes mellitus (T2DM), 559 Type I analphylactic hypersensitivity, 408 Type I cytokine receptors, 344–345 Type I interferons (IFN-α, IFN-β), 368 Type II antibody-mediated hypersensitivity, 423 Type II interferon. See Interferon γ Type III immune complex-mediated hypersensitivity, 425 Type IV cell-mediated hypersensitivity, 428 Typhoid vaccination, 778 Typhoid vaccine, 778 Typhus vaccination, 781 Typhus vaccine, 781 Tyrosinase, 894 Tyrosine kinase, 106–107

U U antigen, 516 µ chain, 250

K10141_IDX.indd 938

Index U1 snRNP autoantibodies, 490 U1-RNP, 477 U2-RNP, 477 U2snRNP autoantibodies, 470 Ubiquitin, 96 autoantibodies, 483 Ubiquitin antibody, 891 Ubiquitination, 96 UCHL1 antihuman T cell, 883 Ulcerative colitis, 554 Ultracentrifugation, 827 Ultrafiltration, 827 Umbrella effect, 587 Undifferentiated connective tissue disease, 492 Ungulate immunity, 811 Unidentified reading frame (URF), 158 Unitarian hypothesis, 286, 306 Univalent, 236 Univalent antibody, 236 Universal donor, 523 Universal recipient, 523 Unprimed, 218 Unproductive rearrangements, 226 Unresponsiveness, 437 Uromodulin Tamm-Horsfall protein, 664 Uropod, 107 Uroshiols, 435 Urticaria, 421 US28, 349 Usual interstitial pneumonitis, 547 Uveitis, 569

V V gene, 226, 229, 282 V gene segment, 226, 229 V region. See Variable (V) region V region groups, 284 V region subgroups, 283 V(D)J recombinase, 226 V(D)J recombination, 225 V(D)J recombination class switching, 225 V(J) recombination, 226 V-myb oncogene, 537, 702, 755 V28, 349 Vaccinable, 769 Vaccinate, 769 Vaccination, 769 Vaccine extraimmunization, 773 Vaccine standardization, 769 Vaccines against bacteria, 778–784 Vaccines against parasites, 784 Vaccines against viruses, 773–778 Vaccinia, 773 Vaccinia gangrenosa (historical), 774 Vaccinia immune globulin, 774 Vaccinia virus, 773 Vaginal mucous agglutination test, 831 Valence, 285 van der Waals forces (London forces), 288 Van Rood, J.J., 59 Variability plot. See Wu-Kabat plot Variable (V) domain, 283 Variable (V) exon, 282 Variable (V) gene segment, 282 Variable (V) region, 282–283 Variable lymphocyte receptors (VLRs), 803 Variable surface glycoproteins, 768 Varicella, 760 Varicella (chickenpox) vaccine, 777 Varicella-zoster virus immunity, 760–761

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939

Index Variola (smallpox), 761 Vascular addressins, 91 Vascular cell adhesion molecule-1 (VCAM-1), 86 Vascular permeability factors, 416 Vasculature, immunological diseases and immunopathology of, 540–543 Vasculitis, 425 Vasectomy, 466 Vasoactive amines, 411 Vasoconstriction, 411 Vasodilation, 411 Vaughan, Victor Clarence, 68 VDRL (Venereal Disease Research Laboratory), 736–737 Vector, 226 Venom, 164, 420 Venoocclusive disease (VOD), 698 Vermiform appendix, 146 Very late activation antigens. See VLA molecules Vesiculation, 434 Veto cells, 108 VH, 245 Vi antigen, 744 Vibrio cholerae immunity, 737 Videx, 647 Vif, 642 Vimentin, 880 Vinblastine, 796 Vincristine, 796 Vinyl chloride (VD), 573 Viracept, 647 Viral capsids, 752 Viral hemagglutination, 745 Viral immunity, 745 Viral interference, 745 Viramune, 647 Virgin B cells, 223 Virion, 745 Viroid, 745 Viropathic, 745 Virosome, 771 Virulence, 717 Virulence genes, 737 Virus, 744–745 Virus infection associated autoantibodies, 492 Virus neutralization test, 862 Virus-associated hemophagocytic syndrome, 745–746 Virus-neutralizing capacity, 746 Viscosity, 146–147 Vitamin A, immunity and, 154–155 Vitamin B, immunity and, 155 Vitamin C, immunity and, 155 Vitamin D, immunity and, 155 Vitamin E, immunity and, 155 Vitiligo, 474 Vitronectin, 88 V L region, 245 VLA molecules, 86 VLA receptors, 86 VLA-4, 498 VLIA (virus-like infectious agent), 641 Vogt-Koyanagi-Harada (VKH) syndrome, 568–569 Vollmer test (historical), 853 Voltage-gated-calcium channel autoantibodies, 468 Von Behring, Emil Adolph, 11 Von Krough equation, 403 Von Pirquet, Clemens Freiherr, 31 Von Wassermann, August, 63 Vosoactive intestinal peptide (VIP), 147 Vpr, 643 Vpre-B, 217 Vpu, 643

K10141_IDX.indd 939

VT region, 329 Vλ, 245

W W, 667 W,X,Y boxes (class II MHC promoter), 669 Waaler-Rose test. See Rose-Waaler test Waldenström’s macroglobulinemia, 589 Waldenström, Jan Gosta, 22 Waldeyer’s ring, 146 Warm antibody, 519 Warm-antibody autoimmune hemolytic anemia, 453 Wassermann reaction, 833 Wasting disease, 663 Wax D, 181 Wegener’s granulomatosis, 541–542 Weibel-Palade bodies, 90 Weil-Felix reaction, 831 Western blot (immunoblot), 842 Wheal and flare reaction, 149, 420 Whipple’s disease, 554 White graft rejection, 690 White pulp, 145 disease, 530 Whooping cough vaccine, 780 Widal reaction, 831 Window, 640–641 Winn assay, 715, 859 Wire loop lesion, 573 Wiskott-Aldrich syndrome, 629 Witebsky’s criteria, 448 Witebsky, Ernest, 39 Worms, 801 WT1, 894 Wu-Kabat plot, 255–256

X X cell. See XYZ cell theory X-linked (congenital) agammaglobulinemia, 617 X-linked agammaglobulinemia, 618–619 X-linked hyper-IgM syndrome. See Hyperimmunoglobulin M syndrome X-linked lymphoproliferative disease (XLP), 626–627 X-linked severe combined immunodeficiency (XSCID), 626 X-ray crystallography, 827 X4 viruses, 639 Xenoantibody, 682 Xenoantigen, 682 Xenobiotics, exposure of humans to, 491–492 Xenogeneic, 682 Xenograft, 682 Xenopus, 807 Xenoreactive, 682 Xenotransplantation, 682 Xenotype, 682–683 Xenozoonosis, 682 Xeroderma pigmentosum (XP), 624 Xga, 520 Xid gene, 174 XYZ cell theory (historical), 264

Y Y cell. See XYZ cell theory Yalow, Rosalyn Sussman, 65 Yellow fever vaccine, 775 Yersinia immunity, 737

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940 Z ζ (zeta) chain, 330 Z cell. See XYZ cell theory ZAP, 326–327 ZAP-70 deficiency, 625–626 Zeta potential, 513 Zeta-associated protein of 70 kDa (ZAP-70), 327 Ziagen, 646 Zidovudine (3'-azido-3'-deoxythymidine), 646 Zinc, 155 immunity and, 155–156

K10141_IDX.indd 940

Index Zinkernagel, Rolf, 59 Zinsser, Hans, 32–33 Zippering, 127 Zirconium granuloma, 429 Zonal centrifugation, 828 Zone electrophoresis, 827 Zone of equivalence, 295 Zoonosis, 682 Zygosity, 865 Zymogen, 96 Zymosan, 395

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