I II III Robbins and Cotran PATHOLOGIC BASIS OF DISEASE Seventh Edition VINAY KUMAR MBBS, MD, FRCPath Alice Hogge and Arthur Baer Professor Chairman, Department of Pathology The University of Chicago, Pritzker School of Medicine Chicago, Illinois ABUL K. ABBAS MBBS Chair, Department of Pathology University of California, San Francisco San Francisco, California NELSON FAUSTO MD Chairman, Department of Pathology University of Washington School of Medicine Seattle, Washington With Illustrations by James A. Perkins, MS, MFA ELSEVIER SAUNDERS IV ELSEVIER SAUNDERS An Imprint of Elsevier The Curtis Center 170 S Independence Mall W 300E Philadelphia, Pennsylvania 19106 ROBBINS AND COTRAN PATHOLOGIC BASIS OF DISEASE, 7/E 0-7216-0187-1 International Edition ISBN 0-8089-2302-1 Copyright © 2005, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Health Sciences Rights Department in Philadelphia, PA, USA: phone: (+1) 215 238 7869, fax: (+1) 215 238 2239, e-mail: healthpermissions@elsevier.com. You may also complete your request on-line via the Elsevier homepage (http://www.elsevier.com), by selecting 'Customer Support' and then 'Obtaining Permissions'. NOTICE Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication. Previous editions copyrighted 1999, 1994, 1989, 1984, 1979, 1974 Library of Congress Cataloging-in-Publication Data Robbins and Cotran pathologic basis of disease.—7th ed./[edited by] Vinay Kumar, Abul K. Abbas, Nelson Fausto ; with illustrations by James A. Perkins. p. ; cm. Rev. ed. of: Robbins pathologic basis of disease, 1999. ISBN 0-7216-0187-1 1. Pathology. [DNLM: 1. Pathology. QZ 4 R6354 2004] I. Title: Pathologic basis of disease. II. Kumar, Vinay. III. Abbas, Abul K. IV. Fausto, Nelson. V. Robbins, Stanley L. (Stanley Leonard). VI. Cotran, Ramzi S. Robbins pathologic basis of disease. RB111.R62 2004 616.07—dc22 2004046835 Publishing Director: William Schmitt Managing Editor: Rebecca Gruliow Design Manager: Ellen Zanolle Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1 V IN MEMORY OF Dr. Stanley L. Robbins (1915–2003) and Dr. Ramzi S. Cotran (1932–2000) Dear friends, respected colleagues, and dedicated teachers They leave a legacy of excellence that will enrich the lives of generations of future physicians. VI VII Contributors Charles E. Alpers MD Professor of Pathology, Adjunct Professor of Medicine, University of Washington School of Medicine; Pathologist, University of Washington Medical Center, Seattle, WA The Kidney Douglas C. Anthony MD, PhD Professor and Chair, Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO Peripheral Nerve and Skeletal Muscle; The Central Nervous System Jon C. Aster MD, PhD Associate Professor of Pathology, Harvard Medical School; Staff Pathologist, Brigham and Women's Hospital, Boston, MA Red Blood Cell and Bleeding Disorders; Diseases of White Blood Cells, Lymph Nodes, Spleen, and Thymus James M. Crawford MD, PhD Professor and Chair, Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine; Professor and Chair, Shands Hospital at the University of Florida, Gainesville, FL The Gastrointestinal Tract; Liver and Biliary Tract Christopher P. Crum MD Professor of Pathology, Harvard Medical School; Director, Women's and Perinatal Pathology, Brigham and Women's Hospital, Boston, MA The Female Genital Tract Umberto De Girolami MD Professor of Pathology, Harvard Medical School, Boston; Director of Neuropathology, Brigham and Women's Hospital, Boston, MA Peripheral Nerve and Skeletal Muscle; The Central Nervous System Jonathan I. Epstein MD Professor of Pathology, Urology, and Oncology; The Rinehard Professor of Urologic Pathology, The Johns Hopkins University School of Medicine, Baltimore; Director of Surgical Pathology, The Johns Hopkins Hospital, Baltimore, MD The Lower Urinary Tract and Male Genital System Robert Folberg MD Frances B. Greever Professor and Head, Department of Pathology, University of Illinois at Chicago, Chicago, IL The Eye Matthew P. Frosch MD, PhD Assistant Professor of Pathology, Harvard Medical School Boston; Assistant Pathologist, C.S. Kubik Laboratory for Neuropathology, Massachusetts General Hospital, Boston, MA Peripheral Nerve and Skeletal Muscle; The Central Nervous System Ralph H. Hruban MD Professor of Pathology and Oncology, The Johns Hopkins University School of Medicine; Attending Pathologist, The Johns Hopkins Hospital, Baltimore, MD The Pancreas Aliya N. Husain MBBS Professor, Department of Pathology, Pritzker School of Medicine, University of Chicago, Chicago, IL The Lung Agnes B. Kane MD, PhD Professor and Chair, Department of Pathology and Laboratory Medicine, Brown University Medical School, Providence, RI Environmental and Nutritional Pathology Susan C. Lester MD, PhD Assistant Professor of Pathology, Harvard Medical School; Chief, Breast Pathology, Brigham and Women's Hospital, Boston, MA The Breast VIII Mark W. Lingen DDS, PhD Associate Professor, Department of Pathology, University of Chicago, Chicago, IL Head and Neck Chen Liu MD, PhD Assistant Professor of Pathology, University of Florida College of Medicine, Gainesville, FL The Gastrointestinal Tract Anirban Maitra MBBS Assistant Professor, Department of Pathology, The Johns Hopkins University School of Medicine; Pathologist, The Johns Hopkins Hospital, Baltimore, MD Diseases of Infancy and Childhood; The Endocrine System Alexander J. McAdam MD, PhD Assistant Professor of Pathology, Harvard Medical School; Medical Director, Infectious Diseases Diagnostic Laboratory, Children's Hospital Boston, Boston, MA Infectious Diseases Martin C. Mihm Jr. MD Clinical Professor of Pathology, Harvard Medical School; Pathologist and Associate Dermatologist, Massachusetts General Hospital, Boston, MA The Skin Richard N. Mitchell MD Associate Professor, Department of Pathology, Harvard Medical School; Director, Human Pathology, Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School; Staff Pathologist, Brigham and Women's Hospital, Boston, MA Hemodynamic Disorders, Thromboembolic Disease, and Shock George F. Murphy MD Professor of Pathology, Harvard Medical School; Director of Dermatopathology, Brigham and Women's Hospital, Boston, MA The Skin Andrew E. Rosenberg MD Associate Professor of Pathology, Harvard Medical School; Associate Pathologist, James Homer Wright Laboratories, Department of Pathology, Massachusetts General Hospital, Boston, MA Bones, Joints, and Soft Tissue Tumors Frederick J. Schoen MD, PhD Professor of Pathology and Health Sciences and Technology, Harvard Medical School; Director, Cardiac Pathology and Executive Vice Chairman, Department of Pathology, Brigham and Women's Hospital, Boston, MA Blood Vessels; The Heart Klaus Sellheyer MD Assistant Professor of Pathology, Thomas Jefferson University; Attending Dermatopathologist, Jefferson Medical College, Philadelphia, PA The Skin Arlene H. Sharpe MD, PhD Professor of Pathology, Harvard Medical School; Chief, Immunology Research Division, Department of Pathology, Brigham and Women's Hospital, Boston, MA Infectious Diseases Robb E. Wilentz MD Voluntary Faculty, Department of Dermatology, University of Miami School of Medicine; Laboratory Director, Division of Pathology, Skin and Cancer Associates, Miami, FL The Pancreas IX Preface We launch the seventh edition of Pathologic Basis of Disease with mixed emotions, excitement and enthusiasm, as we enter the new millennium, tempered by sadness over the loss of our dear colleagues Drs. Stanley Robbins and Ramzi Cotran. To acknowledge their immeasurable and everlasting contribution to this text, the book is now renamed Robbins and Cotran Pathologic Basis of Disease. This edition, like all previous ones, has been extensively revised, and some areas completely rewritten. Some of the more significant changes are as follows: • Chapter 1 has been completely reorganized to include the entire spectrum of cellular responses to injury, from adaptations and sublethal injury to cell death. This was accomplished by combining the first two chapters of the sixth edition. We believe that this integrated and extensively revised chapter will allow a better understanding of cell injury, the most fundamental process in disease causation. • Chapter 3 , covering tissue repair and wound healing, has been extensively revised to include new and exciting information in stem cell biology and the emerging field of regenerative medicine. • Chapter 8 , dealing with infectious diseases, has been organized taxonomically with emphasis on mechanisms of tissue injury by different categories of infectious agents. While examples of infections by prototypic microorganisms have been retained, most of the organ-specific infectious diseases have been moved to later chapters where other diseases of the organ are described. • Discussion of diabetes mellitus has been moved from the chapter on pancreatic diseases to the chapter on endocrine disorders, where it blends more logically with other hormonal diseases. • Discussions of the lower urinary tract and the male genital system have been combined and grouped into a single chapter in recognition of the fact that there is overlap in diseases and diagnostic considerations. • A new feature best described as "boxes" has been introduced in selected chapters. For boxes, we have selected topics at the cutting edge of science that are worthy of a more detailed presentation than is essential for a student textbook. In doing so, we hope that we have presented the excitement of the topic without encumbering the body of the text with details that may appear overwhelming to the beginning reader. • The chapter on ocular diseases has been rewritten and reorganized to facilitate an understanding of ophthalmic pathology by the non-specialist. • In addition to the revision and reorganization of the text, there have been significant changes in illustrations. Many new photographs and schematics have been added and a large number of the older "gems" have been enhanced by digital technology. Thus we hope that even the veterans of the Robbins Pathology titles who have seen many previous editions of the book will find the color illustrations more sparkling and fresh. Approximately 50 new pages of illustrations have been added. In the 5 years since the previous edition, spectacular advances, including the completion of the human genome project, have occurred. Whenever appropriate we have blended the new discoveries into the discussion of pathogenesis and pathophysiology, yet never losing sight X that the "state of the art" has little value if it does not enhance the understanding of disease mechanisms. As in the past, we have not avoided discussions of "unsolved" problems because of our belief that many who read the text might be encouraged to embark on a path of discovery. Despite the changes outlined above and extensive revisions, our goals remain essentially the same. • To integrate into the discussion of pathologic processes and disorders the newest established information available—morphologic and molecular. • To organize the presentations into logical and uniform approaches, thereby facilitating readability, comprehension, and learning. • To maintain a reasonable size of the book, and yet to provide adequate discussion of the significant lesions, processes, and disorders, allotting space in proportion to their clinical and biologic importance. • To place great emphasis on clarity of writing and good usage of language in the recognition that struggling to comprehend is time-consuming and wearisome and gets in the way of the learning process. • To make this first and foremost a student text—used by students throughout their 4 years of medical school and into their residencies—but, at the same time, to provide sufficient detail and depth to meet the needs of more advanced readers. We have been repeatedly told by the readers that one of the features they value most in this book is its up-to-dateness. We have strived to maintain such timeliness by providing references from recent literature, many published in 2003 and some from the early part of 2004. However, older classics have also been retained to provide original source material for advanced readers. With this edition, we also move into the digital age: the text will be available online to those who own the print version. This online access gives the reader the ability to search across the entire text, bookmark passages, add personal notes, use PubMed to view references, and many other exciting features, including timely updates. In addition, included in the text is a CD-ROM of case studies, previously available separately as the Interactive Case Study Companion developed by one of us (VK) in collaboration with Herb Hagler, PhD, and Nancy Schneider, MD, PhD, at the University of Texas, Southwestern Medical School in Dallas. This will enhance and reinforce learning by challenging students to apply their knowledge in solving clinical cases. A virtual microscope feature enables the viewing of selected images at various powers. This edition is also marked by the addition of two new "seasoned" coauthors. All three of us have reviewed, critiqued, and edited each chapter to ensure uniformity of style and flow that have been the hallmarks of the text. Together, we hope that we have succeeded in bringing to the reader the excitement of the study of disease mechanisms and the desire to learn more than what can be offered in any textbook. VK AKA NF XI Acknowledgments The authors are grateful to a large number of individuals who have contributed in many ways toward the completion of this textbook. First and foremost, all three of us offer our tributes and gratitude to two stalwarts of American pathology, Dr. Stanley Robbins and Dr. Ramzi Cotran. Their passion for excellence and uncompromising standards have made this book what it is. While neither of the two will see this edition in its completed form, their stamp on Pathologic Basis of Disease is indelible. Second, we thank our contributing authors for their commitment to this textbook. Many are veterans of previous editions; others are new to the seventh edition. All are acknowledged in the Table of Contents. Their names lend authority to this book, for which we are grateful. Many colleagues have enhanced the text by reading various chapters and providing helpful critiques in their area of expertise. They include (at the University of Chicago): Drs. Todd Kroll, Michelle LeBeau, Olaf Schneewind, Josephine Morello, Megan McNerney, Fred Wondisford, Aliya Husain, Jonathan Miller, Julian Solway, John Hart, Amy Noffsinger, Thomas Krausz, Raminder Kumar, Joanne Yocum, Christopher Weber, Elizabeth McNally, and Manny Utset; (at the University of California at San Francisco): Drs. Steve Gitelman, Jonathan Lin, David Wofsy, Patrick Treseler, Mark Anderson, and Aaron Tward; (at the University of Washington, Seattle): Drs. Zsolt Argenyi, Peter Beyers, Ann DeLancey, Charles Murry, Thomas Norwood, Brian Rubin, Paul Swanson, Melissa Upton, and Mathew Yeh. Dr. David Walker, at the University of Texas Medical Branch at Galveston provided a thorough critique of the chapter on infectious disease. Dr. Lora Hendrick Ellenson at Cornell University (Weill Medical College) provided a critique of the chapter on the female genital tract. Dr. Arlene Herzberg and Kelly McGuigan provided help with the chapter on skin diseases. Special thanks are owed to Dr. Henry Sanchez at the University of California at San Francisco for his painstaking review and revision of the older color illustrations and for his magic touch in enhancing them digitally. Their freshness will be obvious to the readers. Many colleagues provided photographic gems from their collection. They are individually acknowledged in the text. Our administrative staff needs to be acknowledged since they maintain order in the chaotic lives of the authors and have willingly chipped in when needed for multiple tasks relating to the text. At the University of Chicago, they include Ms. Vera Davis and Ms. Ruthie Cornelius; at The University of California at San Francisco, Ms. Ana Narvaez; and at the University of Washington, Seattle, Ms. Catherine Alexander, Steven Berard, Carlton Kim, Ms. Genevieve Thomas, and Ms. Vicki Tolbert. Ms. Beverly Shackelford at the University of Texas at Dallas, who has helped one of us (VK) for 21 years, deserves special mention since she coordinated the submission of all manuscripts, proofread many of them, and maintained liaison with the contributors and publisher. Without her dedication to this book and her meticulous attention to detail, our task would have been much more difficult. Most of the graphic art in this book was created by Mr. James Perkins, Assistant Professor of Medical Illustration at Rochester Institute of Technology. His ability to convert complex ideas into simple and aesthetically pleasing sketches has considerably enhanced this book. Many individuals associated with our publisher, Elsevier (under the imprint of W.B. Saunders), need our special thanks. Outstanding among them is Ellen Sklar, Production Editor, supervising the production of this book. Her understanding of the needs of the authors and the complexity of publishing a textbook went a long way in making our lives less complicated. XII Mr. William Schmitt, Publishing Director of Medical Textbooks, has always been our cheerleader and is now a dear friend. Our thanks also go to Managing Editor Rebecca Gruliow and Design Manager Ellen Zanolle at Elsevier. Undoubtedly there are many other "heroes" who may have been left out unwittingly—to them we say "thank you" and tender apologies for not acknowledging you individually. Efforts of this magnitude take a heavy toll on the families of the authors. We thank our spouses Raminder Kumar, Ann Abbas, and Ann DeLancey for their patience, love, and support of this venture, and for their tolerance of our absences. Finally, Vinay Kumar wishes to express his deep appreciation to Drs. Abul Abbas and Nelson Fausto for joining the team, and together we salute each other for shared vision and dedication to medical education. Despite differences in our vantage points, opinions, and individual styles, our common goal made this an exciting and rewarding partnership. VK AA NF 1 Section I - General Pathology 2 3 Chapter 1 - Cellular Adaptations, Cell Injury, and Cell Death 4 Introduction to Pathology Pathology is literally the study (logos) of suffering (pathos). More specifically, it is abridging discipline involving both basic science and clinical practice and is devoted to the study of the structural and functional changes in cells, tissues, and organs that underlie disease. By the use of molecular, microbiologic, immunologic, and morphologic techniques, pathology attempts to explain the whys and wherefores of the signs and symptoms manifested by patients while providing a sound foundation for rational clinical care and therapy. Traditionally, the study of pathology is divided into general pathology and special, or systemic, pathology. The former is concerned with the basic reactions of cells and tissues to abnormal stimuli that underlie all diseases. The latter examines the specific responses of specialized organs and tissues to more or less well-defined stimuli. In this book, we first cover the principles of general pathology and then proceed to specific disease processes as they affect particular organs or systems. The four aspects of a disease process that form the core of pathology are its cause (etiology), the mechanisms of its development (pathogenesis), the structural alterations induced in the cells and organs of the body (morphologic changes), and the functional consequences of the morphologic changes (clinical significance). Etiology or Cause. The concept that certain abnormal symptoms or diseases are "caused" is as ancient as recorded history. For the Arcadians (2500 BC), if someone became ill, it was the patient's own fault (for 1 having sinned) or the makings of outside agents, such as bad smells, cold, evil spirits, or gods.[ ] In modern terms, there are two major classes of etiologic factors: intrinsic or genetic, and acquired (e.g., infectious, nutritional, chemical, physical). The concept, however, of one etiologic agent for one disease — developed from the study of infections or single-gene disorders — is no longer sufficient. Genetic factors are clearly involved in some of the common environmentally induced maladies, such as atherosclerosis and cancer, and the environment may also have profound influences on certain genetic diseases. Knowledge or discovery of the primary cause remains the backbone on which a diagnosis can be made, a disease understood, or a treatment developed. Pathogenesis. Pathogenesis refers to the sequence of events in the response of cells or tissues to the etiologic agent, from the initial stimulus to the ultimate expression of the disease. The study of pathogenesis remains one of the main domains of pathology. Even when the initial infectious or molecular cause is known, it is many steps removed from the expression of the disease. For example, to understand cystic fibrosis is to know not only the defective gene and gene product, but also the biochemical, immunologic, and morphologic events leading to the formation of cysts and fibrosis in the lung, pancreas, and other organs. Indeed, as we shall see throughout the book, the molecular revolution has already identified mutant genes underlying a great number of diseases, and the entire human genome has been mapped. Nevertheless, the functions of the encoded proteins and how mutations induce disease are often still obscure. Because of technologic advances, it is becoming increasingly feasible to link specific molecular abnormalities to disease manifestations and to use this knowledge to design new therapeutic approaches. For these reasons, the study of pathogenesis has never been more exciting scientifically or more relevant to medicine. Morphologic Changes. The morphologic changes refer to the structural alterations in cells or tissues that are either characteristic of the disease or diagnostic of the etiologic process. The practice of diagnostic pathology is devoted to identifying the nature and progression of disease by studying morphologic changes in tissues and chemical alterations in patients. More recently, the limitations of morphology for diagnosing diseases have become increasingly evident, and the field of diagnostic pathology has expanded to encompass molecular biologic and immunologic approaches for analyzing disease states. Nowhere is this more striking than in the study of tumors — breast cancers and tumors of lymphocytes that look morphologically identical may have widely different courses, therapeutic responses, and prognosis. Molecular analysis by techniques such as DNA microarrays has begun to reveal genetic differences that bear on the behavior of the tumors. Increasingly, such techniques are being used to extend and even supplant traditional morphologic methods. Functional Derangements and Clinical Manifestations. The nature of the morphologic changes and their distribution in different organs or tissues influence normal function and determine the clinical features (symptoms and signs), course, and prognosis of the disease. Virtually all forms of organ injury start with molecular or structural alterations in cells, a concept first put forth in the nineteenth century by Rudolf Virchow, known as the father of modern pathology. We therefore begin our consideration of pathology with the study of the origins, molecular mechanisms, and structural changes of cell injury. Yet different cells in tissues constantly interact with each other, and an elaborate system of extracellular matrix is necessary for the integrity of organs. Cell-cell and cell-matrix interactions contribute significantly to the response to injury, leading collectively to tissue and organ injury, which are as important as cell injury in defining the morphologic and clinical patterns of disease. Overview: Cellular Responses to Stress and Noxious Stimuli The normal cell is confined to a fairly narrow range of function and structure by its genetic programs of metabolism, differentiation, and specialization; by constraints of neighboring cells; and by the availability of metabolic substrates. It is nevertheless able to handle normal physiologic demands, maintaining a steady state called homeostasis. More severe physiologic stresses and some pathologic stimuli may bring about a number of physiologic and morphologic cellular adaptations, during which new but altered steady states are achieved, preserving the viability of the cell and modulating its function as it responds to such stimuli ( Fig. 1-1 and Table 1-1 ). The adaptive response may consist of an increase in the number of cells, called hyperplasia, or an increase in the sizes of individual cells, called hypertrophy. Conversely, atrophy is an adaptive response in which there is a decrease in the size and function of cells. 5 Figure 1-1 Stages in the cellular response to stress and injurious stimuli. TABLE 1-1 -- Cellular Responses to Injury Nature and Severity of Injurious Stimulus Cellular Response Altered physiologic stimuli: Cellular adaptations: • Increased demand, increased trophic stimulation (e.g. growth factors, hormones) • Hyperplasia, hypertrophy • Decreased nutrients, stimulation • Atrophy • Chronic irritation (chemical or physical) • Metaplasia Reduced oxygen supply; chemical injury; microbial infection Cell injury: • Acute and self-limited • Acute reversible injury • Progessive and severe (including DNA damage) • Irreversible injury → cell death •••••Necrosis •••••Apoptosis • Mild chronic injury • Subcellular alterations in various organelles Metabolic alterations, genetic or acquired Intracellular accumulations; calcifications Prolonged life span with cumulative sublethal injury Cellular aging hormone. It also occurs in certain pathologic conditions, when cells are damaged beyond repair, and especially if the damage affects the cell's nuclear DNA. We will return to a detailed discussion of these pathways of cell death later in the chapter. Stresses of different types may induce changes in cells and tissues other than adaptations, cell injury, and death (see Table 1-1 ). Cells that are exposed to sublethal or chronic stimuli may not be damaged but may show a variety of subcellular alterations. Metabolic derangements in cells may be associated with intracellular accumulations of a number of substances, including proteins, lipids, and carbohydrates. Calcium is often deposited at sites of cell death, resulting in pathologic calcification. Finally, cell aging is also accompanied by characteristic morphologic and functional changes. In this chapter, we discuss first how cells adapt to stresses, and then the causes, mechanisms, and consequences of the various forms of acute cell damage, including cell injury and cell death. We conclude with subcellular alterations induced by sublethal stimuli, intracellular accumulations, pathologic calcification, and cell aging. Cellular Adaptations of Growth and Differentiation Cells respond to increased demand and external stimulation by hyperplasia or hypertrophy, and they respond to reduced supply of nutrients and growth factors by atrophy. In some situations, cells change from one type to another, a process called metaplasia. There are numerous molecular mechanisms for cellular adaptations. Some adaptations are induced by direct stimulation of cells by factors produced by the responding cells themselves or by other cells in the environment. Others are due to activation of various cell surface receptors and downstream signaling pathways. Adaptations may be associated with the induction of new protein synthesis by the target cells, as in the response of muscle cells to increased physical demand, and the induction of cellular proliferation, as in responses of the endometrium to estrogens. Adaptations can also involve a switch by cells from producing one type of proteins to another or markedly overproducing one protein; such is the case in cells producing various types of collagens and extracellular matrix proteins in chronic inflammation and fibrosis ( Chapter 2 and Chapter 3 ). 6 Figure 1-2 The relationships between normal, adapted, reversibly injured, and dead myocardial cells. The cellular adaptation depicted here is hypertrophy, and the type of cell death is ischemic necrosis. In reversibly injured myocardium, generally effects are only functional, without any readily apparent gross or even microscopic changes. In the example of myocardial hypertrophy, the left ventricular wall is more than 2 cm in thickness (normal is 1 to 1.5 cm). In the specimen showing necrosis, the transmural light area in the posterolateral left ventricle represents an acute myocardial infarction. All three transverse sections have been stained with triphenyltetrazolium chloride, an enzyme substrate that colors viable myocardium magenta. Failure to stain is due to enzyme leakage after cell death. Figure 1-3 Physiologic hypertrophy of the uterus during pregnancy. A, Gross appearance of a normal uterus (right) and a gravid uterus (removed for postpartum bleeding) (left). B, Small spindle-shaped uterine smooth muscle cells from a normal uterus (left) compared with large plump cells in gravid uterus (right). Figure 1-4 Changes in the expression of selected genes and proteins during myocardial hypertrophy. Figure 1-5 A, Atrophy of the brain in an 82-year-old male with atherosclerotic disease. Atrophy of the brain is due to aging and reduced blood supply. The meninges have been stripped. B, Normal brain of a 36-year-old male. Note that loss of brain substance narrows the gyri and widens the sulci. Figure 1-6 Metaplasia. A, Schematic diagram of columnar to squamous metaplasia. B, Metaplastic transformation of esophageal stratified squamous epithelium (left) to mature columnar epithelium (so-called Barrett metaplasia). Irreversible injury and cell death. With continuing damage, the injury becomes irreversible, at which time the cell cannot recover. Is there a critical biochemical event (the "lethal hit") responsible for the point of no return? There are no clear answers to this question. However, as discussed later, in ischemic tissues such as the myocardium, certain structural changes (e.g., amorphous densities in mitochondria, indicative of severe mitochondrial damage) and functional changes (e.g., loss of membrane permeability) are indicative of cells that have suffered irreversible injury. • Irreversibly injured cells invariably undergo morphologic changes that are recognized as cell death. There are two types of cell death, necrosis and apoptosis, which differ in their morphology, mechanisms, and roles in disease and physiology ( Fig. 1-9 and Table 1-2 ). When damage to membranes is severe, lysosomal enzymes enter the cytoplasm and digest the cell, and cellular contents leak out, resulting in necrosis. Some noxious stimuli, especially those that damage DNA, induce another type of death, apoptosis, which is characterized by nuclear dissolution without complete loss of membrane integrity. Whereas necrosis is always a pathologic process, apoptosis serves many normal functions and is not necessarily associated with cell injury. Although we emphasize the distinctions between necrosis and apoptosis, there may be some overlaps and common mechanisms between these two pathways. In addition, at least some types of stimuli may induce either apoptosis or necrosis, depending on the intensity and duration of the stimulus, the rapidity of the death process, and the biochemical derangements induced in the injured cell. The mechanisms and significance of these two death pathways are discussed later in the chapter. Figure 1-7 Stages in the evolution of cell injury and death. Figure 1-8 Schematic representation of a normal cell and the changes in reversible and irreversible cell injury. Depicted are morphologic changes, which are described in the following pages and shown in electron micrographs in Figure 1-17 . Reversible injury is characterized by generalized swelling of the cell and its organelles; blebbing of the plasma membrane; detachment of ribosomes from the endoplasmic reticulum; and clumping of nuclear chromatin. Transition to irreversible injury is characterized by increasing swelling of the cell; swelling and disruption of lysosomes; presence of large amorphous densities in swollen mitochondria; disruption of cellular membranes; and profound nuclear changes. The latter include nuclear codensation (pyknosis), followed by fragmentation (karyorrhexis) and dissolution of the nucleus (karyolysis). Laminated structures (myelin figures) derived from damaged membranes of organelles and the plasma membrane first appear during the reversible stage and become more pronounced in irreversibly damaged cells. The mechanisms underlying these changes are discussed in the text that follows. Figure 1-9 The sequential ultrastructural changes seen in necrosis (left) and apoptosis (right). In apoptosis, the initial changes consist of nuclear chromatin condensation and fragmentation, followed by cytoplasmic budding and phagocytosis of the extruded apoptotic bodies. Signs of cytoplasmic blebs, and digestion and leakage of cellular components. (Adapted from Walker NI, et al: Patterns of cell death. Methods Archiv Exp Pathol 13:18–32, 1988. Reproduced with permission of S. Karger AG, Basel.) TABLE 1-2 -- Features of Necrosis and Apoptosis Feature Necrosis Apoptosis Cell size Enlarged (swelling) Reduced (shrinkage) Nucelus Pyknosis → karyorrhexis → karyolysis Fragmentation into nucleosome size fragments Plasma membrane Disrupted Intact; altered structure, especially orientation of lipids Cellular contents Enzymatic digestion; may leak out of cell Intact; may be released in apoptotic bodies Adjacent inflammation Frequent No Physiologic or pathologic role Invariably pathologic (culmination of irreversible cell injury) Often physiologic, means of eliminating unwanted cells; may be pathologic after some forms of cell injury, especially DNA damage however, are our daily companions: environmental and air pollutants, insecticides, and herbicides; industrial and occupational hazards, such as carbon monoxide and asbestos; social stimuli, such as alcohol and narcotic drugs; and the ever-increasing variety of therapeutic drugs. Infectious Agents. These agents range from the submicroscopic viruses to the large tapeworms. In between are the rickettsiae, bacteria, fungi, and higher forms of parasites. The ways by which this heterogeneous group of biologic agents cause injury are diverse and are discussed in Chapter 8 . Immunologic Reactions. Although the immune system serves an essential function in defense against infectious pathogens, immune reactions may, in fact, cause cell injury. The anaphylactic reaction to a foreign protein or a drug is a prime example, and reactions to endogenous self-antigens are responsible for a number of autoimmune diseases ( Chapter 6 ). Genetic Derangements. Genetic defects as causes of cell injury are of major interest to scientists and physicians today ( Chapter 5 ). The genetic injury may result in a defect as severe as the congenital malformations associated with Down syndrome, caused by a chromosomal abnormality, or as subtle as the decreased life of red blood cells caused by a single amino acid substitution in hemoglobin S in sickle cell anemia. The many inborn errors of metabolism arising from enzymatic abnormalities, usually an enzyme lack, are excellent examples of cell damage due to subtle alterations at the level of DNA. Variations in genetic makeup can also influence the susceptibility of cells to injury by chemicals and other environmental insults. Nutritional Imbalances. Nutritional imbalances continue to be major causes of cell injury. Protein-calorie deficiencies cause an appalling number of deaths, chiefly among underprivileged populations. Deficiencies of specific vitamins are found throughout the world ( Chapter 9 ). Nutritional problems can be self-imposed, as in anorexia nervosa or self-induced starvation. Ironically, nutritional excesses have also become important causes of cell injury. Excesses of lipids predispose to atherosclerosis, and obesity is a manifestation of the overloading of some cells in the body with fats. Atherosclerosis is virtually endemic in the United States, and obesity is rampant. In addition to the problems of undernutrition and overnutrition, the composition of the diet makes a significant 14 Figure 1-10 Cellular and biochemical sites of damage in cell injury. Figure 1-11 Functional and morphologic consequences of decreased intracellular ATP during cell injury. Figure 1-12 Mitochondrial dysfunction in cell injury. Figure 1-13 Sources and consequences of increased cytosolic calcium in cell injury. ATP, adenosine triphosphate. Figure 1-14 The role of reactive oxygen species in cell injury. O2 is converted to superoxide (O2 - ) by oxidative enzymes in the endoplasmic reticulum (ER), mitochondria, plasma membrane, peroxisomes, and cytosol. O2 - is converted to H2 O2 by dismutation and thence to OH by the Cu2+ /Fe2+ -catalyzed Fenton reaction. H2 O2 is also derived directly from oxidases in peroxisomes. Not shown is another potentially injurious radical, singlet oxygen. Resultant free radical damage to lipid (peroxidation), proteins, and DNA leads to various forms of cell injury. Note that superoxide catalyzes the reduction of Fe3+ to Fe2+ , thus enhancing OH generation by the Fenton reaction. The major antioxidant enzymes are superoxide dismutase (SOD), catalase, and glutathione peroxidase. GSH, reduced glutathione; GSSG, oxidized glutathione; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate. Figure 1-15 Mechanisms of membrane damage in cell injury. Decreased O2 and increased cytosolic Ca2+ are typically seen in ischemia but may accompany other forms of cell injury. Reactive oxygen species, which are often produced on reperfusion of ischemic tissues, also cause membrane damage (not shown). Figure 1-16 Timing of biochemical and morphologic changes in cell injury. Figure 1-17 Morphologic changes in reversible and irreversible cell injury. A, Electron micrograph of a normal epithelial cell of the proximal kidney tubule. Note abundant microvilli (mv), lining the lumen (L). N, nucleus; V, apical vacuoles (which are normal structures in this cell type). B, Epithelial cell of the proximal tubule showing reversible ischemic changes. The microvilli (mv) are lost and have been incorporated in apical cytoplasm; blebs have formed and are extruded in the lumen (L). Mitochondria are slightly dilated. (Compare with A.) C, Proximal tubular cell showing irreversible ischemic injury. Note the markedly swollen mitochondria containing amorphous densities, disrupted cell membranes, and dense pyknotic nucleus. (Courtesy of Dr. M. A. Venkatachalam, University of Texas, San Antonio, TX.) Figure 1-18 Ischemic necrosis of the myocardium. A, Normal myocardium. B, Myocardium with coagulation necrosis (upper two thirds of figure), showing strongly eosinophilic anucleate myocardial fibers. Leukocytes in the interstitium are an early reaction to necrotic muscle. Compare with A and with normal fibers in the lower part of the figure. Figure 1-19 Coagulative and liquefactive necrosis. A, Kidney infarct exhibiting coagulative necrosis, with loss of nuclei and clumping of cytoplasm but with preservation of basic outlines of glomerular and tubular architecture. B, A focus of liquefactive necrosis in the kidney caused by fungal infection. The focus is filled with white cells and cellular debris, creating a renal abscess that obliterates the normal architecture. Figure 1-20 A tuberculous lung with a large area of caseous necrosis. The caseous debris is yellow-white and cheesy. Figure 1-21 Foci of fat necrosis with saponification in the mesentery. The areas of white chalky deposits represent calcium soap formation at sites of lipid breakdown. Figure 1-22 Postulated sequence of events in reversible and irreversible ischemic cell injury. Note that although reduced oxidative phosphorylation and ATP levels have a central role, ischemia can cause direct membrane damage. ER, endoplasmic reticulum; CK, creatine kinase; LDH, lactate dehydrogenase; RNP, ribonucleoprotein. Figure 1-23 Sequence of events leading to fatty change and cell necrosis in carbon tetrachloride (CCl4 ) toxicity. RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. Figure 1-24 Rat liver cell 4 hours after carbon tetrachloride intoxication, with swelling of endoplasmic reticulum and shedding of ribosomes. At this stage, mitochondria are unaltered. (Courtesy of Dr. O. Iseri, University of Maryland, Baltimore, MD.) Figure 1-25 Ultrastructural features of apoptosis. Some nuclear fragments show peripheral crescents of compacted chromatin, whereas others are uniformly dense. (From Kerr JFR, Harmon BV: Definition and incidence of apoptosis: a historical perspective. In Tomei LD, Cope FO (eds): Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1991, pp 5–29.) Figure 1-26 A, Apoptosis of epidermal cells in an immune-mediated reaction. The apoptotic cells are visible in the epidermis with intensely eosinophilic cytoplasm and small, dense nuclei. H&E stain. (Courtesy of Dr. Scott Granter, Brigham and Women's Hospital, Boston, AM.) B, High power of apoptotic cell in liver in immune-mediated hepatic cell injury. (Courtesy of Dr. Dhanpat Jain, Yale University, New Haven, CT.) Figure 1-27 Agarose gel electrophoresis of DNA extracted from culture cells. Ethidium bromide stain; photographed under ultraviolet illumination. Lane A, Control culture. Lane B, Culture of cells exposed to heat showing extensive apoptosis; note ladder pattern of DNA fragments, which represent multiples of oligonucleosomes. Lane C, Culture showing massive necrosis; note diffuse smearing of DNA. The ladder pattern is produced by enzymatic cleavage of nuclear DNA into nucleosome-sized fragments, usually multiples of 180–200 base pairs. These patterns are characteristic of but not specific for apoptosis and necrosis, respectively. (From Kerr JFR, Harmon BV: Definition and incidence of apoptosis: a historical perspective. In Tomei LD, Cope FO [eds]: Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1991, p 13.) Figure 1-28 Mechanisms of apoptosis. Labeled (1) are some of the major inducers of apoptosis. These include specific death ligands (tumor necrosis factor [TNF] and Fas ligand), withdrawal of growth factors or hormones, and injurious agents (e.g., radiation). Some stimuli (such as cytotoxic cells) directly activate execution caspases (right). Others act by way of adapter proteins and initiator caspases, or by mitochondrial events involving cytochrome c. (2) Control and regulation are influenced by members of the Bcl-2 family of proteins, which can either inhibit or promote the cell's death. (3) Executioner caspases activate latent cytoplasmic endonucleases and proteases that degrade nuclear and cytoskeletal proteins. This results in a cascade of intracellular degradation, including fragmentation of nuclear chromatin and breakdown of the cytoskeleton. (4) The end result is formation of apoptotic bodies containing intracellular organelles and other cytosolic components; these bodies also express new ligands for binding and uptake by phagocytic cells. Figure 1-29 The extrinsic (death receptor-initiated) pathway of apoptosis, illustrated by the events following Fas engagement (see text). Figure 1-30 The intrinsic (mitochondrial) pathway of apoptosis. Death agonists cause changes in the inner mitochondrial membrane, resulting in the mitochondrial permeability transition (MPT) and release of cytochrome c and other pro-apoptotic proteins into the cytosol, which activate caspases (see text). Figure 1-31 A, schematic representation of heterophagy (left) and autophagy (right). (Redrawn from Fawcett DW: A Textbook of Histology, 11th ed. Philadelphia, WB Saunders, 1986, p 17.) B, Electron micrograph of an autophagolysosome containing a degenerating mitochondrion and amorphous material. Figure 1-32 Electron micrograph of liver from phenobarbital-treated rat showing marked increase in smooth endoplasmic reticulum. (From Jones AL, Fawcett DW: Hypertrophy of the agranular endoplasmic reticulum in hamster liver induced by Phenobarbital. J Histochem Cytochem 14:215, 1966. Courtesy of Dr. Fawcett.) Figure 1-33 Enlarged, abnormally shaped mitochondria from the liver of a patient with alcoholic cirrhosis. Note also crystalline formations in the mitochondria. Figure 1-34 A, The liver of alcohol abuse (chronic alcoholism). Hyaline inclusions in the hepatic parenchymal cell in the center appear as eosinophilic networks disposed about the nuclei (arrow). B, Electron micrograph of alcoholic hyalin. The material is composed of intermediate (prekeratin) filaments and an amorphous matrix. Figure 1-35 Mechanisms of intracellular accumulations: (1) abnormal metabolism, as in fatty change in the liver; (2) mutations causing alterations in protein folding and transport, as in alpha1 antitrypsin deficiency; (3) deficiency of critical enzymes that prevent breakdown of substrates that accumulate in lysosomes, as in lysosomal storage diseases; and (4) inability to degrade phagocytosed particles, as in hemosiderosis and carbon pigment accumulation. Figure 1-36 Fatty liver. A, Schematic diagram of the possible mechanisms leading to accumulation of triglycerides in fatty liver. Defects in any of the steps of uptake, catabolism, or secretion can result in lipid accumulation. B, High-power detail of fatty change of the liver. In most cells, the well-preserved nucleus is squeezed into the displaced rim of cytoplasm about the fat vacuole. (B, Courtesy of Dr. James Crawford, Department of Pathology, Yale University School of Medicine, New Haven, CT.) Figure 1-37 Cholesterolosis. Cholesterol-laden macrophages (foam cells) from a focus of gallbladder cholesterolosis (arrow). (Courtesy of Dr. Matthew Yeh, University of Washington, Seattle, WA.) Figure 1-38 Protein reabsorption droplets in the renal tubular epithelium. (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 1-39 Mechanisms of protein folding and the role of chaperones. A, Chaperones, such as heat shock proteins (Hsp), protect unfolded or partially folded protein from degradation and guide proteins into organelles. B, Chaperones repair misfolded proteins; when this process is ineffective, proteins are targeted for degradation in the proteasome, and if misfolded proteins accumulate they trigger apoptosis. Figure 1-40 Lipofuscin granules in a cardiac myocyte as shown by A, light microscopy (deposits indicated by arrows), and B, electron microscopy (note the perinuclear, intralysosomal location). Figure 1-41 Hemosiderin granules in liver cells. A, H&E section showing golden-brown, finely granular pigment. B, Prussian blue reaction, specific for iron. Figure 1-42 View looking down onto the unopened aortic valve in a heart with calcific aortic stenosis. The semilunar cusps are thickened and fibrotic. Behind each cusp are seen irregular masses of piled-up dystrophic calcification. Figure 1-43 Mechanisms of cellular aging. Genetic factors and environmental insults combine to produce the cellular abnormalities characteristic of aging. Figure 1-44 Finite population doublings of primary human fibroblasts derived from a newborn, a 100-year-old person, and a 20-year-old patient with Werner's syndrome. The ability of cells to grow to a confluent monolayer decreases with increasing population-doubling levels. (From Dice JF: Cellular and molecular mechanisms of aging. Physiol Rev 73:150, 1993.) Figure 1-45 The role of telomeres and telomerase in replicative senescence of cells. A, Telomerase directs RNA template-dependent DNA synthesis, in which nucleotides are added to one strand at the end of a chromosome. The lagging strand is presumably filled in by DNA polymerase α. The RNA sequence in the telomerase is different in different species. (Modified from Alberts BR, et al: Molecular Biology of the Cell, 2002, Garland Science, New York.) B, Telomere-telomerase hypothesis and proliferative capacity. Telomere length is plotted against the number of cell divisions. 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Inflammation agents such as microbes and damaged, usually necrotic, cells that consists of vascular responses, migration and activation of leukocytes, and systemic reactions. Invertebrates with no vascular system, and even single-celled organisms, are able to get rid of injurious agents such as microbes by a variety of mechanisms. These mechanisms include entrapment and phagocytosis of the offending agent, sometimes by specialized cells (hemocytes), and neutralization of noxious stimuli by hypertrophy of the host cell or one of its organelles. These cellular reactions have been retained through evolution, and the more potent defensive reaction of inflammation has been added in higher species. The unique feature of the inflammatory process is the reaction of blood vessels, leading to the accumulation of fluid and leukocytes in extravascular tissues. The inflammatory response is closely intertwined with the process of repair. Inflammation serves to destroy, dilute, or wall off the injurious agent, and it sets into motion a series of events that try to heal and reconstitute the damaged tissue. Repair begins during the early phases of inflammation but reaches completion usually after the injurious influence has been neutralized. During repair, the injured tissue is replaced through regeneration of native parenchymal cells, by filling of the defect with fibrous tissue (scarring) or, most commonly, by a combination of these two processes. Figure 2-1 The components of acute and chronic inflammatory responses: circulating cells and proteins, cells of blood vessels, and cells and proteins of the extracellular matrix. Figure 2-2 The major local manifestations of acute inflammation, compared to normal. (1) Vascular dilation and increased blood flow (causing erythema and warmth), (2) extravasation and deposition of plasma fluid and proteins (edema), and (3) leukocyte emigration and accumulation in the site of injury. Figure 2-3 Blood pressure and plasma colloid osmotic forces in normal and inflamed microcirculation. A, Normal hydrostatic pressure (red arrows) is about 32 mm Hg at the arterial end of a capillary bed and 12 mm Hg at the venous end; the mean colloid osmotic pressure of tissues is approximately 25 mm Hg (green arrows), which is equal to the mean capillary pressure. Although fluid tends to leave the precapillary arteriole, it is returned in equal amounts via the postcapillary venule, so that the net flow (black arrows) in or out is zero. B, Acute inflammation. Arteriole pressure is increased to 50 mm Hg, the mean capillary pressure is increased because of arteriolar dilation, and the venous pressure increases to approximately 30 mm Hg. At the same time, osmotic pressure is reduced (averaging 20 mm Hg) because of protein leakage across the venule. The net result is an excess of extravasated fluid. Figure 2-4 Diagrammatic representation of five mechanisms of increased vascular permeability in inflammation (see text). Figure 2-5 Vascular leakage induced by chemical mediators. A, This is a fixed and cleared preparation of a rat cremaster muscle examined unstained by transillumination. One hour before sacrifice, bradykinin was injected over this muscle, and colloidal carbon was given intravenously. Plasma, loaded with carbon, escaped, but most of the carbon particles were retained by the basement membrane of the leaking vessels, with the result that these became "labeled" black. Note that not all the vessels leak—only the venules. In B, a higher power, the capillary network is faintly visible in the background. (Courtesy of Dr. Guido Majno, University of Massachusetts Medical School, Worcester, MA.) Figure 2-6 The multistep process of leukocyte migration through blood vessels, shown here for neutrophils. The leukocytes first roll, then become activated and adhere to endothelium, then transmigrate across the endothelium, pierce the basement membrane, and migrate toward chemoattractants emanating from the source of injury. Different molecules play predominant roles in different steps of this process—selectins in rolling; chemokines in activating the neutrophils to increase avidity of integrins (in green); integrins in firm adhesion; and CD31 (PECAM-1) in transmigration. TABLE 2-1 -- Endothelial/Leukocyte Adhesion Molecules Endothelial Molecule P-selectin Leukocyte Receptor Sialyl-Lewis X Major Role Rolling (neutrophils, monocytes, lymphocytes) PSGL-1 E-selectin Sialyl-Lewis X Rolling, adhesion to activated endothelium (neutrophils, monocytes, T cells) ICAM-1 CD11/CD18 (integrins) Adhesion, arrest, transmigration (all leukocytes) (LFA-1, Mac-1) VCAM-1 α4β1 (VLA4) (integrins) Adhesion (eosinophils, monocytes, lymphocytes) α4β7 (LPAM-1) GlyCam-1 L-selection Lymphocyte homing to high endothelial venules CD31 (PECAM) CD31 Leukocyte migration through endothelium *ICAM-1, VCAM-1, and CD31 belong to the immunoglobulin family of proteins; PSGL-1, P-selectin glycoprotein ligand 1. Leukocyte Adhesion and Transmigration Leukocyte adhesion and transmigration are regulated largely by the binding of complementary adhesion molecules on the leukocyte and endothelial surfaces, and chemical mediators— 16 17 chemoattractants and certain cytokines—affect these processes by modulating the surface expression or avidity of such adhesion molecules.[ ] [ ] The adhesion receptors involved belong to four molecular families—the selectins, the immunoglobulin superfamily, the integrins, and mucin-like glycoproteins. The most important of these are listed in Table 2-1 . • Selectins, so called because they are characterized by an extracellular N-terminal domain related to sugar-binding mammalian lectins, consist of E-selectin (CD62E, previously known as ELAM-1), which is confined to endothelium; P-selectin (CD62P, previously called GMP140 or PADGEM), which is present in endothelium and platelets; and L-selectin (CD62L, 18 19 previously known by many names, including LAM-1), which is expressed on most leukocyte types ( Box 2-1 ).[ ] [ ] Selectins bind, through their lectin domain, to sialylated forms of oligosaccharides (e.g., sialylated Lewis X), which themselves are covalently bound to various mucin-like glycoproteins (GlyCAM-1, PSGL-1, ESL-1, and CD34). • The immunoglobulin family molecules include two endothelial adhesion molecules: ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1). Both these molecules serve as ligands for integrins found on leukocytes. • Integrins are transmembrane heterodimeric glycoproteins, made up of α and β chains, that are expressed on many cell types and bind to ligands on endothelial cells, other leukocytes, and the extracellular matrix ( Box 2-1 ).[ 20] The β2 integrins LFA-1 and Mac-1 (CD11a/CD18 and CD11b/CD18) bind to ICAM-1, and the β1 integrins (such as VLA-4) bind VCAM-1. • Mucin-like glycoproteins, such as heparan sulfate, serve as ligands for the leukocyte adhesion molecule called CD44. These glycoproteins are found in the extracellular matrix and on cell surfaces. The recruitment of leukocytes to sites of injury and infection is a multistep process involving attachment of circulating leukocytes to endothelial cells and their migration through the endothelium (see Fig. 2-6 ). The first events are the induction of adhesion molecules on endothelial cells, by a number of mechanisms ( Fig. 2-7 ). Mediators such as histamine, thrombin, and platelet activating factor (PAF) stimulate the redistribution of P-selectin from its normal intracellular stores in granules (Weibel-Palade bodies) to the cell surface. Resident tissue macrophages, mast cells, and endothelial cells respond to injurious agents by secreting the cytokines TNF, IL-1, and chemokines (chemoattractant cytokines). (Cytokines are described in more detail below and in Chapter 6 .) TNF and IL-1 act on the endothelial cells of postcapillary venules adjacent to the infection and induce the expression of several adhesion molecules. Within 1 to 2 hours, the endothelial cells begin to express E-selectin. Leukocytes express at the tips of their microvilli carbohydrate ligands for the selectins, which bind to the endothelial selectins. These are lowaffinity interactions with a fast off-rate, and they are easily disrupted by the flowing blood. As a result, the bound leukocytes detach and bind again, and thus begin to roll along the endothelial surface. TNF and IL-1 also induce endothelial expression of ligands for integrins, mainly VCAM-1 (the ligand for the VLA-4 integrin) and ICAM-1 (the ligand for the LFA-1 and Mac-1 integrins). Leukocytes normally express these integrins in a low-affinity state. Meanwhile, chemokines that were produced at the site of injury enter the blood vessel, bind to endothelial cell heparan 21 sulfate glycosaminoglycans (labeled "proteoglycan" in Figure 2-6 ), and are displayed at high concentrations on the endothelial surface. [ ] These chemokines act on the rolling leukocytes and activate the leukocytes. One of the consequences of activation is the conversion of VLA-4 and LFA-1 integrins on the leukocytes to a high-affinity state. The combination of induced expression of integrin ligands on the endothelium and activation of integrins on the leukocytes results in firm integrin-mediated binding of the leukocytes to the endothelium at the site of infection. The leukocytes stop rolling, their cytoskeleton is reorganized, and they spread out on the endothelial surface. The next step in the process is migration of the leukocytes through the endothelium, called transmigration or diapedesis. Chemokines act on the adherent leukocytes and stimulate the cells to migrate through interendothelial spaces toward the chemical concentration gradient, that is, toward the site of injury or infection. Certain homophilic adhesion molecules (i.e., adhesion molecules that bind to each other) present in the intercellular junction of endothelium are involved in 55 Box 2-1. Selectins and Integrins: Adhesion Molecules Involved in the Inflammatory Response The specific (nonrandom) adhesion of cells to other cells or to extracellular matrices is a basic component of cell migration and recognition and underlies many biologic processes, including embryogenesis, tissue repair, and immune and inflammatory responses. It is, therefore, not surprising that many different genes have evolved that encode proteins with specific adhesive functions. Two families of adhesive proteins that are especially important in inflammation are the selectins and the integrins. Selectins The selectins are a family of three closely related proteins that differ in their cellular distribution but all function in adhesion of leukocytes to endothelial cells. All selectins are single-chain transmembrane glycoproteins with an amino terminus that is related to carbohydrate-binding proteins known as C-type lectins. Like other C-type lectins, ligand binding by selectins is calcium-dependent (hence the name C-type). The binding of selectins to their ligands has a fast on rate but also has a fast off rate and is of low affinity; this property allows selectins to mediate initial attachment and subsequent rolling of leukocytes on endothelium in the face of flowing blood. L-selectin, or CD62L, is expressed on lymphocytes and other leukocytes. It serves as a homing receptor for lymphocytes to enter lymph nodes by binding to high endothelial venules (HEVs). It also serves to bind neutrophils to cytokine-activated endothelial cells at sites of inflammation. L-selectin is located on the tips of microvillus projections of leukocytes, facilitating its interaction with ligands on endothelium. At least three endothelial cell ligands can bind L-selectin—glycan-bearing cell adhesion molecule-1 (GlyCAM-1), a secreted proteoglycan found on HEVs of lymph node; mucosal addressin cell adhesion molecule-1 (MadCAM-1), expressed on endothelial cells in gut-associated lymphoid tissues; and CD34, a proteoglycan on endothelial cells (and bone marrow cells). The protein backbones of all these ligands are modified by specific carbohydrates, which are the molecules actually recognized by the selectin. E-selectin, or CD62E, previously known as endothelial leukocyte adhesion molecule-1 (ELAM-1), is expressed only on cytokine-activated endothelial cells, hence the designation E. Eselectin recognizes complex sialylated carbohydrate groups related to the Lewis X or Lewis A family found on various surface proteins of granulocytes, monocytes, and previously activated effector and memory T cells. E-selectin is important in the homing of effector and memory T cells to some peripheral sites of inflammation, particularly in the skin. Endothelial cell expression of E-selectin is a hallmark of acute cytokine-mediated inflammation, and antibodies to E-selectin can block leukocyte accumulation in vivo. P-selectin (CD62P) was first identified in the secretory granules of platelets, hence the designation P. It has since been found in secretory granules of endothelial cells, called Weibel-Palade bodies. When endothelial cells or platelets are stimulated, P-selectin is translocated within minutes to the cell surface. On reaching the cell surface, P-selectin mediates binding of neutrophils, T lymphocytes, and monocytes. The complex carbohydrate ligands recognized by P-selectin appear similar to those recognized by E-selectin. The essential physiologic roles of selectins have been reinforced by studies of gene knockout mice. L-selectin-deficient mice have small, poorly formed lymph nodes with few T cells. Mice lacking either E-selectin or P-selectin have only mild defects in leukocyte recruitment, suggesting that these two molecules are functionally redundant. Double knockout mice lacking both Eselectin and P-selectin have significantly impaired leukocyte recruitment and increased susceptibility to infections. Humans who lack one of the enzymes needed to express the carbohydrate ligands for E-selectin and P-selectin on neutrophils have similar problems, resulting in a syndrome called leukocyte adhesion deficiency-2 (LAD-2) (see text). Integrins The integrin superfamily consists of about 30 structurally homologous proteins that promote cell-cell or cell-matrix interactions. The name of this family of proteins derives from the hypothesis that they coordinate (i.e., "integrate") signals from extracellular ligands with cytoskeleton-dependent motility, shape change, and phagocytic responses. All integrins are heterodimeric cell surface proteins composed of two noncovalently linked polypeptide chains, α and β. The extracellular domains of the two chains bind to various ligands, including extracellular matrix glycoproteins, activated complement components, and proteins on the surfaces of other cells. Several integrins bind to Arg-Gly-Asp (RGD) sequences in the fibronectin and vitronectin molecules. The cytoplasmic domains of the integrins interact with cytoskeletal components (including vinculin, talin, actin, α-actinin, and tropomyosin). Three integrin subfamilies were originally defined on the basis of which of three β subunits were used to form the heterodimers. More recently, five additional β chains have been identified. The β1 -containing integrins are also called VLA molecules, referring to "very late activation" molecules, because α1 β1 and α2 β1 were first shown to be expressed on T cells 2 to 4 weeks after repetitive stimulation in vitro. In fact, other VLA integrins are constitutively expressed on some leukocytes and rapidly induced on others. The β1 integrins are also called CD49ahCD29, CD49a-h referring to different α chains (α1 -α8 ) and CD29 referring to the common β1 subunit. Most of the β1 integrins are widely expressed on leukocytes and other cells and mediate attachment of cells to extracellular matrices. VLA-4 (α4 β1 ) is expressed only on leukocytes and can mediate attachment of these cells to endothelium by interacting with vascular cell adhesion molecule-1 (VCAM-1). VLA-4 is one of the principal surface proteins that mediate homing of lymphocytes to endothelium at peripheral sites of inflammation. The β2 integrins are also called CD11a-cCD18, or the leukocyte function-associated antigen-1 (LFA-1) family, CD11a-c referring to different α chains and CD18 to the common β2 subunit. LFA-1 (CD11aCD18) plays an important role in the adhesion of lymphocytes and other leukocytes with other cells, such as antigen-presenting cells and vascular endothelium. Other members of the family include CD11bCD18 (Mac-1 or CR3) and CD11cCD18 (p150,95 or CR4), which mediate leukocyte attachment to endothelial cells and subsequent extravasation. CD11bCD18 also functions as a fibrinogen receptor and as a complement receptor on phagocytic cells, binding particles opsonized with a by-product of complement activation called the inactivated C3b (iC3b) fragment. The other integrins are expressed on platelets and other cell types, and bind to extracellular matrix proteins as well as proteins involved in coagulation. 56 Figure 2-7 Regulation of endothelial and leukocyte adhesion molecules. A, Redistribution of P-selectin. B, Cytokine activation of endothelium. C, Increased binding avidity of integrins (see text). Figure 2-8 Schematic and histologic sequence of events following acute injury. The photomicrographs are representative of the early (neutrophilic) (left) and later (mononuclear) cellular infiltrates (right) of infarcted myocardium. The kinetics of edema and cellular infiltration are approximations. For sake of simplicity, edema is shown as an acute transient response, although secondary waves of delayed edema and neutrophil infiltration can also occur. Figure 2-9 Scanning electron micrograph of a moving leukocyte in culture showing a filopodium (upper left) and a trailing tail. (Courtesy of Dr. Morris J. Karnovsky, Harvard Medical School, Boston, MA.) Figure 2-10 Leukocyte activation. Different classes of cell surface receptors of leukocytes recognize different stimuli. The receptors initiate responses that mediate the functions of the leukocytes. Only some receptors are depicted (see text for details). Figure 2-11 A, Phagocytosis of a particle (e.g., bacterium) involves attachment and binding of Fc and C3b to receptors on the leukocyte membrane, engulfment, and fusion of lysosomes with phagocytic vacuoles, followed by destruction of ingested particles within the phagolysosomes. Note that during phagocytosis, granule contents may be released into extracellular tissues. B, Production of microbicidal reactive oxygen intermediates within phagocytic vesicles. ). Superoxide is then converted into hydrogen peroxide (H2 O2 ), mostly by spontaneous dismutation. Hydrogen peroxide can also be further reduced to the highly reactive hydroxyl radical (OH). Most of the H2 O2 is eventually broken down by catalase into H2 O and O2 , and some is destroyed by the action of glutathione oxidase. This process and its regulation were described in detail in Chapter 1 . NADPH oxidase is an enzyme complex consisting of at least seven proteins.[ 32] In resting neutrophils, different NADPH 61 oxidase protein components are located in the plasma membrane and the cytoplasm. In response to activating stimuli, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane, where they assemble and form the functional enzyme complex (see Fig. 2-11B ). Thus, the reactive oxygen intermediates are produced within the lysosome where the ingested substances are segregated, and the cell's own organelles are protected from the harmful effects of the ROIs. A similar enzyme system generates reactive nitrogen intermediates, notably nitric oxide, which also helps to kill microbes. The H2 O2 generated by the NADPH oxidase system is generally not able to efficiently kill microbes by itself. However, the azurophilic granules of neutrophils contain the enzyme myeloperoxidase (MPO), which, in the presence of a halide such as Cl- , converts H2 O2 to hypochlorite (HOCl). The latter is a potent antimicrobial agent that destroys microbes by halogenation (in which the halide is bound covalently to cellular constituents) or by oxidation of proteins and lipids (lipid peroxidation).[ 33] The H2 O2 -MPO-halide system is the most efficient bactericidal system in neutrophils. MPO-deficient leukocytes are capable of killing bacteria (albeit more slowly than normal cells), by virtue of the formation of superoxide, hydroxyl radicals, and singlet-oxygen. 34 Bacterial killing can also occur by oxygen-independent mechanisms, through the action of substances in leukocyte granules.[ ] These include bactericidal permeability increasing protein (BPI), a highly cationic granule-associated protein that causes phospholipase activation, phospholipid degradation, and increased permeability in the outer membrane of the microorganisms; lysozyme, which hydrolyzes the muramic acid-N-acetyl-glucosamine bond, found in the glycopeptide coat of all bacteria; lactoferrin, an iron-binding protein present in specific granules; major basic protein, a cationic protein of eosinophils, which has limited bactericidal activity but is cytotoxic to many parasites; and defensins, cationic arginine-rich granule peptides that are cytotoxic 35] to microbes (and certain mammalian cells).[ In addition, neutrophil granules contain many enzymes, such as elastase, that also contribute to microbial killing (discussed later in the chapter). After killing, acid hydrolases, which are normally stored in lysosomes, degrade the microbes within phagolysosomes. The pH of the phagolysosome drops to between 4 and 5 after phagocytosis, this being the optimal pH for the action of these enzymes. Release of Leukocyte Products and Leukocyte-Induced Tissue Injury During activation and phagocytosis, leukocytes release microbicidal and other products not only within the phagolysosome but also into the extracellular space. The most important of these substances in neutrophils and macrophages are lysosomal enzymes, present in the granules; reactive oxygen intermediates; and products of arachidonic acid metabolism, including prostaglandins and leukotrienes. These products are capable of causing endothelial injury and tissue damage and may thus amplify the effects of the initial injurious agent. Products of monocytes/macrophages and other leukocyte types have additional potentially harmful products, which are described in the discussion of chronic inflammation. Thus, if persistent and 36 unchecked, the leukocyte infiltrate itself becomes the offender,[ ] and leukocyte-dependent tissue injury underlies many acute and chronic human diseases ( Table 2-2 ). This fact becomes evident in the discussion of specific disorders throughout this book. Regulated secretion of lysosomal proteins is a peculiarity of leukocytes and other hematopoietic cells. (Recall that in most secretory cells, the proteins that are secreted are not stored within lysosomes.) The contents of lysosomal granules are secreted by leukocytes into the extracellular milieu by diverse mechanisms. Release may occur if the phagocytic vacuole remains transiently open to the outside before complete closure of the phagolysosome (regurgitation during feeding). If cells are exposed to potentially ingestible materials, such as immune complexes deposited on immovable flat surfaces (e.g., glomerular basement membrane), attachment of leukocytes to the immune complexes triggers leukocyte activation, but the fixed immune complexes cannot be phagocytosed, and lysosomal enzymes are released into the medium (frustrated phagocytosis). Cytotoxic release occurs after phagocytosis of potentially membranolytic substances, such as urate crystals, which damage the membrane of the phagolysosome. In addition, there is some evidence that proteins in certain granules, particularly the specific (secondary) granules of 37] [38] neutrophils, may be directly secreted by exocytosis.[ After phagocytosis, neutrophils rapidly undergo apoptotic cell death and are ingested by macrophages. Defects in Leukocyte Function From the preceding discussion, it is obvious that leukocytes play a central role in host defense. Not surprisingly, therefore, defects in leukocyte function, both genetic and acquired, lead to increased vulnerability to infections ( Table 2-3 ). Impairments of virtually every phase of leukocyte function—from adherence to vascular endothelium to microbicidal activity—have been identified, and the existence of clinical genetic deficiencies in each of the critical steps in the process has been described. These include the following: • Defects in leukocyte adhesion. We previously mentioned the genetic deficiencies in leukocyte adhesion molecules (LAD types 1 and 2). LAD 1 is characterized by recurrent bacterial infections and impaired wound healing. LAD 2 is clinically milder than LAD 1 but is also characterized by recurrent bacterial infections. • Defects in phagolysosome function. One such disorder is Chédiak-Higashi syndrome, an autosomal recessive condition 62 characterized by neutropenia (decreased numbers of neutrophils), defective degranulation, and delayed microbial killing. The neutrophils (and other leukocytes) have giant granules, which can 39 be readily seen in peripheral blood smears and which are thought to result from aberrant organelle fusion.[ ]In this syndrome, there is reduced transfer of lysosomal enzymes to phagocytic vacuoles in phagocytes (causing susceptibility to infections) and abnormalities in melanocytes (leading to albinism), cells of the nervous system (associated with nerve defects), and platelets (generating bleeding disorders). The gene associated with this disorder encodes a large cytosolic protein that is apparently involved in vesicular traffic but whose precise function is not yet known. The secretion of granule proteins by cytotoxic T cells is also affected, accounting for part of the immunodeficiency seen in the disorder. • Defects in microbicidal activity. The importance of oxygen-dependent bactericidal mechanisms is shown by the existence of a group of congenital disorders with defects in bacterial killing called chronic granulomatous disease, which render patients susceptible to recurrent bacterial infection. Chronic granulomatous disease results from inherited defects in the genes encoding several components of NADPH oxidase, which generates superoxide. The most common variants are an X-linked defect in one of the plasma membrane-bound 40 41 components (gp91phox) and autosomal recessive defects in the genes encoding two of the cytoplasmic components (p47phox and p67phox).[ ] [ ] • Clinically, the most frequent cause of leukocyte defects is bone marrow suppression, leading to reduced production of leukocytes. This is seen following therapies for cancer (radiation and chemotherapy) and when the marrow space is compromised by tumor metastases to bone. TABLE 2-2 -- Clinical Examples of Leukocyte-Induced Injury Acute Chronic Acute respiratory distress syndrome Arthritis Acute transplant rejection Asthma Asthma Atherosclerosis Glomerulonephritis Chronic lung disease Reperfusion injury Chronic rejection Septic shock Vasculitis TABLE 2-3 -- Defects in Leukocyte Functions Disease Defect Genetic Leukocyte adhesion deficiency 1 β chain of CD11/CD18 integrins Leukocyte adhesion deficiency 2 Fucosyl transferase required for synthesis of sialylated oligosaccharide (receptor for selectin) Chronic granulomatous disease Decreased oxidative burst ••X-linked ••NADPH oxidase (membrane component) ••Autosomal recessive ••NADPH oxidase (cytoplasmic components) Myeloperoxidase deficiency Absent MPO-H2 O2 system Chédiak-Higashi syndrome Protein involved in organelle membrane docking and fusion Acquired Thermal injury, diabetes, malignancy, sepsis, immunodeficiencies Chemotaxis Hemodialysis, diabetes mellitus Adhesion Leukemia, anemia, sepsis, diabetes, neonates, malnutrition Phagocytosis and microbicidal activity Data from Gallin JI: Disorders of phagocytic cells. In Gallin JI, et al (eds): Inflammation: Basic Principles and Clinical Correlates, 2nd ed. New York, Raven Press, 1992, pp 860, 861. Although we have emphasized the role of leukocytes recruited from the circulation in the acute inflammatory response, cells resident in tissues also serve important functions in initiating acute inflammation. The two most important of these cell types are mast cells and tissue macrophages. Mast cells react to physical trauma, breakdown products of complement, microbial products, and neuropeptides. The cells release histamine, leukotrienes, enzymes, and many cytokines (including TNF, IL-1, and chemokines), all of which contribute to inflammation. The functions of mast cells are discussed in more detail in Chapter 6 . Macrophages recognize microbial products and secrete most of the cytokines important in acute inflammation. These cells are stationed in tissues to rapidly recognize potentially injurious stimuli and initiate the host defense reaction. TERMINATION OF THE ACUTE INFLAMMATORY RESPONSE It is predictable that such a powerful system of host defense, with its inherent capacity to cause tissue damage, needs tight controls to minimize the damage. In part, inflammation declines simply because the mediators of inflammation have short half-lives, are degraded after their release, and are produced in quick bursts, only as long as the stimulus persists. In addition as 42 inflammation develops, the process also triggers a variety of stop signals that serve to actively terminate the reaction.[ ] These active mechanisms include a switch in the production of proinflammatory leukotrienes to anti-inflammatory lipoxins from arachidonic acid (described below); the liberation of an anti-inflammatory cytokine, transforming growth factor-β (TGF-β), from 43 macrophages and other cells; and neural impulses (cholinergic discharge) that inhibit the production of TNF in macrophages.[ ] There are, in addition, many other controls whose existence is suspected from the phenotypes of mice in which genes encoding putative regulatory molecules have been knocked out—these mice develop uncontrolled inflammation, but precisely how the regulation works normally is not yet defined.[ 42] Not surprisingly, there is great interest in defining the molecular basis 63 of the brakes on inflammation, since this knowledge could be used to design powerful anti-inflammatory drugs. Chemical Mediators of Inflammation Having described the events in acute inflammation, we can now turn to a discussion of the chemical mediators that are responsible for the events. Many mediators have been identified, and how they function in a coordinated manner is still not fully understood. Here we review general principles and highlight some of the major mediators ( Fig. 2-12 ). • Mediators originate either from plasma or from cells. Plasma-derived mediators (e.g., complement proteins, kinins) are present in plasma in precursor forms that must be activated, usually by a series of proteolytic cleavages, to acquire their biologic properties. Cell-derived mediators are normally sequestered in intracellular granules that need to be secreted (e.g., histamine in mast cell granules) or are synthesized de novo (e.g., prostaglandins, cytokines) in response to a stimulus. The major cellular sources are platelets, neutrophils, monocytes/ macrophages, and mast cells, but mesenchymal cells (endothelium, smooth muscle, fibroblasts) and most epithelia can also be induced to elaborate some of the mediators. • The production of active mediators is triggered by microbial products or by host proteins, such as the proteins of the complement, kinin, and coagulation systems, that are themselves activated by microbes and damaged tissues. • Most mediators perform their biologic activity by initially binding to specific receptors on target cells. Some, however, have direct enzymatic activity (e.g., lysosomal proteases) or mediate oxidative damage (e.g., reactive oxygen and nitrogen intermediates). • One mediator can stimulate the release of other mediators by target cells themselves. These secondary mediators may be identical or similar to the initial mediators but may also have opposing activities. They provide mechanisms for amplifying—or in certain instances counteracting—the initial mediator action. • Mediators can act on one or few target cell types, have diverse targets, or may even have differing effects on different types of cells. • Once activated and released from the cell, most of these mediators are short-lived. They quickly decay (e.g., arachidonic acid metabolites) or are inactivated by enzymes (e.g., kininase inactivates bradykinin), or they are otherwise scavenged (e.g., antioxidants scavenge toxic oxygen metabolites) or inhibited (e.g., complement regulatory proteins break up and degrade activated complement components). There is thus a system of checks and balances in the regulation of mediator actions. • Most mediators have the potential to cause harmful effects. Figure 2-12 Chemical mediators of inflammation. EC, endothelial cells. Figure 2-13 A flat spread of omentum showing mast cells around blood vessels and in the interstitial tissue. Stained with metachromatic stain to identify the mast cell granules (dark blue or purple). The red structures are fat globules stained with fat stain. (Courtesy of Dr. G. Majno, University of Massachusetts Medical School, Worcester, MA.) Figure 2-14 The activation and functions of the complement system. Activation of complement by different pathways leads to cleavage of C3. The functions of the complement system are mediated by breakdown products of C3 and other complement proteins, and by the membrane attack complex (MAC). The steps in the activation and regulation of complement are described in Box 2-2 . Box 2-2. The Complement System in Health and Disease The activation of the complement cascade may be divided into early and late steps. In the early steps, three different pathways lead to the proteolytic cleavage of C3. In the late steps, all three pathways converge, and the major breakdown product of C3, C3b, activates a series of other complement components. The Early Steps of Complement Activation The pathways of early complement activation are the following (see Figure ): The classical pathway is triggered by fixation of C1 to antibody (IgM or IgG) that has combined with antigen, and proteolysis of C2 and C4, and subsequent formation of a C4b2b complex that functions as a C3 convertase. The alternative pathway can be triggered by microbial surface molecules (e.g., endotoxin, or LPS), complex polysaccharides, and cobra venom. It involves a distinct set of plasma components (properdin, and factors B and D). In this pathway, the spontaneous cleavage of C3 that occurs normally is enhanced and stabilized by a complex of C3b and a breakdown product of Factor B called Bb; the C3bBb complex is a C3 convertase. In the lectin pathway, mannose-binding lectin, a plasma collectin, binds to carbohydrate-containing proteins on bacteria and viruses and directly activates C1; the remaining steps are as in the classical pathway. The C3 convertases break down C3 into C3b, which remains attached to the surface where complement is activated, and a smaller C3a fragment that diffuses away. The Late Steps of Complement Activation The C3b that is generated by any of the pathways binds to the C3 convertase and produces a C5 convertase, which cleaves C5. C5b remains attached to the complex and forms a substrate for the subsequent binding of the C6-C9 components. Polymerized C9 forms a channel in lipid membranes, called the Figure 2- 67 49 receptors (PARs) because they bind multiple trypsin-like serine proteases in addition to thrombin.[ ] These receptors are seven-transmembrane G protein-coupled receptors that are expressed on platelets, endothelial and smooth muscle cells, and many other cell types. Engagement of the so-called type Figure 2-15 Interrelationships between the four plasma mediator systems triggered by activation of factor XII (Hageman factor). Note that thrombin induces inflammation by binding to protease-activated receptors (principally PAR-1) on platelets, endothelium, smooth muscle cells, and other cells. The prostaglandins are also involved in the pathogenesis of pain and fever in inflammation. PGE2 is hyperalgesic in that it makes the skin hypersensitive to painful stimuli. It causes a marked increase in pain produced by intradermal injection of suboptimal concentrations of histamine and bradykinin and is involved in cytokine-induced fever during infections (described later). PGD2 is the major metabolite of the cyclooxygenase pathway in mast cells; 69 along with PGE2 and PGF2α (which are more widely distributed), it causes vasodilation and increases the permeability of postcapillary venules, thus potentiating edema formation. There has been great interest in the COX-2 enzyme because it is induced by a variety of inflammatory stimuli and is absent in most tissues under normal "resting" conditions. COX-1, by contrast, is produced in response to inflammatory stimuli and is also constitutively expressed in most tissues. This difference has led to the notion that COX-1 is responsible for the production of prostaglandins that are involved in inflammation but also serve a homeostatic function (e.g., fluid and electrolyte balance in the kidneys, cytoprotection in the gastrointestinal tract). In contrast, COX-2 stimulates the production of the prostaglandins that are involved in inflammatory reactions. • In the lipoxygenase pathway, the initial products are generated by three different lipoxygenases, which are present in only a few types of cells. 5-lipoxygenase (5-LO) is the predominant enzyme in neutrophils. The main product, 5-HETE, which is chemotactic for neutrophils, is converted into a family of compounds collectively called leukotrienes. LTB4 is a potent chemotactic agent and activator of neutrophil functional responses, such as aggregation and adhesion of leukocytes to venular endothelium, generation of oxygen free radicals, and release of lysosomal enzymes. The cysteinyl-containing leukotrienes C4 , D4 , and E4 (LTC4 , LTD4 , and LTE4 ) cause intense vasoconstriction, bronchospasm, and increased vascular permeability. The vascular leakage, as with histamine, is restricted to venules. Leukotrienes are several orders of magnitude more potent than histamine in increasing vascular permeability and causing bronchospasm. Leukotrienes mediate their actions by binding to cysteiny leukotreine 1 (CysLT1) and CysLT2 receptors. They are important in the pathogenesis of bronchial asthma. • Lipoxins are a recent addition to the family of bioactive products generated from AA, and transcellular biosynthetic mechanisms (involving two cell populations) are key to their production. Leukocytes, particularly neutrophils, produce intermediates in lipoxin synthesis, and these are converted to lipoxins by platelets interacting with the leukocytes. Lipoxins A4 and B4 (LXA4 , LXB4 ) are generated by the action of platelet 12-lipoxygenase on neutrophil-derived LTA4 ( Fig. 2-17 ). Cell-cell contact enhances transcellular metabolism, and blocking adhesion inhibits lipoxin 70 production. The principal actions of lipoxins are to inhibit leukocyte recruitment and the cellular components of inflammation. They inhibit neutrophil chemotaxis and adhesion to 55 endothelium.[ ] There is an inverse relationship between the amount of lipoxin and leukotrienes formed, suggesting that the lipoxins may be endogenous negative regulators of leukotriene action and may thus play a role in the resolution of inflammation. 54 • A new class of arachidonic acid-derived mediators, called resolvins, have been identified in experimental animals treated with aspirin.[ ] These mediators inhibit leukocyte recruitment and activation, in part by inhibiting the production of cytokines. Thus, the anti-inflammatory activity of aspirin is likely attributable to its ability to inhibit cyclooxygenases (see below) and, perhaps, to stimulate the production of resolvins. Figure 2-16 Generation of arachidonic acid metabolites and their roles in inflammation. The molecular targets of action of some anti-inflammatory drugs are indicated by a red X. COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid. TABLE 2-4 -- Inflammatory Actions of Eicosanoids Action Vasoconstriction Metabolite Thromboxane A2 , leukotrienes C4 , D4 , E4 Vasodilation PGI2 , PGE1 , PGE2 , PGD2 Increased vascular permeability Leukotrienes C4 , D4 , E4 Chemotaxis, leukocyte adhesion Leukotriene B4 , HETE, lipoxins Figure 2-17 Biosynthesis of leukotrienes and lipoxins by cell-cell interaction. Activated neutrophils generate LTB4 from arachidonic acid-derived LTA4 by the action of 5-lipoxygenase, but they do not possess LTC4 -synthase activity and consequently do not produce LTC4 . In contrast, platelets cannot form LTC4 from endogenous substrates, but they can generate LTC4 and lipoxins from neutrophil-derived LTA4 . (Courtesy of Dr. C. Serhan, Brigham and Women's Hospital, Boston, MA.) Figure 2-18 Major effects of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in inflammation. Figure 2-19 Functions of nitric oxide (NO) in blood vessels and macrophages, produced by two NO synthase enzymes. NO causes vasodilation, and NO free radicals are toxic to microbial and mammalian cells. NOS, nitric oxide synthase. Figure 2-20 Ultrastructure and contents of neutrophil granules, stained for peroxidase activity. The large peroxidase-containing granules are the azurophil granules; the smaller peroxidasenegative ones are the specific granules (SG). N, portion of nucleus; BPI, bactericidal permeability increasing protein. ), hydrogen peroxide (H2 O2 ), and hydroxyl radical (OH) are the major species produced within the cell, and these metabolites can combine with NO to form other reactive nitrogen intermediates.[ 70] Extracellular release of low levels of these potent mediators can increase the expression of chemokines (e.g., IL-8), cytokines, and endothelial leukocyte adhesion molecules, amplifying the cascade that elicits the inflammatory response.[ microbes. At higher levels, release of 71] As mentioned earlier, the physiologic function of these reactive oxygen intermediates is to destroy phagocytosed 74 these potent mediators can be damaging to the host. They are implicated in the following responses: • Endothelial cell damage, with resultant increased vascular permeability. Adherent neutrophils, when activated, not only produce their own toxic species, but also stimulate xanthine oxidation in endothelial cells themselves, thus elaborating more superoxide. • Inactivation of antiprotease, such as α1 -antitrypsin. This leads to unopposed protease activity, with increased destruction of extracellular matrix. • Injury to other cell types (parenchymal cells, red blood cells). Serum, tissue fluids, and host cells possess antioxidant mechanisms that protect against these potentially harmful oxygen-derived radicals. These antioxidants were discussed in Chapter 1 ; they include: (1) the copper-containing serum protein ceruloplasmin; (2) the iron-free fraction of serum, transferrin; (3) the enzyme superoxide dismutase, which is found or can be activated in a variety of cell types; (4) the enzyme catalase, which detoxifies H2 O2 ; and (5) glutathione peroxidase, another powerful H2 O2 detoxifier. Thus, the influence of oxygen-derived free radicals in any given inflammatory reaction depends on the balance between the production and the inactivation of these metabolites by cells and tissues. NEUROPEPTIDES Neuropeptides, similar to the vasoactive amines and the eicosanoids previously discussed, play a role in the initiation and propagation of an inflammatory response. The small peptides, such 72 as substance P and neurokinin A, belong to a family of tachykinin neuropeptides produced in the central and peripheral nervous systems.[ ] Nerve fibers containing substance P are prominent in the lung and gastrointestinal tract. Substance P has many biologic functions, including the transmission of pain signals, regulation of blood pressure, TABLE 2-5 -- Summary of Mediators of Acute Inflammation Action Mediator Source Vascular Leakage Chemotaxis Other Histamine and serotonin Mast cells, platelets + - Bradykinin Plasma substrate + - Pain C3a Plasma protein via liver + - Opsonic fragment (C3b) C5a Macrophages + + Leukocyte adhesion, activation Prostaglandins Mast cells, from membrane phospholipids - Vasodilation, pain, fever Leukotriene B4 Leukocytes - + Leukocyte adhesion, activation Leukotriene C4 , D4 , E4 Leukocytes, mast cells + - Bronchoconstriction, vasoconstriction Oxygen metabolites Leukocytes + - Endothelial damage, tissue damage PAF Leukocytes, mast cells + + Bronchoconstriction, leukocyte priming IL-1 and TNF Macrophages, other - + Acute-phase reactions, endothelial activation Chemokines Leukocytes, others - + Leukocyte activation Nitric oxide Macrophages, endothelium + + Vasodilation, cytotoxicity Potentiate other mediators stimulation of secretion by endocrine cells, and increasing vascular permeability. [ sensing of dangerous stimuli to the development of protective host 73] Sensory neurons appear to produce other pro-inflammatory molecules, which are thought to link the 74 responses.[ ] OTHER MEDIATORS The mediators described above account for inflammatory reactions to microbes, toxins, and many types of injury, but may not explain why inflammation develops in some specific situations. Recent studies are providing clues about the mechanisms of inflammation in two frequently encountered pathologic conditions. • Response to hypoxia. In Chapter 1 we described the role of hypoxia in causing cell injury and necrosis. Hypoxia by itself is also an inducer of the inflammatory response. This response is mediated largely by a protein called hypoxia-induced factor 1α, which is produced by cells deprived of oxygen and activates many genes involved in inflammation, 75 including VEGF, which increases vascular permeability.[ ] • Response to necrotic cells. Although it has been known for many years that necrotic cells elicit inflammatory reactions that serve to eliminate these cells, the molecular basis of this reaction has been largely unknown. One participant may be uric acid, which is a product of DNA breakdown, and crystallizes when present at sufficiently high concentrations in extracellular tissues. Uric acid crystals stimulate inflammation and subsequent immune response. [ which excessive amounts of uric acid are produced and crystals deposit in joints and other tissues. 76] This proinflammatory action of uric acid is the basis of the disease gout, in SUMMARY OF CHEMICAL MEDIATORS OF ACUTE INFLAMMATION Table 2-5 summarizes the major actions of the principal mediators. When Lewis discovered the role of histamine in 75 inflammation, one mediator was thought to be enough. Now, we are wallowing in them! Yet, from this menu of substances we can emphasize a few mediators that may be particularly relevant in vivo ( Table 2-6 ). Vasodilation, an early event in inflammation, is caused by histamine, prostaglandins, and nitric oxide. Increased vascular permeability is caused by histamine; the anaphylatoxins (C3a and C5a); the kinins; leukotrienes C, D, and E; PAF; and substance P. For chemotaxis, the most likely contributors are complement fragment C5a, lipoxygenase products (LTB4 ), and chemokines. Prostaglandins play an important role in vasodilation, pain, and fever, and in potentiating edema. IL-1 and TNF are critical for endothelial-leukocyte interactions and subsequent leukocyte recruitment, and for the production of acute-phase reactants. Lysosomal products and oxygen-derived radicals are the most likely candidates responsible for the ensuing tissue destruction. NO is involved in vasodilation and also causes tissue damage. Outcomes of Acute Inflammation The discussion of mediators completes the description of the basic, relatively uniform pattern of the inflammatory reaction encountered in most injuries. Although hemodynamic, permeability, and leukocyte changes have been described sequentially and may be initiated in this order, all these phenomena may be concurrent in the fully evolved reaction to injury. As might be expected, many variables may modify this basic process, including the nature and intensity of the injury, Figure 2-21 Outcomes of acute inflammation: resolution, healing by fibrosis, or chronic inflammation (see text). TABLE 2-6 -- Role of Mediators in Different Reactions of Inflammation Vasodilation Prostaglandins Nitric oxide Histamine Increased vascular permeability Vasoactive amines C3a and C5a (through liberating amines) Bradykinin Leukotrienes C4 , D4 , E4 PAF Substance P Chemotaxis, leukocyte recruitment and activation C5a Leukotriene B4 Chemokines IL-1, TNF Bacterial products Fever IL-1, TNF Prostaglandins Pain Prostaglandins Bradykinin Tissue damage Neutrophil and macrophage lysosomal enzymes Oxygen metabolites Nitric oxide 76 the site and tissue affected, and the responsiveness of the host. In general, however, acute inflammation may have one of three outcomes ( Fig. 2-21 ): 1. Complete resolution. In a perfect world, all inflammatory reactions, once they have succeeded in neutralizing and eliminating the injurious stimulus, should end with restoration of the site of acute inflammation to normal. This is called resolution and is the usual outcome when the injury is limited or short-lived or when there has been little tissue destruction and the damaged parenchymal cells can regenerate. Resolution involves neutralization or spontaneous decay of the chemical mediators, with subsequent return of normal vascular permeability, cessation of leukocytic infiltration, death (largely by apoptosis) of neutrophils, and finally removal of edema fluid and protein, leukocytes, foreign agents, and necrotic debris from the site ( Fig. 2-22 ). Lymphatics and phagocytes play a role in these events, as described later in this Chapter and in Chapter 3 . 2. Healing by connective tissue replacement (fibrosis). This occurs after substantial tissue destruction, when the inflammatory injury involves tissues that are incapable of regeneration, or when there is abundant fibrin exudation. When the fibrinous exudate in tissue or serous cavities (pleura, peritoneum) cannot be adequately cleared, connective tissue grows into the area of exudate, converting it into a mass of fibrous tissue—a process also called organization. In many pyogenic infections there may be intense neutrophil infiltration and liquefaction of tissues, leading to pus formation. The destroyed tissue is resorbed and eventually replaced by fibrosis. 3. Progression of the tissue response to chronic inflammation (discussed below). This may follow acute inflammation, or the response may be chronic almost from the onset. Acute to chronic transition occurs when the acute inflammatory response cannot be resolved, owing either to the persistence of the injurious agent or to some interference with the normal process of healing. For example, bacterial infection of the lung may begin as a focus of acute inflammation (pneumonia), but its failure to resolve may lead to extensive tissue destruction and formation of a cavity in which the inflammation continues to smolder, leading eventually to a chronic lung abscess. Another example of chronic inflammation with a persisting stimulus is peptic ulcer of the duodenum or stomach. Peptic ulcers may persist for months or years and, as discussed below, are manifested by both acute and chronic inflammatory reactions. Figure 2-22 Events in the resolution of inflammation: (1) return to normal vascular permeability; (2) drainage of edema fluid and proteins into lymphatics or (3) by pinocytosis into macrophages; (4) phagocytosis of apoptotic neutrophils and (5) phagocytosis of necrotic debris; and (6) disposal of macrophages. Macrophages also produce growth factors that initiate the subsequent process of repair. Note the central role of macrophages in resolution. (Modified from Haslett C, Henson PM: In Clark R, Henson PM (eds): The Molecular and Cellular Biology of Wound Repair. New York, Plenum Press, 1996.) Figure 2-23 Serous inflammation. Low-power view of a cross-section of a skin blister showing the epidermis separated from the dermis by a focal collection of serous effusion. Figure 2-24 Fibrinous pericarditis. A, Deposits of fibrin on the pericardium. B, A pink meshwork of fibrin exudate (F) overlies the pericardial surface (P). Figure 2-25 Suppurative inflammation. A, A subcutaneous bacterial abscess with collections of pus. B, The abscess contains neutrophils, edema fluid, and cellular debris. Figure 2-26 The morphology of an ulcer. A, A chronic duodenal ulcer. B, Low-power cross-section of a duodenal ulcer crater with an acute inflammatory exudate in the base. Figure 2-27 Maturation of mononuclear phagocytes. (From Abbas AK, et al: Cellular and Molecular Immunology, 5th ed. Philadelphia, Saunders, 2003.) Figure 2-28 The roles of activated macrophages in chronic inflammation. Macrophages are activated by cytokines from immune-activated T cells (particularly IFN-γ) or by nonimmunologic stimuli such as endotoxin. The products made by activated macrophages that cause tissue injury and fibrosis are indicated. AA, arachidonic acid; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; TGFβ, transforming growth factor β. Figure 2-29 A, Chronic inflammation in the lung, showing all three characteristic histologic features: (1) collection of chronic inflammatory cells (*), (2) destruction of parenchyma (normal alveoli are replaced by spaces lined by cuboidal epithelium, arrowheads), and (3) replacement by connective tissue (fibrosis, arrows). B, By contrast, in acute inflammation of the lung (acute bronchopneumonia), neutrophils fill the alveolar spaces and blood vessels are congested. Figure 2-30 Mechanisms of macrophage accumulation in tissues. The most important is continued recruitment from the microcirculation. (Adapted from Ryan G, Majno G: Inflammation. Kalamazoo, MI, Upjohn, 1977.) Lymphocytes and macrophages interact in a bidirectional way, and these reactions play an important role in chronic inflammation ( Fig. 2-31 ). Macrophages display antigens to T cells, and produce membrane molecules (costimulators) and cytokines (notably IL-12) that stimulate T-cell responses ( Chapter 6 ). Activated T lymphocytes produce cytokines, and one of these, IFNγ, is a major activator of macrophages. Plasma cells develop from activated B lymphocytes and produce antibody directed either against persistent antigen in the inflammatory site or against altered tissue components. In some strong chronic inflammatory reactions, the accumulation of lymphocytes, antigen-presenting cells, and 82 plasma cells may assume the morphologic features of lymphoid organs, particularly lymph nodes, even containing well-formed germinal centers. This pattern of lymphoid organogenesis is often seen in the synovium of patients with long-standing rheumatoid arthritis. • Eosinophils are abundant in immune reactions mediated by IgE and in parasitic infections ( Fig. 2-32 ). The recruitment of eosinophils involves extravasation from the blood and their migration into tissue by processes similar to those for other leukocytes. One of the chemokines that is especially important for eosinophil recruitment is eotaxin. Eosinophils have granules that contain major basic protein, a highly cationic protein that is toxic to parasites but also causes lysis of mammalian epithelial cells. They may thus be of benefit in controlling 79 parasitic infections but they contribute to tissue damage in immune reactions ( Chapter 6 ).[ ] • Mast cells are widely distributed in connective tissues and participate in both acute and persistent inflammatory reactions. Mast cells express on their surface the receptor that binds the Fc portion of IgE antibody (FcepsilonRI). In acute reactions, IgE antibodies bound to the cells' Fc receptors specifically recognize antigen, and the cells degranulate and release mediators, such as histamine and products of AA oxidation ( Chapter 6 ). This type of response occurs during anaphylactic reactions to foods, insect venom, or drugs, frequently with catastrophic results. When properly regulated, this response can benefit the host. Mast cells are also present in chronic inflammatory reactions, and may produce cytokines that contribute to fibrosis. Figure 2-31 Macrophage-lymphocyte interactions in chronic inflammation. Activated lymphocytes and macrophages influence each other and also release inflammatory mediators that affect other cells. Figure 2-32 A focus of inflammation showing numerous eosinophils. TABLE 2-7 -- Examples of Diseases with Granulomatous Inflammations Disease Cause Tissue Reaction Tuberculosis Mycobacterium tuberculosis Noncaseating tubercle (granuloma prototype): a focus of epithelioid cells, rimmed by fibroblasts, lymphocytes, histiocytes, occasional Langhans giant cell; caseating tubercle: central amorphous granular debris, loss of all cellular detail; acid-fast bacilli Leprosy Mycobacterium leprae Acid-fast bacilli in macrophages; non-caseating granulomas Syphilis Treponema pallidum Gumma: microscopic to grossly visible lesion, enclosing wall of histiocytes; plasma cell infiltrate; central cells are necrotic without loss of cellular outline Cat-scratch disease Gram-negative bacillus Rounded or stellate granuloma containing central granular debris and recognizable neutrophils; giant cells uncommon fibroblasts and connective tissue. Frequently, epithelioid cells fuse to form giant cells in the periphery or sometimes in the center of granulomas. These giant cells may attain diameters of 40 to 50 µm. They have a large mass of cytoplasm containing 20 or more small nuclei arranged either peripherally (Langhans-type giant cell) or haphazardly (foreign body-type giant cell) ( Fig. 2-33 ). There is no known functional difference between these two types of giant cells, a fact that does not deter students from remembering the morphologic differences! There are two types of granulomas, which differ in their pathogenesis. Foreign body granulomas are incited by relatively inert foreign bodies. Typically, foreign body granulomas form when material such as talc (associated with intravenous drug abuse) ( Chapter 9 ), sutures, or other fibers are large enough to preclude phagocytosis by a single macrophage and do not incite any specific inflammatory or immune response. Epithelioid cells and giant cells form and are apposed to the surface and encompass the foreign body. The foreign material can usually be identified in the center of the granuloma, particularly if viewed with polarized light, in which it appears refractile. Immune granulomas are caused by insoluble particles, typically microbes, that are capable of inducing a cell-mediated immune response ( Chapter 6 ). This type of immune response does not necessarily produce granulomas but it does so when Figure 2-33 Typical tuberculous granuloma showing an area of central necrosis, epithelioid cells, multiple Langhans-type giant cells, and lymphocytes. References 1. Diamond J: Guns, Germs and Steel: The Fates of Human Societies. WW Norton, New York. 2. Majno G: The Healing Hand: Man and Wound in the Ancient World. Cambridge, MA: Harvard University Press, 1975. 3. Weissman G: Inflammation: historical perspectives. In Gallin JI, et al (eds.): Inflammation: Basic Principles and Clinical Correlates. 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Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 348:138, 2003. 87 Chapter 3 - Tissue Renewal and Repair: Regeneration, Healing, and Fibrosis 88 The body's ability to replace injured or dead cells and to repair tissues after inflammation is critical to survival. When injurious agents damage cells and tissues, the host responds by setting in motion a series of events that serve to eliminate these agents, contain the damage, and prepare the surviving cells for replication. The repair of tissue damage caused by surgical resection, wounds, and diverse types of chronic injury can be broadly separated into two processes, regeneration and healing ( Fig. 3-1 ). Regeneration results in restitution of lost tissues; healing may restore original structures but involves collagen deposition and scar formation. Definitions The mechanisms of regeneration and healing will be discussed later in this chapter, but it is important from the outset to establish some important distinctions between these processes and to become familiar with terms used to describe them. Figure 3-1 Tissue response to injury. Repair after injury can occur by regeneration, which restores normal tissue, or by healing, which leads to scar formation and fibrosis. Figure 3-2 Mechanisms regulating cell populations. Cell numbers can be altered by increased or decreased rates of stem cell input, by cell death due to apoptosis, or by changes in the rates of proliferation or differentiation. (Modified from McCarthy NJ et al: Apoptosis in the development of the immune system: growth factors, clonal selection and bcl-2. Cancer Metastasis Rev 11:157, 1992.) Figure 3-3 Cell-cycle landmarks. The figure shows the cell-cycle phases (G0 , G1 , G2 , S, and M), the location of the G1 restriction point, and the G1 /S and G2 /M cell-cycle checkpoints. Cells from labile tissues such as the epidermis and the gastrointestinal tract may cycle continuously; stable cells such as hepatocytes are quiescent but can enter the cell cycle; permanent cells such as neurons and cardiac myocytes have lost the capacity to proliferate. (Modified from Pollard TD and Earnshaw WC: Cell Biology. Philadelphia, Saunders, 2002.) Figure 3-4 Steps involved in therapeutic cloning, using embryonic stem cells (ES cells) for cell therapy. The diploid nucleus of an adult cell from a patient is introduced into an enucleated oocyte. The oocyte is activated, and the zygote divides to become a blastocyst that contains the donor DNA. The blastocyst is dissociated to obtain ES. These cells are capable of differentiating into various tissues, either in culture or after transplantation into the donor. The goal of the procedure is to reconstitute or repopulate damaged organs of a patient, using the cells of the same patient to avoid immunologic rejection. (Modified from Hochedlinger K, Jaenisch R: Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med 349:275–286, 2003.) Figure 3-5 Stem-cell niches in various tissues. A, Epidermal stem cells located in the bulge area of the hair follicle serve as a stem cells for the hair follicle and the epidermis. B, Intestinal stem cells are located at the base of a colon crypt, above Paneth cells. C, Liver stem cells (commonly known as oval cells) are located in the canals of Hering (thick arrow), structures that connect bile ductules (thin arrow) with parenchymal hepatocytes (bile duct and Hering canals are stained for cytokeratin 7; courtesy of Tania Roskams, M.D., University of Leuven). D, Corneal stem cells are located in the limbus region, between the conjunctiva and the cornea. (Courtesy of T-T Sun, New York University, New York, NY.) Figure 3-6 Differentiation pathways for pluripotent bone marrow stromal cells. Activation of key regulatory proteins by growth factors, cytokines, or matrix components leads to commitment of stem cells to differentiate into specific cellular lineages. Differentiation of myotubes requires the combined action of several factors (e.g., myoD, myogenin); fat cells require PPARγ, the osteogenic lineage requires CBFA1 (also known as RUNX2), cartilage formation requires Sox9, and endothelial cells require VEGF and FGF-2. (Adapted and redrawn from Rodan GA, Harada S: The missing bone. Cell 89:677, 1997.) Figure 3-7 Differentiation of embryonic cells and generation of tissue cells by bone marrow precursors. During embryonic development the three germ layers—endoderm, mesoderm, and ectoderm—are formed, generating all tissues of the body. Adult stem cells localized in organs derived from these layers produce cells that are specific for the organs at which they reside. However, some adult bone marrow stem cells, in addition to producing the blood lineages (mesodermal derived), can also generate cells for tissues that originated from the endoderm and ectoderm (indicated by the red lines). (Modified from Korbling M, Estrov Z: Adult stem cells for tissue repair—a new theropeutic concept? N Engl J Med 349:570–582, 2003.) TABLE 3-1 -- Growth Factors and Cytokines Involved in Regeneration and Wound Healing Cytokine Symbol Source Functions Epidermal growth factor EGF Platelets, macrophages, saliva, urine, milk, plasma Mitogenic for keratinocytes and fibroblasts; stimulates keratinocyte migration and granulation tissue formation Transforming growth factor alpha TGF-α Macrophages, T lymphocytes, keratinocytes, and many tissues Similar to EGF; stimulates replication of hepatocytes and certain epithelial cells Hepatocyte growth factor/scatter HGF factor Mesenchymal cells Enhances proliferation of epithelial and endothelial cells, and of hepatocytes; increases cell motility Vascular endothelial cell growth factor (isoforms A, B, C, D) VEGF Mesenchymal cells Increases vascular permeability; mitogenic for endothelial cells (see Table 3-3 ) Platelet-derived growth factor (isoforms A, B, C, D) PDGF Platelets, macrophages, endothelial cells, keratinocytes, smooth muscle cells Chemotactic for PMNs, marcrophages, fibroblasts, and smooth muscle cells; activates PMNs, macrophages, and fibroblasts; mitogenic for fibroblasts, endothelial cells, and smooth muscle cells; stimulates production of MMPs, fibronectin, and HA; stimulates angiogenesis and wound contraction; remodeling; inhibits platelet aggregation; regulates integrin expression Fibroblast growth factor-1 (acidic), -2 (basic) and family FGF Macrophages, mast cells, T lymphocytes, Chemotactic for fibroblasts; mitogenic for fibroblasts and keratinocytes; stimulates endothelial cells, fibroblasts, and many tissues keratinocyte migration, angiogenesis, fam wound contraction and matrix deposition Transforming growth factor beta (isoforms 1, 2, 3); other members of the family are BMP and activin TGF-β Platelets, T lymphocytes, macrophages, endothelial cells, keratinocytes, smooth muscle cells, fibroblasts Chemotactic for PMNs, macrophages, lymphocytes, fibroblasts, and smooth muscle cells; stimulates TIMP synthesis, keratinocyte migration, angiogenesis, and fibroplasia; inhibits production of MMPs and keratinocyte proliferation; regulates integrin expression and other cytokines; induces TGF-β production Keratinocyte growth factor (also called FGF-7) KGF Fibroblasts Stimulates keratinocyte migration, proliferation, and differentiation Insulin-like growth factor-1 IGF-1 Macrophages, fibroblasts and other cells Stimulates synthesis of sulfated proteoglycans, collagen, keratinocyte migration, and fibroblast proliferation; endocrine effects similar to growth hormone Tumor necrosis factor TNF Macrophages, mast cells, T lymphocytes Activates macrophages; regulates other cytokines; multiple functions Interleukins IL-1, etc. Macrophages, mast cells, keratinocytes, lymphocytes, and many tissues Many functions. Some examples: chemotactic for PMNs (IL-1) and fibroblasts (IL-4), stimulation of MMP-1 synthesis (IL-1), angiogenesis (IL-8), TIMP synthesis (IL-6); regulation of other cytokines Interferons IFN-α, etc. Lymphocytes and fibroblasts Activates macrophages; inhibits fibroblast proliferation and synthesis of MMPs; regulates other cytokines BMP, bone morphogenetic proteins; PMNs, polymorphonuclear leukocytes; MMPs, matrix metalloproteinases; HA, hyaluronic acid; TIMP, tissue inhibitor of matrix metalloproteinase. Modified from Schwartz SI: Principles of Surgery, McGraw Hill, New York, 1999. those that have major roles in these processes. Other growth factors are alluded to in various sections of the book. Epidermal Growth Factor (EGF) and Transforming Growth Factor-α (TGF-α). These two factors belong to the EGF family and share a common receptor. EGF was discovered by its ability to cause precocious tooth eruption and eyelid opening in newborn mice. EGF is mitogenic for a variety of epithelial cells, hepatocytes, and fibroblasts. It is widely distributed in tissue secretions and fluids, such as sweat, saliva, urine, and intestinal contents. In healing wounds of the skin, EGF is produced by keratinocytes, macrophages, and other inflammatory cells that migrate into the area. EGF binds to a receptor (EGFR) with intrinsic tyrosine kinase activity, triggering the signal transduction events described later. TGF-α was originally extracted from sarcoma 96 virus-transformed cells and is involved in epithelial cell proliferation in embryos and adults and malignant transformation of normal cells to cancer. TGF-α has homology with EGF, binds to EGFR, and produces most of the biologic activities of EGF. The "EGF receptor" is actually a family of membrane tyrosine kinase receptors that respond to EGF, TGF-α, and other ligands of 44 the EGF family.[ ] The main EGFR is referred to as EGFR1, or ERB B1. The ERB B2 receptor (also known as HER-2/Neu) has received great attention because it is overexpressed in breast cancers and is a therapeutic target. Hepatocyte Growth Factor (HGF). HGF was originally isolated from platelets and serum. Subsequent studies demonstrated that it is identical to a previously identified growth factor known as scatter factor (HGF is also referred to as HGF/scatter factor). It has mitogenic effects in most epithelial cells, including hepatocytes and cells of the biliary epithelium in the liver, and epithelial cells of the lungs, 45 mammary gland, skin, and other tissues.[ ] Besides its mitogenic effects, HGF acts as a morphogen in embryonic development and promotes cell scattering and migration. This factor is produced by fibroblasts, endothelial cells, and liver nonparenchymal cells. The receptor for HGF is the product of the proto-oncogene c-MET, which is frequently overexpressed in human tumors. HGF signaling is required for survival during embryonic development, as demonstrated by the lethality of knockout mice lacking c-MET. Vascular Endothelial Growth Factor (VEGF). VEGF is a family of peptides that includes VEGF-A (referred throughout as VEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor. VEGF is a potent inducer of blood vessel 46 formation in early development (vasculogenesis) and has a central role in the growth of new blood vessels (angiogenesis) in adults (see Table 3-3 ). [ ] It promotes angiogenesis in tumors, chronic inflammation, and healing of wounds. Mice that lack a single allele of the gene (heterozygous VEGF knockout mice) die during embryonic development with defective vasculogenesis and hematopoiesis. VEGF family members signal through three tyrosine kinase receptors: VEGFR-1, VEGFR-2, and VEGFR-3. VEGFR-2 is located in endothelial cells and is the main receptor for the vasculogenic and angiogenic effects of VEGF. The role of VEGFR-1 is less well understood, but it may facilitate the mobilization of endothelial stem cells and has a role in inflammation. VEGF-C and VEGF-D bind to VEGFR-3 and act on lymphatic endothelial cells to induce the production of lymphatic vessels (lymphangiogenesis). VEGF-B binds exclusively to VEGFR-1. It is not required for vasculogenesis or angiogenesis, but may play a role in maintenance of myocardial function. Platelet-Derived Growth Factor (PDGF). PDGF is a family of several closely related proteins, each consisting of two chains designated A and B. All three isoforms of PDGF (AA, AB, and BB) are secreted and are biologically active. Recently, two new isoforms—PDGF-C and PDGF-D—have been identified. PDGF isoforms exert their effects by binding to two cell-surface receptors, designated PDGFR α and β, 47 which have different ligand specificities.[ ] PDGF is stored in platelet α granules and is released on platelet activation. It can also be produced by a variety of other cells, including activated macrophages, endothelial cells, smooth muscle cells, and many tumor cells. PDGF causes migration and proliferation of fibroblasts, smooth muscle cells, and monocytes, as demonstrated by defects in these functions in mice deficient in either the A or the B chain of PDGF. It also participates in the activation of hepatic stellate cells in the initial steps of liver fibrosis ( Chapter 18 ). Fibroblast Growth Factor (FGF). This is a family of growth factors containing more than 10 members, of which acidic FGF (aFGF, or FGF-1) and basic FGF (bFGF, or FGF-2) are the best characterized. FGF-1 and FGF-2 are made by a variety of cells. Released FGFs associate with heparan sulfate in the ECM, which can serve as a reservoir for storing inactive factors. FGFs are recognized by a family of cellsurface receptors that have intrinsic tyrosine kinase activity. A large number of functions are attributed to FGFs, including the following: • New blood vessel formation (angiogenesis): FGF-2, in particular, has the ability to induce the steps necessary for new blood vessel formation both in vivo and in vitro (see below). • Wound repair: FGFs participate in macrophage, fibroblast, and endothelial cell migration in damaged tissues and migration of epithelium to form new epidermis. • Development: FGFs play a role in skeletal muscle development and in lung maturation. For example, FGF-6 and its receptor induce myoblast proliferation and suppress myocyte differentiation, providing a supply of proliferating myocytes. FGF-2 is also thought to be involved in the generation of angioblasts during embryogenesis. FGF-1 and FGF-2 are 48 involved in the specification of the liver from endodermal cells.[ ] • Hematopoiesis: FGFs have been implicated in the differentiation of specific lineages of blood cells and development of bone marrow stroma. TGF-β and Related Growth Factors. TGF-β belongs to a family of homologous polypeptides that includes three TGF-β isoforms (TGF-β1, TGF-β2, TGF-β3) and factors with wide-ranging functions, such as bone 49 morphogenetic proteins (BMPs), activins, inhibins, and mullerian inhibiting substance.[ ] TGF-β1 has the most widespread distribution in mammals and will be referred to as TGF-β. It is a homodimeric protein produced by a variety of different cell types, including platelets, endothelial cells, lymphocytes, and macrophages. Native TGF-βs are synthesized as precursor proteins, which are secreted and then proteolytically cleaved to yield the biologically active growth factor and a second latent component. Active TGF-β binds to two cell surface receptors (types I and 50 II) with serine/threonine kinase activity and triggers the phosphorylation of cytoplasmic transcription factors called Smads. [ ] TGF-β first binds to a type II receptor, which then forms a complex with a type I receptor, leading to the phosphorylation of Smad 2 and 3. Phosphorylated Smad2 and 3 form heterodimers with Smad4, which enter the nucleus and associate with other DNA-binding proteins to activate or inhibit gene transcription. TGF-β has multiple and often opposing effects depending on the tissue and the type of injury. Agents that have multiple effects are called pleiotropic; because of the large diversity of TGF-β effects, it has been said that TGF-β is pleiotropic with a vengeance. • TGF-β is a growth inhibitor for most epithelial cell types and for leukocytes.[ ARF families (see Chapter 7 ). Loss of TGF-β receptors 51] It blocks the cell cycle by increasing the expression of cell-cycle inhibitors of the Cip/Kip and INK4/ 97 frequently occurs in human tumors, providing a proliferative advantage to tumor cells. • The effects of TGF-β on mesenchymal cells depend on concentration and culture conditions, it generally stimulates the proliferation of fibroblasts and smooth muscle cells. • TGF-β is a potent fibrogenic agent that stimulates fibroblast chemotaxis, enhances the production of collagen, fibronectin, and proteoglycans. It inhibits collagen degradation by decreasing matrix proteases and increasing protease inhibitor activities. TGF-β is involved in the development of fibrosis in a variety of chronic inflammatory conditions particularly in the lungs, kidney, and liver. • TGF-β has a strong anti-inflammatory effect. Knockout mice lacking the TGF-β1 gene have widespread inflammation and abundant lymphocyte proliferation, presumably because of unregulated T-cell proliferation and macrophage activation. Cytokines. Cytokines have important functions as mediators of inflammation and immune responses ( Chapter 6 ). Some of these proteins can be placed into the larger functional group of polypeptide growth factors because they have growth-promoting activities for a variety of cells. These are discussed in the appropriate chapters. SIGNALING MECHANISMS IN CELL GROWTH All growth factors function by binding to specific receptors, which deliver signals to the target cells. These signals have two general effects: (1) they stimulate the transcription of many genes that were silent in the resting cells, and (2) several of these genes regulate the entry of the cells into the cell cycle and their passage through the various stages of the cell cycle. In this section we review the process of receptor-initiated signal transduction as it applies to growth factors and signaling molecules in general, and their role in regulating the cell cycle. Cell proliferation is a tightly regulated process that involves a large number of molecules and interrelated pathways. The first event that initiates cell proliferation is, usually, the binding of a signaling molecule, the ligand, to a specific cell receptor. As we shall see, typical ligands are growth factors and proteins of the ECM. We describe different classes of receptor molecules and the pathways by which receptor activation initiates a cascade of events leading to expression of specific genes. We end this section with brief comments about transcription factors. Based on the source of the ligand and the location of its receptors—in the same, adjacent, or distant cells—three general modes of signaling, named autocrine, paracrine, and endocrine, can be distinguished ( Fig. 3-8 ). • Autocrine signaling: Cells respond to the signaling molecules that they themselves secrete, thus establishing an autocrine loop. Several polypeptide growth factors and cytokines act in this manner. Autocrine growth regulation plays a role in liver regeneration, proliferation of antigen-stimulated lymphocytes, and the growth of some tumors. Tumors frequently overproduce growth factors and their receptors, thus stimulating their own proliferation through an autocrine loop. • Paracrine signaling: One cell type produces the ligand, which then acts on adjacent target cells that express the appropriate receptors. The responding cells are in close proximity to the ligand-producing cell and are generally of a different type. Paracrine stimulation is common in connective tissue repair of healing wounds, in which a factor produced by one cell type (e.g., a macrophage) has its growth effect on adjacent cells (e.g., a fibroblast). Paracrine signaling is also necessary for hepatocyte replication during liver regeneration (see below). A special type of paracrine signaling, called juxtacrine, occurs when the signaling molecule (e.g., tumor necrosis factor, TGF-α, and heparin-binding epidermal growth factor) is anchored in the cell membrane and binds a receptor in the plasma membrane of another cell. In this type of signaling, receptor-ligand 98 interaction is dependent on and promotes cell-cell adhesion. • Endocrine signaling: Hormones are synthesized by cells of endocrine organs and act on target cells distant from their site of synthesis, being usually carried by the blood. Growth factors may also circulate and act at distant sites, as is the case for HGF. Several cytokines, such as those associated with the systemic aspects of inflammation discussed in Chapter 2 , also act as endocrine agents. Figure 3-8 General patterns of intercellular signaling demonstrating autocrine, paracrine, and endocrine signaling (see text). (Modified from Lodish H, et al. [eds]: Molecular Cell Biology, 3rd ed. New York, WH Freeman, 1995, p. 855. © 1995 by Scientific American Books. Used with permission of WH Freeman and Company.) Figure 3-9 Examples of signal transduction systems that require cell-surface receptors. Shown are receptors with intrinsic tyrosine kinase activity, seven transmembrane G-protein-coupled receptors, and receptors without intrinsic tyrosine kinase activity. The figure also shows important signaling pathways transduced by the activation of these receptors through ligand binding. Figure 3-10 Signaling from tyrosine kinase receptors. Binding of the growth factor (ligand) causes receptor dimerization and autophosphorylation of tyrosine residues. Attachment of adapter (or bridging) proteins (e.g., GRB2 and SOS) couples the receptor to inactive RAS. Cycling of RAS between its inactive and active forms is regulated by GAP. Activated RAS interacts with and activates RAF (also known as MAP kinase kinase kinase). This kinase then phosphorylates a component of the MAP kinase signaling pathway, MEK (also known as MAP kinase kinase), which then phosphorylates ERK (MAP kinase). Activated MAP kinase phosphorylates other cytoplasmic proteins and nuclear transcription factors, generating cellular responses. The phosphorylated tyrosine kinase receptor can also bind other components, such as PI-3 kinase, which activates distinct signaling systems. Figure 3-11 Liver regeneration after partial hepatectomy. Upper panel, The lobes of the liver of a rat are shown (M, median; RL and LL, right and left lateral lobes; C, caudate lobe). Partial hepatectomy removes two thirds of the liver (median and left lateral lobes), and only the right lateral and caudate lobes remain. After 3 weeks, the right lateral and caudate lobes enlarge to reach a mass equivalent to that of the original liver. Note that there is no regrowth of the median and left lateral lobes removed after partial hepatectomy. (From Goss RJ: Regeneration versus repair. In Cohen IK, Diegelman RF, Lindblad WJ (eds): Wound Healing. Biochemical and Clinical Aspects. Philadelphia, W. B. Saunders Co., 1992, pp. 20–39.) Lower panel, Timing of hepatocyte DNA replication, hepatocyte mitosis, and expression of messenger RNAs during liver regeneration. DNA replication is shown as the incorporation of tritiated thymidine × 10-4 (right-side scale). Mitosis presented as the percentage of hepatocytes undergoing mitosis (right-side scale). The expression of some of the many mRNAs in the regenerating rat liver is presented as fold elevation above normal (left-side scale). Expression of the proto-oncogenes c-fos, c-jun, and c-myc corresponds to the immediate early gene phase of gene expression during liver regeneration. Figure 3-12 Regeneration of human liver. Computed tomography (CT) scans of the donor liver in living-donor hepatic transplantation. Upper panel, The liver of the donor before the operation. The right lobe, which will be used as a transplant, is outlined. Lower panel, A scan of the liver 1 week after performance of partial hepatectomy to remove the right lobe. Note the great enlargement of the left lobe (outlined in the panel) without regrowth of the right lobe (Courtesy of R. Troisi, M.D. Ghent University city; reproduced in part from Fausto; Liver Regeneration. In Arias, et al: The Liver: Biology and Pathobiology, 4th ed. Philadelphia, Lippincott Williams & Wilkins, 2001.) Figure 3-13 Priming and cell-cycle progression in hepatocyte replication during liver regeneration. Quiescent hepatocytes become competent to enter the cell cycle through a priming phase mostly mediated by the cytokines TNF and IL-6 (upper panel). Growth factors, mainly HGF and TGF-α, act on primed hepatocytes to make them progress through the cell cycle and undergo DNA replication (lower panel). Norepinephrine, insulin, thyroid hormone, and growth hormone act as adjuvants for liver regeneration. The factors that determine the termination of cell replication are not known but are likely to involve cell cycle inhibitors, shut-off of growth factor production, and decreased metabolic demand on the liver. Figure 3-14 Major components of the extracellular matrix (ECM), including collagens, proteoglycans, and adhesive glycoproteins. Both epithelial and mesenchymal cells (e.g., fibroblasts) interact with ECM via integrins. To simplify the diagram, many ECM components (e.g., elastin, fibrillin, hyaluronan, syndecan) are not included. TABLE 3-2 -- Main Types of Collagens, Tissue Distribution, and Genetic Disorders Collagen Type Tissue Distribution Genetic Disorders Fibrillar Collagens I Ubiquitous in hard and soft tissues Osteogenesis Imperfecta Ehlers-Danlos syndrome—arthrochalasias type II Cartilage, intervertebral disk, vitreous Achondrogenesis type II, spondyloepiphyseal dysplasia syndrome III Hollow organs, soft tissues Vascular Ehlers-Danlos syndrome V Soft tissues, blood vessels Classical Ehlers-Danlos syndrome IX Cartilage, vitreous Stickler syndrome Basement Membrane Collagens IV Basement membranes Alport syndrome VI Ubiquitous in microfibrils Bethlem myopathy VII Anchoring fibrils at dermal-epidermal junctions Dystrophic epidermolysis bullosa IX Cartilage, intervertebral disks Multiple epiphyseal dysplasias XVII Transmembrane collagen in epidermal cells Benign atrophic generalized epidermolysis bullosa XV and XVIII Endostatin-forming collagens, endothelial cells Knobloch syndrome (type XVIII collagen) Other Collagens Courtesy of Dr. Peter H. Byers, Department of Pathology, University of Washington, Seattle, WA original size after release of the tension. Morphologically, elastic fibers consist of a central core made of elastin, surrounded by a peripheral network of microfibrils. Substantial amounts of elastin are found in the walls of large blood vessels, such as the aorta, and in the uterus, skin, and ligaments. The peripheral microfibrillar network that surrounds the core consists largely of fibrillin, a 350-kD secreted glycoprotein, which associates either with itself or with other components of the ECM. The microfibrils serve as scaffolding for deposition of elastin and the 86 assembly of elastic fibers. Inherited defects in fibrillin[ ] result in formation of abnormal elastic fibers in a fairly common familial disorder, Marfan syndrome, manifested by changes in the cardiovascular system (aortic dissection) and the skeleton ( Chapter 5 ). CELL ADHESION PROTEINS Most adhesion proteins, also called CAMs (cell adhesion molecules), can be classified into four main families: immunoglobulin family CAMs, cadherins, integrins, and selectins. These proteins are located in the cell membrane, where they function as receptors, or they are stored in the cytoplasm. As receptors, CAMs can bind to similar or different molecules in other cells, providing for interaction between the same cells (homotypic interaction) or different cell types (heterotypic interaction). Cadherins are generally involved in calcium-dependent homotypic interactions, while immunoglobulin family CAMs, because of the types of ligands they can bind, participate in both homotypic and heterotypic cell-to-cell interactions. The integrins have broader ligand specificity and are responsible for many events involving cell adhesion.[ 87] [88] Integrins bind both to matrix proteins such as fibronectin and laminin, mediating adhesiveness between cells and ECM, as well as to adhesive proteins in other cells, establishing cell-to-cell contacts (see Box 2-1 , Chapter 2). Fibronectin is a larger protein that binds to many molecules, such as collagen, fibrin, proteoglycans, and cell-surface receptors. It consists of two glycoprotein chains, held together by disulfide bonds. Fibronectin mRNA has two splice forms, giving rise to tissue fibronectin and plasma fibronectin. The tissue fibronectin forms fibrillar aggregates at wound healing sites. The plasma form binds to fibrin, forming the provisional blood clot that 105 Figure 3-15 Steps in collagen synthesis (see text). Figure 3-16 Mechanisms by which ECM (e.g., fibronectin and laminin) and growth factors can influence cell growth, motility, differentiation, and protein synthesis. Integrins bind ECM components and interact with the cytoskeleton at focal adhesion complexes (protein aggregates that include vinculin, α-actin, and talin). This can initiate the production of intracellular messengers or can directly mediate nuclear signals. Cell-surface receptors for growth factors may activate signal transduction pathways that overlap with those activated by integrins. Collectively, these are integrated by the cell to yield various responses, including changes in cell growth, locomotion, and differentiation. Figure 3-17 A, Granulation tissue showing numerous blood vessels, edema, and a loose ECM containing occasional inflammatory cells. This is a trichrome stain that stains collagen blue; minimal mature collagen can be seen at this point. B, Trichrome stain of mature scar, showing dense collagen, with only scattered vascular channels. Figure 3-18 Angiogenesis by mobilization of endothelial precursor cells (EPCs) from the bone marrow and from pre-existing vessels (capillary growth). EPCs are mobilized from the bone marrow and may migrate to a site of injury or tumor growth (upper panel). The homing mechanisms have not yet been defined. At these sites, EPCs differentiate and form a mature network by linking with existing vessels. In angiogenesis from pre-existing vessels, endothelial cells from these vessels become motile and proliferate to form capillary sprouts (lower panel). Regardless of the initiating mechanism, vessel maturation (stabilization) involves the recruitment of pericytes and smooth muscle cells to form the periendothelial layer. (Modified from Conway EM, Collen D, Carmeliet P: Molecular mechanisms of blood vessel growth. Cardiovasc Res 49:507, 2001.) TABLE 3-3 -- Vascular Endothelial Growth Factor (VEGF) Proteins Family members: VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D Dimeric glycoprotein with multiple isoforms Targeted mutations in VEGF result in defective vasculogenesis and angiogenesis Production Expressed at low levels in a variety of adult tissues and at higher levels in a few sites, such as podocytes in the glomerulus and cardiac myocytes Inducing Agents Hypoxia TGF-β PDGF TGF-α Receptors VEGFR-1 VEGFR-2 (restricted to endothelial cells) VEGFR-3 (lymphatic endothelial cells) Targeted mutations in the receptors result in lack of vasculogenesis Functions Promotes angiogenesis Increases vascular permeability Stimulates endothelial cell migration Stimulates endothelial cell proliferation VEGF-C selectively induces hyperplasia of lymphatic vasculature Up-regulates endothelial expression of plasminogen activator, plasminogen activator inhibitor-1, tissue factor, and interstitial collagenase in the absence of VEGF, more responsive to inhibitors of angiogenesis. A telling proof of the importance of these molecules is the existence of a genetic disorder caused by mutations in Tie2 103 that is characterized by venous malformations.[ ] Both physiologic and pathologic angiogenesis can be influenced by agents or conditions that stimulate VEGF expression, such as certain cytokines and growth factors (e.g., TGF-β, PDGF, TGF-α) and, notably, tissue hypoxia, which has long been associated with angiogenesis (see Table 3-3 ). Despite the diversity of factors that may participate at various steps in angiogenesis, VEGF emerges as the most important growth factor in adult tissues undergoing physiologic angiogenesis (e.g., proliferating endometrium) as well as pathologic angiogenesis seen in chronic inflammation, wound healing, tumors, and diabetic retinopathy. ECM Proteins as Regulators of Angiogenesis A key component of angiogenesis is the motility and directed migration of endothelial cells, required for the formation of new blood vessels. These processes are controlled by several classes of proteins, including (1) integrins, especially αv β3 , which is critical for the formation and maintenance of newly formed blood vessels,[ 104] (2) matricellular proteins, including 105 thrombospondin 1, SPARC, and tenascin C, which destabilize cell-matrix interactions and therefore promote angiogenesis,[ ] and (3) proteinases, such as the plasminogen activators and matrix metalloproteinases, which are important in tissue remodeling during endothelial invasion. Additionally, these proteinases cleave extracellular proteins, releasing matrix-bound growth factors such as VEGF and FGF-2 that stimulate angiogenesis. Proteinases can also release inhibitors such as endostatin, a small fragment of collagen that inhibits endothelial proliferation and 106] angiogenesis.[ αv β3 integrin expression in endothelial cells is stimulated by hypoxia. This integrin has multiple effects on angiogenesis: it directly interacts with a metalloproteinase 104] (MMP-2, discussed below), it binds to and regulates the activity of VEGFR-2, and it mediates adhesion to ECM components such as fibronectin, thrombospondin, and osteopontin. [ 110 SCAR FORMATION Growth factors and cytokines released at the site of injury induce fibroblast proliferation and migration into the granulation tissue framework of new blood vessels and loose ECM that initially forms at the repair site. We discuss three processes that participate in the formation of a scar: (1) emigration and proliferation of fibroblasts in the site of injury, (2) deposition of ECM, and (3) tissue remodeling. Fibroblast Migration and Proliferation Granulation tissue contains numerous newly formed blood vessels. As discussed previously, VEGF promotes angiogenesis but is also responsible for a marked increase in vascular 107 permeability (VEGF was first named vascular permeability factor).[ ] The latter activity leads to exudation and deposition of plasma proteins, such as fibrinogen and plasma fibronectin, in the ECM and provides a provisional stroma for fibroblast and endothelial cell ingrowth. Migration of fibroblasts to the site of injury and their subsequent proliferation are triggered by multiple growth factors, including TGF-β, PDGF, EGF, FGF, and the cytokines IL-1 and TNF (see Table 3-5 ). The sources of these growth factors and cytokines include platelets, a variety of inflammatory cells (notably macrophages), and activated endothelium. Macrophages are important cellular constituents of granulation tissue, clearing extracellular debris, fibrin, and other 108 foreign material at the site of repair. These cells also elaborate TGF-β, PDGF, and FGF and there-fore promote fibroblast migration and proliferation.[ ] If the appropriate chemotactic stimuli are present, mast cells, eosinophils, and lymphocytes may also accumulate. Each of these cells can contribute directly or indirectly to fibroblast migration and proliferation. Of the growth factors involved in inflammatory fibrosis, TGF-β appears to be the most important because of the multitude of effects that favor fibrous tissue deposition. TGF-β is produced by most of the cells in granulation tissue and causes fibroblast migration and proliferation, increased synthesis of collagen and fibronectin, and decreased degradation of ECM by metalloproteinases (discussed later). TGF-β is also chemotactic for monocytes and causes angiogenesis in vivo, possibly by inducing macrophage influx. TGF-β expression is increased in tissues in a number of chronic fibrotic diseases in humans and experimental animals. ECM Deposition and Scar Formation As repair continues, the number of proliferating endothelial cells and fibroblasts decreases. Fibroblasts progressively deposit increased amounts of ECM. Fibrillar collagens form a major portion of the connective tissue in repair sites and are important for the development of strength in healing wounds. As described later in the discussion of cutaneous wound healing, collagen synthesis by fibroblasts begins within 3 to 5 days after injury and continues for several weeks, depending on the size of wound. Many of the same growth factors that regulate fibroblast proliferation also stimulate ECM synthesis (see Table 3-4 (Table Not Available) ). For example, collagen synthesis is enhanced by several factors, including growth factors (PDGF, FGF, TGF-β) and cytokines (IL-1, IL-13), which are secreted by leukocytes and fibroblasts in healing wounds. Net collagen accumulation, however, depends not only on increased collagen synthesis but also on decreased degradation. Ultimately, the granulation tissue scaffolding is converted into a scar composed of spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, and other ECM components. As the scar matures, vascular regression continues, eventually transforming the richly vascularized granulation tissue into a pale, avascular scar. Tissue Remodeling The replacement of granulation tissue with a scar involves transitions in the composition of the ECM. Some of the growth factors that stimulate synthesis of collagen and other connective tissue molecules also modulate the synthesis and activation of metalloproteinases, enzymes that degrade these ECM components. The balance between ECM synthesis and degradation results in remodeling of the connective tissue framework—an important feature of both chronic inflammation and wound repair. Degradation of collagen and other ECM proteins is achieved by a family of matrix metalloproteinases (MMPs), which are dependent on zinc ions for their activity[ 109] [110] ( Fig. 3-19 ). This Figure 3-19 Matrix metalloproteinase regulation. Four mechanisms are shown: (1) regulation of synthesis by growth factors or cytokines, (2) inhibition of synthesis by corticosteroids or TGF-β, (3) regulation of the activation of the secreted but inactive precursors, and (4) blockage of the enzymes by specific tissue inhibitors of metalloproteinase (TIMPs). (Modified from Matrisian LM: Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet 6:122, 1990, with permission from Elsevier Science.) Figure 3-20 Phases of wound healing. (Modified from Clark RAF: Wound repair. In Clark RAF (ed): The molecular and cellular biology of wound repair, 2nd ed, New York, Plenum Press, 1996, p. 3.) TABLE 3-4 -- Growth Factors and Cytokines Affecting Various Steps in Wound Healing (Not Available) 112 Figure 3-21 Steps in wound healing by first intention (left) and second intention (right). Note large amounts of granulation tissue and wound contraction in healing by second intention. Figure 3-22 Healing of skin ulcers. A, Pressure ulcer of the skin, commonly found in diabetic patients. The histology slides show B, a skin ulcer with a large gap between the edges of the lesion; C, a thin layer of epidermal reepithelialization and extensive granulation tissue formation in the dermis; and D, continuing reepithelialization of the epidermis and wound contraction. (Courtesy of Z. Argenyi, M.D., University of Washington.) TABLE 3-5 -- Factors That Retard Wound Healing Local Factors Blood supply Mechanical stress Denervation Necrotic tissue Local infection Protection (dressings) Foreign body Surgical techniques Hematoma Type of tissue Systemic Factors Age Malnutrition Anemia Obesity Drugs (steroids, cytotoxic medications, intensive antibiotic therapy) Systemic infection Temperature Trauma, hypovolemia, and hypoxia Genetic disorders (osteogenesis imperfecta, Ehlers-Danlos syndromes, Marfan syndrome) Uremia Vitamin deficiency (vitamin C) Hormones Trace metal deficiency (zinc, copper) Diabetes Malignant disease Adapted from Schwartz SI: Principles of Surgery. New York, McGraw Hill, 1999. • Infection is the single most important cause of delay in healing because it results in persistent tissue injury and inflammation. • Mechanical factors, such as early motion of wounds, can delay healing, by compressing blood vessels and separating the edges of the wound. • Foreign bodies, such as unnecessary sutures or fragments of steel, glass, or even bone, constitute impediments to healing. • Size, location, and type of wound influence healing. Wounds in richly vascularized areas, such as the face, heal faster than those in poorly vascularized ones, such as the foot. As we have discussed, small incisional injuries heal faster and with less scar formation than large excisional wounds or wounds caused by blunt trauma. SUMMARY OF CUTANEOUS WOUND HEALING Various stages in the healing of an ulcer of the skin are shown in Figure 3-22 . The healing wound, as a prototype of tissue repair, is a dynamic and changing process. The early phase is one of inflammation, followed by formation of granulation tissue and subsequent tissue remodeling and scarring. Simple cutaneous incisional wounds heal by first intention. Large cutaneous wounds heal by second intention, generating a significant amount of scar tissue. Different mechanisms occurring at different times trigger the release of chemical signals that modulate the orderly migration, proliferation, and differentiation of cells and the synthesis and degradation of ECM proteins. These proteins, in turn, directly affect cellular events and modulate cell responsiveness to soluble growth factors. The magic behind the precise orchestration of these events under normal conditions remains beyond our grasp. It almost certainly lies in the regulation of specific soluble and membrane-anchored mediators and their receptors on particular cells, cell-matrix interactions, and the effect of physical factors, including ECM remodeling forces generated by changes in cell shape. COMPLICATIONS IN CUTANEOUS WOUND HEALING Complications in wound healing can arise from abnormalities in any of the basic components of the repair process. These aberrations can be grouped into three general categories: (1) deficient scar formation, (2) excessive formation of the repair components, and (3) formation of contractures. Inadequate formation of granulation tissue or assembly of a scar can lead to two types of complications: wound dehiscence and ulceration. Dehiscence or rupture of a wound is most common after abdominal surgery and is due to increased abdominal pressure. This mechanical stress on the abdominal wound can be generated by vomiting, coughing, or ileus. Wounds can ulcerate because of inadequate vascularization during healing. For example, lower extremity wounds in individuals with atherosclerotic peripheral vascular disease typically ulcerate ( Chapter 11 ). Nonhealing wounds also form in areas devoid of sensation. These neuropathic ulcers are occasionally seen in patients with diabetic peripheral neuropathy ( Chapter 24 and Chapter 27 ). 115 Excessive formation of the components of the repair process can also complicate wound healing. Aberrations of growth may occur even in what may begin initially as normal wound healing. The accumulation of excessive amounts of collagen may give rise to a raised scar known as a hypertrophic scar; if the scar tissue grows beyond the boundaries of the original wound and does not regress, it is called a keloid ( Fig. 3-23 ). Keloid formation appears to be an individual predisposition, and for unknown reasons this aberration is somewhat more common in AfricanAmericans. The mechanisms of keloid formation are still unknown. Another deviation in wound healing is the formation of excessive amounts of granulation tissue, which protrudes above the level of the surrounding skin and blocks re-epithelialization. This has been called exuberant granulation (or, with more literary fervor, proud flesh). Excessive granulation must be removed by cautery or surgical excision to permit restoration of the continuity of the epithelium. Finally (fortunately rarely), incisional scars or traumatic injuries may be followed by exuberant proliferation of fibroblasts and other connective tissue elements that may, in fact, recur after excision. Called desmoids, or aggressive fibromatoses, these lie in the interface between benign proliferations and malignant (though low-grade) tumors. The line between the benign hyperplasias characteristic of repair and neoplasia is frequently finely drawn ( Chapter 7 ). Contraction in the size of a wound is an important part of the normal healing process. An exaggeration of this process is called a contracture and results in deformities of the wound and the surrounding tissues. Contractures are particularly prone to develop on the palms, the soles, and the anterior aspect of the thorax. Contractures are commonly seen after serious burns and can compromise the movement of joints. Impaired wound contraction occurs in stromelysin-1 (MMP3)—deficient mice, suggesting that proteolysis by this metalloproteinase is required for the assembly of fibroblasts containing actin filaments, needed for the contraction of early wounds. [ 120] Figure 3-23 A, Keloid. Excess collagen deposition in the skin forming a raised scar known as keloid. (From Murphy GF, Herzberg AJ: Atlas of Dermatopathology. Philadelphia, Saunders, W.B. 1996, p. 219.) B, Note the thick connective tissue deposition in the dermis. (Slide courtesy of Z. Argenyi, M.D., University of Washington, Seattle, WA.) Figure 3-24 Development of fibrosis in chronic inflammation. The persistent stimulus of chronic inflammation activates macrophages and lymphocytes, leading to the production of growth factors and cytokines, which increase the synthesis of collagen. Deposition of collagen is enhanced by decreased activity of metalloproteinases. Figure 3-25 Repair responses after injury and inflammation. Repair after acute injury has several outcomes, including normal tissue restitution and healing with scar formation. Healing in chronic injury involves scar formation and fibrosis (see text). References 1. Goss RJ: Regeneration versus repair. In Cohen IK, Diegelman RF, Lindblad WJ (eds): Wound Healing. Biochemical and Clinical Aspects. Philadelphia, W.B. Saunders, 1992, pp 20–39. 2. Clark RAF: Wound repair. In Clark RAF (ed): The Molecular and Cellular Biology of Wound Repair, 2nd ed. New York, Plenum Press, 1996, pp 3–50. 3. 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However, the well-being of tissues also requires normal fluid balance; abnormalities in vascular permeability or hemostasis can result in injury even in the setting of an intact blood supply. This chapter will describe major disturbances involving hemodynamics and the maintenance of blood flow, including edema, hemorrhage, thrombosis, embolism, infarction, and shock. Normal fluid homeostasis encompasses maintenance of vessel wall integrity as well as intravascular pressure and osmolarity within certain physiologic ranges. Changes in vascular volume, pressure, or protein content, or alterations in endothelial function, all affect the net movement of water across the vascular wall. Such water extravasation into the interstitial spaces is called edema and has different manifestations depending on its location. In the lower extremities, edema mainly causes swelling; in the lungs, edema causes water to fill alveoli, leading to difficulty in breathing. Normal fluid homeostasis also means maintaining blood as a liquid until such time as injury necessitates clot formation. Clotting at inappropriate sites (thrombosis) or migration of clots (embolism) obstructs blood flow to tissues and leads to cell death (infarction). Conversely, inability to clot after vascular injury results in hemorrhage; local bleeding can compromise regional tissue perfusion, while more extensive hemorrhage can result in hypotension (shock) and death. Some of the failures of fluid homeostasis reflect a primary pathology in a discrete vascular bed (e.g., hemorrhage due to local trauma) or in systemic coagulation (thrombosis due to hypercoagulability disorders); others may represent a *The contributions of the late Dr. Ramzi Cotran to this chapter in previous editions are gratefully acknowledged. 120 secondary manifestation of some other disease process. Thus, pulmonary edema due to increased hydrostatic pressure may be a terminal complication of ischemic or valvular heart disease. Similarly, shock may be the fatal sequela of infection. Overall, disturbances in normal blood flow are major sources of human morbidity and mortality; thrombosis, embolism, and infarction underlie three of the most important causes of pathology in Western society—myocardial infarction, pulmonary embolism, and cerebrovascular accident (stroke). Thus, the hemodynamic disorders described in this chapter are important in a wide spectrum of human disease. Edema Approximately 60% of lean body weight is water; two thirds of this water is intracellular, and the remainder is found in the extracellular space, mostly as interstitial fluid (only about 5% of total body water is in blood plasma). The term edema signifies increased fluid in the interstitial tissue spaces. In addition, depending on the site, fluid collections in the different body cavities are variously designated hydrothorax, hydropericardium, and hydroperitoneum (the last is more commonly called ascites). Anasarca is a severe and generalized edema with profound subcutaneous tissue swelling. Table 4-1 lists the pathophysiologic categories of edema. The mechanisms of inflammatory edema are largely related to TABLE 4-1 -- Pathophysiologic Categories of Edema Increased Hydrostatic Pressure Impaired venous return ••Congestive heart failure ••Constrictive pericarditis ••Ascites (liver cirrhosis) ••Venous obstruction or compression ••••Thrombosis ••••External pressure (e.g., mass) ••••Lower extremity inactivity with prolonged dependency Arteriolar dilation ••Heat ••Neurohumoral dysregulation Reduced Plasma Osmotic Pressure (Hypoproteinemia) Protein-losing glomerulopathies (nephrotic syndrome) Liver cirrhosis (ascites) Malnutrition Protein-losing gastroenteropathy Lymphatic Obstruction Inflammatory Neoplastic Postsurgical Postirradiation Sodium Retention Excessive salt intake with renal insufficiency Increased tubular reabsorption of sodium Renal hypoperfusion Increased renin-angiotensin-aldosterone secretion Inflammation Acute inflammation Chronic inflammation Angiogenesis Modified from Leaf A, Cotran RS: Renal Pathophysiology, 3rd ed., New York, Oxford University Press, 1985, p 146. Used by permission of Oxford Press, Inc. local increases in vascular permeability and are discussed in Chapter 2 . The noninflammatory causes of edema are described in further detail below. Because of increased vascular permeability, inflammatory edema is a protein-rich exudate, with a specific gravity usually over 1.020. Conversely, the edema fluid occurring in hydrodynamic derangements is typically a protein-poor transudate, with a specific gravity below 1.012. In general, the opposing effects of vascular hydrostatic pressure and plasma colloid osmotic pressure are the major factors that govern movement of fluid between vascular and interstitial spaces. Normally the exit of fluid into the interstitium from the arteriolar end of the microcirculation is nearly balanced by inflow at the venular end; a small residuum of excess interstitial fluid is drained by the lymphatics. Either increased capillary pressure or diminished colloid osmotic pressure can result in increased interstitial fluid ( Fig. 4-1 ). As extravascular fluid accumulates, the increased tissue hydrostatic pressure and plasma colloid osmotic pressure eventually achieve a new equilibrium, and water reenters the venules. Any excess interstitial edema fluid is typically removed by lymphatic drainage, ultimately returning to the bloodstream via the thoracic duct (see Fig. 4-1 ); clearly, lymphatic obstruction (e.g., due to scarring or tumor) will also impair fluid drainage and result in edema. Finally, a primary retention of sodium (and its obligatory associated water) in renal disease also leads to edema. Increased Hydrostatic Pressure. Local increases in hydrostatic pressure may result from impaired venous outflow. For example, deep venous thrombosis in the lower extremities leads to edema, which is restricted to the affected leg. Generalized increases in venous pressure, with resulting systemic edema, occur most commonly in congestive heart failure ( Chapter 12 ) affecting right ventricular cardiac function. Figure 4-1 Factors affecting fluid balance across capillary walls. Capillary hydrostatic and osmotic forces are normally balanced so that there is no net loss or gain of fluid across the capillary bed. However, increased hydrostatic pressure or diminished plasma osmotic pressure leads to a net accumulation of extravascular fluid (edema). As the interstitial fluid pressure increases, tissue lymphatics remove much of the excess volume, eventually returning it to the circulation via the thoracic duct. If the ability of the lymphatics to drain tissue is exceeded, persistent tissue edema results. Figure 4-2 Sequence of events leading to systemic edema due to primary heart failure, primary renal failure, or reduced plasma osmotic pressure (as in malnutrition, diminished hepatic protein synthesis, or loss of protein owing to the nephrotic syndrome). ADH, antidiuretic hormone; GFR, glomerular filtration rate. Figure 4-3 Hyperemia versus congestion. In both cases there is an increased volume and pressure of blood in a given tissue with associated capillary dilation and a potential for fluid extravasation. In hyperemia, increased inflow leads to engorgement with oxygenated blood, resulting in erythema. In congestion, diminished outflow leads to a capillary bed swollen with deoxygenated venous blood and resulting in cyanosis. Figure 4-4 Liver with chronic passive congestion and hemorrhagic necrosis. A, Central areas are red and slightly depressed compared with the surrounding tan viable parenchyma, forming the so-called "nutmeg liver" pattern. B, Centrilobular necrosis with degenerating hepatocytes, hemorrhage, and sparse acute inflammation. (Courtesy of Dr. James Crawford, Department of Pathology, University of Florida, Gainesville, FL.) Figure 4-5 A, Punctate petechial hemorrhages of the colonic mucosa, seen here as a consequence of thrombocytopenia. B, Fatal intracerebral bleed. Even relatively inconsequential volumes of hemorrhage in a critical location, or into a closed space (such as the cranium), can have fatal outcomes. Figure 4-6 Diagrammatic representation of the normal hemostatic process. A, After vascular injury, local neurohumoral factors induce a transient vasoconstriction. B, Platelets adhere to exposed extracellular matrix (ECM) via von Willebrand factor (vWF) and are activated, undergoing a shape change and granule release; released adenosine diphosphate (ADP) and thromboxane A2 (TxA2 ) lead to further platelet aggregation to form the primary hemostatic plug. C, Local activation of the coagulation cascade (involving tissue factor and platelet phospholipids) results in fibrin polymerization, "cementing" the platelets into a definitive secondary hemostatic plug. D, Counter-regulatory mechanisms, such as release of tissue type plasminogen activator (t-PA) (fibrinolytic) and thrombomodulin (interfering with the coagulation cascade), limit the hemostatic process to the site of injury. Figure 4-7 Schematic illustration of some of the pro- and anticoagulant activities of endothelial cells. Not shown are the pro- and antifibrinolytic properties. vWF, von Willebrand factor; PGI2 , prostacyclin; NO, nitric oxide; t-PA, tissue plasminogen activator. Thrombin receptor is referred to as protease activated receptor (PAR; see text). Figure 4-8 Platelet adhesion and aggregation. von Willebrand factor functions as an adhesion bridge between subendothelial collagen and the GpIb platelet receptor complex (the functional complex is composed of GpIb in association with factors V and IX). Aggregation involves linking platelets via fibrinogen bridges bound to the platelet GpIIb-IIIa receptors. Figure 4-9 The coagulation cascade. Note the common link between the intrinsic and extrinsic pathways at the level of factor IX activation. Factors in red boxes represent inactive molecules; activated factors are indicated with a lower case "a" and a green box. PL, phospholipid surface; HMWK, high-molecular-weight kininogen. Not shown are the anticoagulant inhibitory pathways (see Fig. 4-7 and Fig. 4-12 ). Figure 4-10 Schematic illustration of the conversion of factor X to factor Xa, which in turn converts factor II (prothrombin) to factor IIa (thrombin). The initial reaction complex consists of an enzyme (factor IXa), a substrate (factor X), and a reaction accelerator (factor VIIIa), which are assembled on the phospholipid surface of platelets. Calcium ions hold the assembled components together and are essential for reaction. Activated factor Xa then becomes the enzyme part of the second adjacent complex in the coagulation cascade, converting the prothrombin substrate (II) to thrombin (IIa), with the cooperation of the reaction accelerator factor Va. (Modified from Mann KG: Clin Lab Med 4:217, 1984.) Figure 4-11 The central roles of thrombin in hemostasis and cellular activation. In addition to a critical function in generating cross-linked fibrin (via cleavage of fibrinogen to fibrin and activation of factor XIII), thrombin also directly induces platelet aggregation and secretion (e.g., of TxA2 ). Thrombin also activates endothelium to generate leukocyte adhesion molecules and a variety of fibrinolytic (t-PA), vasoactive (NO, PGI2 ), or cytokine (PDGF) mediators. Likewise, mononuclear inflammatory cells may be activated by the direct actions of thrombin. ECM, extracellular matrix; NO, nitric oxide; PDGF, platelet-derived growth factor; PGI2 , prostacyclin; TxA2 , thromboxane A2 ; t-PA, tissue type plasminogen activator. See Fig. 4-7 for additional anticoagulant modulators of thrombin activity, such as antithrombin III and thrombomodulin. (Modified with permission from Shaun Coughlin, MD, PhD, Cardiovascular Research Institute, University of California at San Francisco.) Figure 4-12 The fibrinolytic system, illustrating the plasminogen activators and inhibitors. Figure 4-13 Virchow triad in thrombosis. Endothelial integrity is the single most important factor. Note that injury to endothelial cells can affect local blood flow and/or coagulability; abnormal blood flow (stasis or turbulence) can, in turn, cause endothelial injury. The elements of the triad may act independently or may combine to cause thrombus formation. TABLE 4-2 -- Hypercoagulable States Primary (Genetic) Common ••Mutation in factor V gene (factor V Leiden) ••Mutation in prothrombin gene ••Mutation in methyltetrahydrofolate gene Rare ••Antithrombin III deficiency ••Protein C deficiency ••Protein S deficiency Very rare ••Fibrinolysis defects Secondary (Acquired) High risk for thrombosis ••Prolonged bed rest or immobilization ••Myocardial infarction ••Atrial fibrillation ••Tissue damage (surgery, fracture, burns) ••Cancer ••Prosthetic cardiac valves ••Disseminated intravascular coagulation ••Heparin-induced thrombocytopenia ••Antiphospholipid antibody syndrome (lupus anticoagulant syndrome) Lower risk for thrombosis ••Cardiomyopathy ••Nephrotic syndrome ••Hyperestrogenic states (pregnancy) ••Oral contraceptive use ••Sickle cell anemia ••Smoking to increased susceptibility to platelet aggregation and reduced PGI2 release by endothelium. Smoking and obesity promote hypercoagulability by unknown mechanisms. Among the acquired causes of thrombotic diatheses, the so-called heparin-induced thrombocytopenia syndrome and antiphospholipid antibody syndrome (previously called the lupus anticoagulant syndrome) deserve special mention. 33 34 Heparin-induced thrombocytopenia syndrome. [ ] [ ] Seen in upward of 5% of the population, this syndrome occurs when administration of unfractionated heparin (for purposes of therapeutic anticoagulation) induces formation of antibodies that bind to molecular complexes of heparin and platelet factor 4 membrane protein. This antibody can also bind to similar complexes present on platelet and endothelial surfaces; the result is platelet activation, endothelial injury, and a prothrombotic state. To reduce this problem, specially manufactured lowmolecular-weight heparin preparations—which retain anticoagulant activity but do not interact with platelets—are used. These have the additional benefit of a prolonged serum half-life. 35 36 Antiphospholipid antibody syndrome. [ ] [ ] This syndrome has protean clinical presentations, including multiple thromboses; the clinical manifestations are associated with high titers of circulating antibodies directed against anionic phospholipids (e.g., cardiolipin) or, more accurately, against plasma protein epitopes that are unveiled by binding to such phospholipids (e.g., prothrombin). Patients with anticardiolipin antibodies also have a false-positive serologic test for syphilis because the antigen in the standard tests is embedded in cardiolipin. In vitro these antibodies interfere with the assembly of phospholipid complexes and thus inhibit coagulation. However, in vivo, the antibodies induce a hypercoagulable state. Patients with antiphospholipid antibody syndrome fall into two categories. Many have a well-defined autoimmune disease, such as systemic lupus erythematosus ( Chapter 6 ) and have secondary antiphospholipid syndrome (such patients previously carried the designation of lupus anticoagulant syndrome). The remainder show no evidence of other autoimmune disorder and exhibit only the manifestations of a hypercoagulable state (primary antiphospholipid syndrome). Occasionally the syndrome can occur in association with certain drugs or infections. How antiphospholipid antibodies lead to hypercoagulability is not clear, but possible explanations include direct platelet activation, inhibition of PGI2 production by endothelial cells, or interference with protein C synthesis or activity. Although antiphospholipid antibodies are associated with thrombotic diatheses, they have also been identified in 5% to 15% of apparently normal individuals and may therefore be necessary but not sufficient to cause full-blown antiphospholipid antibody syndrome. Individuals with the antiphospholipid antibody syndrome present with an extreme variety of clinical manifestations; these are typically characterized by recurrent venous or arterial thrombi 37 but also include repeated miscarriages, cardiac valvular vegetations, or thrombocytopenia. [ ] Venous thromboses occur most commonly in deep leg veins, but renal, hepatic, and retinal veins are also susceptible. Arterial thromboses typically occur in the cerebral circulation, but coronary, mesenteric, and renal arterial occlusions have also been described. Depending on the vascular bed involved, the clinical presentations can vary from pulmonary embolism (due to a lower extremity venous thrombus), to pulmonary hypertension (from recurrent subclinical pulmonary emboli), to stroke, bowel infarction, or renovascular hypertension. Fetal loss is attributable to antibody-mediated inhibition of t-PA activity necessary for trophoblastic invasion of the uterus. Antiphospholipid antibody syndrome is also a cause of renal microangiopathy, resulting in renal failure owing to multiple capillary and arterial thromboses ( Chapter 20 ). Patients with antiphospholipid antibody syndrome are at increased risk of a fatal event (upward of 7% in one series of patients with lupus erythematosus, particularly with arterial thromboses or thrombocytopenia). Current treatment includes anticoagulation therapy (aspirin, heparin, and warfarin) and immunosuppression in refractory cases. [ 35] [37] [38] Morphology. Thrombi may develop anywhere in the cardiovascular system: within the cardiac chambers; on valve cusps; or in arteries, veins, or capillaries. They are of variable size and shape, depending on the site of origin and the circumstances leading to their development. Arterial or cardiac thrombi usually begin at a site of endothelial injury (e.g., atherosclerotic plaque) or turbulence (vessel bifurcation); venous thrombi characteristically occur in sites of stasis. An area of attachment to the underlying vessel or heart wall, frequently firmest at the point of origin, is characteristic of all thromboses. Arterial thrombi tend to grow in a retrograde direction from the point of attachment, whereas venous thrombi extend in the direction of blood flow (i.e., toward the heart). The propagating tail may not be well attached and, particularly in veins, is prone to fragmentation, creating an embolus. 133 When formed in the heart or aorta, thrombi may have grossly (and microscopically) apparent laminations, called lines of Zahn; these are produced by alternating pale layers of platelets admixed with some fibrin and darker layers containing more red cells. Lines of Zahn are significant only in that they imply thrombosis at a site of blood flow; in veins or in smaller arteries, the laminations are typically not as apparent, and, in fact, thrombi formed in the sluggish flow of venous blood usually resemble statically coagulated blood (similar to blood clotted in a test tube). Nevertheless, careful evaluation generally reveals irregular, somewhat ill-defined laminations. When arterial thrombi arise in heart chambers or in the aortic lumen, they usually adhere to the wall of the underlying structure and are termed mural thrombi. Abnormal myocardial contraction (arrhythmias, dilated cardiomyopathy, or myocardial infarction) leads to cardiac mural thrombi ( Fig. 4-14A ), while ulcerated atherosclerotic plaque and aneurysmal dilation are the precursors of aortic thrombus formation ( Fig. 4-14B ). Arterial thrombi are usually occlusive; the most common sites, in descending order, are coronary, cerebral, and femoral arteries. The thrombus is usually superimposed on an atherosclerotic plaque, although other forms of vascular injury (vasculitis, trauma) may be involved. The thrombi are typically firmly adherent to the injured arterial wall and are gray-white and friable, composed of a tangled mesh of platelets, fibrin, erythrocytes, and degenerating leukocytes. Venous thrombosis, or phlebothrombosis, is almost invariably occlusive; the thrombus often creates a long cast of the vein lumen. Because these thrombi form in a relatively static environment, they tend to contain more enmeshed erythrocytes and are therefore known as red, or stasis, thrombi. Phlebothrombosis most commonly affects the veins of the lower extremities (90% of cases). Less commonly, venous thrombi may develop in the upper extremities, periprostatic plexus, or the ovarian and periuterine veins; under special circumstances, they may be found in the dural sinuses, the portal vein, or the hepatic vein. At autopsy, postmortem clots may be Figure 4-14 Mural thrombi. A, Thrombus in the left and right ventricular apices, overlying a white fibrous scar. B, Laminated thrombus in a dilated abdominal aortic aneurysm. Figure 4-15 Potential outcomes of venous thrombosis. Figure 4-16 Low-power view of a thrombosed artery. A, H&E-stained section. B, Stain for elastic tissue. The original lumen is delineated by the internal elastic lamina (arrows) and is totally filled with organized thrombus, now punctuated by a number of small recanalized channels. Figure 4-17 Large embolus derived from a lower extremity deep venous thrombosis and now impacted in a pulmonary artery branch. Figure 4-18 Bone marrow embolus in the pulmonary circulation. The cleared vacuoles represent marrow fat that is now impacted in a distal vessel along with the cellular hematopoietic precursors. Figure 4-19 Examples of infarcts. A, Hemorrhagic, roughly wedge-shaped pulmonary infarct. B, Sharply demarcated white infarct in the spleen. Figure 4-20 Remote kidney infarct, now replaced by a large fibrotic cortical scar. TABLE 4-3 -- Three Major Types of Shock Type of Shock Clinical Examples Principal Mechanisms Cardiogenic Myocardial infarction Failure of myocardial pump owing to intrinsic myocardial damage, extrinsic pressure, or obstruction to outflow Ventricular rupture Arrhythmia Cardiac tamponade Pulmonary embolism Hypovolemic Hemorrhage Inadequate blood or plasma volume Fluid loss, e.g., vomiting, diarrhea, burns, or trauma Septic Overwhelming microbial infections Peripheral vasodilation and pooling of blood; endothelial activation/injury; leukocyteinduced damage; disseminated intravascular coagulation; activation of cytokine cascades Endotoxic shock Gram-positive septicemia Fungal sepsis Superantigens 53 estimated to account for over 200,000 deaths annually in the United States.[ ] Moreover, the reported incidence of sepsis syndromes has increased dramatically in the past two decades, owing to improved life support for high-risk patients, increasing use of invasive procedures, and growing numbers of immunocompromised hosts (secondary to chemotherapy, immunosuppression, or human immunodeficiency virus infection). Septic shock results from spread and expansion of an initially localized infection (e.g., abscess, peritonitis, pneumonia) into the bloodstream. Most cases of septic shock (approximately 70%) are caused by endotoxin-producing gram-negative bacilli ( Chapter 8 ), hence the term endotoxic shock. Endotoxins are bacterial wall lipopolysaccharides (LPSs) that are released when the cell walls are degraded (e.g., in an inflammatory response). LPS consists of a toxic fatty acid (lipid A) core and a complex polysaccharide coat (including O antigens) unique to each bacterial species. Analogous molecules in the walls of gram-positive bacteria and fungi can also elicit septic shock. All of the cellular and resultant hemodynamic effects of septic shock may be reproduced by injection of LPS alone. Free LPS attaches to a circulating LPS-binding protein, and the complex then binds to a cell-surface receptor (called CD14), followed by binding of the LPS to a signal-transducing protein called mammalian Toll-like receptor protein 4 (TLR-4). (Toll is a Drosophila protein involved in fly development; a variety of molecules with homology to Toll [i.e., "Toll-like"] participate in innate immune responses to different microbial components; see Box 6-1 , Chapter 6.) Signals from TLR-4 can then directly activate vascular wall cells and leukocytes or initiate a cascade of cytokine mediators, which propagates the pathologic state. [ 54] [55] Engagement of TLR-4 on endothelial cells can lead directly to down-regulation of natural anticoagulation mechanisms, including diminished synthesis of tissue factor pathway inhibitor (TFPI) and thrombomodulin. Engagement of the receptor on monocytes and macrophages (even at doses of LPS as minute as 10 picograms/ml) causes profound mononuclear cell activation with the subsequent production of potent effector cytokines such as IL-1 and TNF ( Chapter 6 ). Presumably, this series of responses helps to isolate organisms and to trigger elements of the innate immune system to efficiently eradicate invading microbes. Unfortunately, depending on the dosage and numbers of macrophages that are activated, the secondary effects of LPS release can also cause severe pathologic changes, including fatal shock. • At low doses, LPS predominantly serves to activate monocytes and macrophages, with effects intended to enhance their ability to eliminate invading bacteria. LPS can also directly activate complement, which likewise contributes to local bacterial eradication. The mononuclear phagocytes respond to LPS by producing cytokines, mainly TNF, IL-1, IL-6, and chemokines. TNF and IL-1 both act on endothelial cells to stimulate the expression of adhesion molecules ( Chapter 2 ; Fig. 4-21 ) and the production of other cytokines and chemokines. Thus, the initial release of LPS results in a circumscribed cytokine cascade doubtless intended to enhance the local acute inflammatory response and improve clearance of the infection. • With moderately severe infections, and therefore with higher levels of LPS (and a consequent augmentation of the cytokine cascade), cytokine-induced secondary effectors (e.g., nitric oxide; Chapter 2 ) become significant. In addition, systemic effects of the cytokines such as TNF and IL-1 may begin to be seen; these include fever and increased synthesis of acute phase reactants ( Chapter 2 ; Fig. 4-21 ). LPS at higher doses also results in diminished endothelial cell production of thrombomodulin and TFPI, tipping the coagulation cascade toward thrombosis. • Finally, at still higher levels of LPS, the syndrome of septic shock supervenes ( Fig. 4-22 ); the same cytokines and secondary mediators, now at high levels, result in: • Systemic vasodilation (hypotension) • Diminished myocardial contractility • Widespread endothelial injury and activation, causing systemic leukocyte adhesion and pulmonary alveolar capillary damage (acute respiratory distress syndrome; Chapter 15 ) • Activation of the coagulation system, culminating in DIC The hypoperfusion resulting from the combined effects of widespread vasodilation, myocardial pump failure, and DIC induces multiorgan system failure affecting the liver, kidneys, 141 Figure 4-21 Cytokine cascade in sepsis. After release of lipopolysaccharide (LPS) from invading gram-negative microorganisms, there are successive waves of tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 secretion. (Modified from Abbas AK, et al: Cellular and Molecular Immunology, 4th ed. Philadelphia, WB Saunders, 2000.) A progressive stage characterized by tissue hypoperfusion and onset of worsening circulatory and metabolic imbalances, including acidosis An irreversible stage that sets in after the body has incurred cellular and tissue injury so severe that even if the hemodynamic defects are corrected, survival is not possible. In the early nonprogressive phase of shock, a variety of neurohumoral mechanisms help maintain cardiac output and blood pressure. These include baroreceptor reflexes, release of catecholamines, activation of the renin-angiotensin axis, antidiuretic hormone release, and generalized sympathetic stimulation. The net effect is tachycardia, peripheral vasoconstriction, and renal conservation of fluid. Cutaneous vasoconstriction, for example, is responsible for the characteristic coolness and pallor of skin in well-developed shock (although septic shock may initially cause cutaneous vasodilation and thus present with warm, flushed skin). Coronary and cerebral vessels are less sensitive to this compensatory sympathetic response and thus maintain relatively normal caliber, blood flow, and oxygen delivery to their respective vital organs. If the underlying causes are not corrected, shock passes imperceptibly to the progressive phase, during which there is widespread tissue hypoxia. In the setting of persistent oxygen deficit, intracellular aerobic respiration is replaced by anaerobic glycolysis with excessive production of lactic acid. The resultant metabolic lactic acidosis lowers the tissue pH and blunts the vasomotor response; arterioles dilate, and blood begins to pool in the microcirculation. Peripheral pooling not only worsens the cardiac output, but also puts endothelial cells at risk for developing anoxic injury with subsequent DIC. With widespread tissue hypoxia, vital organs are affected and begin to fail; clinically the patient may become confused, and the urine output declines. Unless there is intervention, the process eventually enters an irreversible stage. Widespread cell injury is reflected in lysosomal enzyme leakage, further aggravating the shock state. Myocardial contractile function worsens in part because of nitric oxide synthesis. If ischemic bowel allows intestinal flora to enter the circulation, endotoxic shock may be superimposed. At this point, the patient has complete renal shutdown owing to acute tubular necrosis ( Chapter 20 ), and despite heroic measures, the downward clinical spiral almost inevitably culminates in death. Morphology. The cellular and tissue changes induced by shock are essentially those of hypoxic injury ( Chapter 1 ); since shock is characterized by failure of multiple organ systems, the cellular changes may appear in any tissue. Nevertheless, they are particularly evident in brain, heart, lungs, kidneys, adrenals, and gastrointestinal tract. The brain may develop so-called ischemic encephalopathy, discussed in Chapter 28 . The heart may undergo focal or widespread coagulation necrosis or 142 Figure 4-22 Effects of lipopolysaccharide (LPS) and secondarily induced effector molecules. LPS initiates the cytokine cascade described in Figure 4-21 ; in addition, LPS and the various factors can directly stimulate downstream cytokine production, as indicated. Secondary effectors that become important include nitric oxide (NO) and platelet-activating factor (PAF). At low levels, only local inflammatory effects are seen. With moderate levels, more systemic events occur in addition to the local vascular effects. At high concentrations, the syndrome of septic shock is seen. DIC, disseminated intravascular coagulation; ARDS, adult respiratory distress syndrome. (Modified from Abbas AK, et al: Cellular and Molecular Immunology, 4th ed. Philadelphia, WB Saunders, 2000.) References 1. Schrier R, Abraham W: Hormones and hemodynamics in heart failure. N Engl J Med 341:577, 1999. 2. Bick R, Murano G: Physiology of hemostasis. Clin Lab Med 14:677, 1994. 3. Hoffman M, Monroe DR: A cell-based model of hemostasis. Thromb Haemost 85:958, 2001. 4. Gross P, Aird W: The endothelium and thrombosis. Semin Thromb Hemost 26:463, 2000. 5. Pearson J: Endothelial cell function and thrombosis. Best Pract Res Clin Haematol 12:329, 1999. 6. 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Cur Opin Immunol 10:465, 1998. 145 Chapter 5 - Genetic Disorders 146 1 Genetic disorders are far more common than is widely appreciated. The lifetime frequency of genetic diseases is estimated to be 670 per 1000.[ ] Included in this figure are not only the "classic" genetic disorders but also cancer and cardiovascular diseases, the two most common causes of death in the Western world. Both of these have major genetic components. Cardiovascular diseases, such as atherosclerosis and hypertension, result from complex interactions of genes and environment, and most cancers are now known to result from an accumulation of mutations in somatic cells ( Chapter 7 ). The genetic diseases encountered in medical practice represent only the tip of the iceberg, that is, those with less extreme genotypic errors permitting full embryonic development and live birth. It is estimated that 50% of spontaneous abortuses during the early months of gestation have a demonstrable chromosomal abnormality; there are, in addition, numerous smaller detectable errors and many others still beyond our range of identification. About 1% of all newborn infants possess a gross chromosomal abnormality, and approximately 5% of individuals under age 25 develop a serious disease with a significant genetic component. How many more mutations remain hidden? 2 The draft sequence of the human genome is complete and much has been learned about the "genetic architecture" of humans. Some of what has been revealed was quite unexpected.[ ] For example, we now know that less than 2% of the human genome codes for proteins, whereas more than one half represents blocks of repetitive nucleotide codes whose functions remain mysterious. What was totally unexpected was that humans have a mere 30,000 genes rather than the 100,000 predicted only recently. Quite remarkably, this figure is not much greater than that of the mustard plant, with 26,000 genes! However, it is also known that by alternative splicing, 30,000 genes can give rise to greater than 100,000 proteins. In addition, very recent 2A studies indicate that fully formed proteins can be sliced and stitched together to give rise to peptides that could not have been predicted from the structure of the gene.[ ] Humans are not so poor, after all. With the completion of the human genome project, a new term, called genomics, has been added to the medical vocabulary. Whereas genetics is the study of single or a few genes and their phenotypic effects, genomics is the study of all the genes in the genome and their interactions. DNA microarray analysis of tumors ( Chapter 7 ) is an excellent example of 3 genomics in current clinical use.[ ] However, the most important contribution of genomics to human health will be in the unraveling of complex multifactorial diseases (discussed later) that arise from the interaction of multiple genes with environmental factors.[ 4] Another surprising revelation from the recent progress in genomics is that, on average, any two individuals share 99.9% of their DNA sequences. Thus, the remarkable diversity of humans is encoded in about 0.1% of our DNA. The secrets to disease predisposition and response to environmental agents and drugs must therefore reside within these variable regions. Although small as compared to the total nucleotide sequences, this 0.1% represents about 3 million base pairs. The most common form of DNA variations in the human genome is the single nucleotide polymorphism (SNP). Typically, the SNPs are biallelic (i.e., only two choices exist at a given site within the population), and they may occur anywhere in the genome—within exons, introns, or intergenic regions. Less than 1% of SNPs occur in coding regions. These could of course alter the gene product and give rise to a disease. Much more commonly, however, the SNP is just a marker that is co-inherited with a disease-causing gene, due to physical proximity. Another way of expressing this is to say that the SNP and the genetic factor are in linkage disequilibrium. 5 Much effort is ongoing to make SNP maps of the human genome so that we can decipher genetic determinants of disease.[ ] Just as genomics involves the study of all the DNA sequences, proteomics concerns itself with the measurement of all proteins expressed in a cell or tissue. Currently, progress in proteomics is lagging behind genomics, because the methodology to identify hundreds of distinct proteins simultaneously is not fully developed, but much effort continues. Although genomics and proteomics are revealing a treasure-trove of information, our ability to organize and mine such a vast array of data is not yet fully developed. To simultaneously analyze patterns of expression involving thousands of genes and proteins has required the parallel development of computer-based techniques that can manage vast collections of data. In response to this, an exciting new discipline called bioinformatics has sprouted. This has involved biologists, computer scientists, physicists, and mathematicians, a true example of a multidisciplinary approach in modern medical practice.[ 6] Much of the progress in medical genetics has resulted from the spectacular advances in molecular biology, involving recombinant DNA technology. The details of these techniques are well known and are not repeated here. Some examples, however, of the impact of recombinant DNA technology on medicine are worthy of attention. • Molecular basis of human disease: Two general strategies have been used to isolate and characterize involved genes ( Fig. 5-1 ). The functional cloning, or classic, approach has been successfully used to study a variety of inborn errors of metabolism, such as phenylketonuria and disorders of hemoglobin synthesis. Common to these genetic diseases is knowledge of the abnormal gene product and the corresponding protein. When the affected protein is known, a variety of methods can be employed to isolate the normal gene, to clone it, and ultimately to determine the molecular changes that affect the gene in patients with the disorder. Because in many common single-gene disorders, such as cystic fibrosis, there was no clue to the nature of the defective gene product, an alternative strategy called positional cloning, or the "candidate gene," approach had to be employed. This strategy initially ignores the biochemical clues from the phenotype and relies instead on mapping the disease phenotype to a particular chromosome location. This mapping is accomplished if the disease is associated with a distinctive cytogenetic change (e.g., fragile-X syndrome) or by linkage analysis. In the latter, the approximate location of the gene is determined by linkage to known "marker genes" or SNPs that are in close proximity to the disease locus. Once the region in which the mutant gene lies has been localized within reasonably narrow limits, the next step is to clone several pieces of DNA from the relevant segment of the genome. Expression of the cloned DNA in vitro, followed by identification of the protein products, can then be used to identify the aberrant protein encoded by the mutant genes. This approach has been used successfully in several diseases, such as cystic fibrosis, neurofibromatosis, Duchenne muscular dystrophy (a hereditary disorder characterized by progressive muscle weakness), polycystic 147 kidney disease, and Huntington disease. In addition to this step-by-step approach to cloning single genes, cDNA microarray analysis allows simultaneous detection of thousands of genes and their RNA products. When normal and diseased tissues are analyzed in this fashion, changes in the expression levels of multiple genes can be detected, thus providing a more comprehensive profile of genetic alterations in diseased tissues. • Production of human biologically active agents: An array of ultrapure biologically active agents can now be produced in virtually unlimited quantities by inserting the requisite gene into bacteria or other suitable cells in tissue culture. Some examples of genetically engineered products already in clinical use include soluble TNF receptor for blocking TNF in treatment of rheumatoid arthritis, tissue plasminogen activator for the treatment of thrombotic states, growth hormone for the treatment of deficiency states, erythropoietin to reverse several types of anemia, and myeloid growth and differentiation factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor) to enhance production of monocytes and neutrophils in states of poor marrow function. • Gene therapy: The goal of treating genetic diseases by transfer of somatic cells transfected with the normal gene, although simple in concept, has yet to succeed on a large scale. Problems include designing appropriate vectors to carry the gene and unexpected complications resulting from random insertion of the normal gene in the host genome. In recent wellpublicized cases, gene therapy in patients with x-linked SCID (severe combined immunodeficiency, Chapter 6 ) who lack the common γ chain of cytokine receptors had to be put on hold because the transduced gene inserted next to a host gene that controls proliferation of cells. The resulting dysregulation gave rise to T-cell leukemia in the patient. • Disease diagnosis: Molecular probes are proving to be extremely useful in the diagnosis of both genetic and non-genetic (e.g., infectious) diseases. The diagnostic applications of recombinant DNA technology are detailed at the end of this chapter. Figure 5-1 Schematic illustration of the strategies employed in functional and positional cloning. Functional cloning begins with relating the clinical phenotype to biochemical-protein abnormalities, followed by isolation of the mutant gene. Positional cloning, also called candidate gene approach, begins by mapping and cloning the disease gene by linkage analysis, without any knowledge of the gene product. Identification of the gene product and the mechanism by which it produces the disease follow the isolation of the mutant gene. Figure 5-2 Schematic illustration of a point mutation resulting from a single base pair change in the DNA. In the example shown, a CTC to CAC change alters the meaning of the genetic code (GAG to GUG in the opposite strand), leading to replacement of glutamic acid by valine in the polypeptide chain. This change, affecting the sixth amino acid of the normal β-globin (βA ) chain, converts it to sickle β-globin (βS ). Figure 5-3 Single-base deletion at the ABO (glycosyltransferase) locus, leading to a frameshift mutation responsible for the O allele. (From Thompson MW, et al: Thompson and Thompson Genetics in Medicine, 5th ed. Philadelphia, WB Saunders, 1991, p 134.) Figure 5-4 Four-base insertion in the hexosaminidase A gene in Tay-Sachs disease, leading to a frameshift mutation. This mutation is the major cause of Tay-Sachs disease in Ashkenazi Jews. (From Nussbaum, RL, et al: Thompson and Thompson Genetics in Medicine, 6th ed. Philadelphia, WB Saunders, 2001, p. 212.) Figure 5-5 Point mutation leading to premature chain termination. Partial mRNA sequence of the β-globin chain of hemoglobin showing codons for amino acids 38 to 40. A point mutation (C→U) in codon 39 changes glutamine (Gln) codon to a stop codon, and hence protein synthesis stops at the 38th amino acid. Figure 5-6 Three-base deletion in the common cystic fibrosis (CF) allele results in synthesis of a protein that is missing amino acid 508 (phenylalanine). Because the deletion is a multiple of three, this is not a frameshift mutation. (From Thompson MW, et al: Thompson and Thompson Genetics in Medicine, 5th ed. Philadelphia, WB Saunders, 1991, p. 135.) TABLE 5-1 -- Autosomal Dominant Disorders System Nervous Disorder Huntington disease Neurofibromatosis * Myotonic dystrophy Tuberous sclerosis Urinary Polycystic kidney disease Gastrointestinal Familial polyposis coli Hematopoietic Hereditary spherocytosis von Willebrand disease Skeletal Marfan syndrome * Ehlers-Danlos syndrome (some variants) * Osteogenesis imperfecta Achondroplasia Metabolic Familial hypercholesterolemia * Acute intermittent porphyria *Discussed in this chapter. Other disorders listed are discussed in appropriate chapters of this book. Autosomal recessive disorders include almost all inborn errors of metabolism. The various consequences of enzyme deficiencies are discussed later. The more common of these conditions are listed in Table 5-2 . Most are presented elsewhere; a few prototypes are discussed later in this chapter. TABLE 5-2 -- Autosomal Recessive Disorders System Metabolic Disorder Cystic fibrosis Phenylketonuria Galactosemia Homocystinuria Lysosomal storage diseases α1 -Antitrypsin deficiency * Wilson disease Hemochromatosis Glycogen storage diseases Hematopoietic * Sickle cell anemia Thalassemias Endocrine Congenital adrenal hyperplasia Skeletal Ehlers-Danlos syndrome (some variants) Alkaptonuria Nervous * * Neurogenic muscular atrophies Friedreich ataxia Spinal muscular atrophy *Discussed in this chapter. Many others are discussed elsewhere in the text. 152 X-Linked Disorders 9 All sex-linked disorders are X-linked, almost all X-linked recessive. Several genes are encoded in the "male-specific region of Y"; all of these are related to spermatogenesis.[ ] Males with mutations affecting the Y-linked genes are usually infertile, and hence there is no Y-linked inheritance. As discussed later, a few additional genes with homologues on the X chromosome have been mapped to the Y chromosome, but no disorders resulting from mutations in such genes have been described. X-linked recessive inheritance accounts for a small number of well-defined clinical conditions. The Y chromosome, for the most part, is not homologous to the X, and so mutant genes on the X are not paired with alleles on the Y. Thus, the male is said to be hemizygous for X-linked mutant genes, so these disorders are expressed in the male. Other features that characterize these disorders are as follows: • An affected male does not transmit the disorder to his sons, but all daughters are carriers. Sons of heterozygous women have, of course, one chance in two of receiving the mutant gene. • The heterozygous female usually does not express the full phenotypic change because of the paired normal allele. Because of the random inactivation of one of the X chromosomes in the female, however, females have a variable proportion of cells in which the mutant X chromosome is active. Thus, it is remotely possible for the normal allele to be inactivated in most cells, permitting full expression of heterozygous X-linked conditions in the female. Much more commonly, the normal allele is inactivated in only some of the cells, and thus the heterozygous female expresses the disorder partially. An illustrative condition is glucose-6-phosphate dehydrogenase (G6PD) deficiency. Transmitted on the X chromosome, this enzyme deficiency, which predisposes to red cell hemolysis in patients receiving certain types of drugs ( Chapter 13 ), is expressed principally in males. In the female, a proportion of the red cells may be derived from marrow cells with inactivation of the normal allele. Such red cells are at the same risk for undergoing hemolysis as are the red cells in the hemizygous male. Thus, the female is not only a carrier of this trait, but also is susceptible to drug-induced hemolytic reactions. Because the proportion of defective red cells in heterozygous females depends on the random inactivation of one of the X chromosomes, however, the severity of the hemolytic reaction is almost always less in heterozygous women than in hemizygous men. Most of the X-linked conditions listed in Table 5-3 are covered elsewhere in the text. TABLE 5-3 -- X-Linked Recessive Disorders System Disease Musculoskeletal Duchenne muscular dystrophy Blood Hemophilia A and B Chronic granulomatous disease Glucose-6-phosphate dehydrogenase deficiency Immune Agammaglobulinemia Wiskott-Aldrich syndrome Metabolic Diabetes insipidus Lesch-Nyhan syndrome Nervous Fragile-X syndrome * *Discussed in this chapter. Others discussed in appropriate chapters in the book. There are only a few X-linked dominant conditions. They are caused by dominant disease alleles on the X chromosome. These disorders are transmitted by an affected heterozygous female to half her sons and half her daughters and by an affected male parent to all his daughters but none of his sons, if the female parent is unaffected. Vitamin D-resistant rickets is an example of this type of inheritance. BIOCHEMICAL AND MOLECULAR BASIS OF SINGLE-GENE (MENDELIAN) DISORDERS Mendelian disorders result from alterations involving single genes. The genetic defect may lead to the formation of an abnormal protein or a reduction in the output of the gene product. As mentioned earlier, mutations may affect protein synthesis by affecting transcription, mRNA processing, or translation. The phenotypic effects of a mutation may result directly, from abnormalities in the protein encoded by the mutant gene, or indirectly, owing to interactions of the mutant protein with other normal proteins. For example, all forms of Ehlers-Danlos syndrome (EDS) are associated with abnormalities of collagen. In some forms (e.g., vascular type), there is a mutation in one of the collagen genes, whereas in others (e.g., kyphoscoliosis type), the collagen genes are normal, but there is a mutation in the gene that encodes lysyl hydroxylase, an enzyme essential for the cross-linking of collagen. In these patients, collagen weakness is secondary to a deficiency of lysyl hydroxylase. Virtually any type of protein may be affected in single-gene disorders and by a variety of mechanisms ( Table 5-4 ). To some extent, the pattern of inheritance of the disease is related to the kind of protein affected by the mutation, as was discussed earlier and is reiterated subsequently. For the purposes of this discussion, the mechanisms involved in single-gene disorders can be classified into four categories: (1) enzyme defects and their consequences; (2) defects in membrane receptors and transport systems; (3) alterations in the structure, function, or quantity of nonenzyme proteins; and (4) mutations resulting in unusual reactions to drugs. Enzyme Defects and Their Consequences Mutations may result in the synthesis of a defective enzyme with reduced activity or in a reduced amount of a normal enzyme. In either case, the consequence is a metabolic block. Figure 5-7 provides an example of an enzyme reaction in which the substrate is converted by intracellular enzymes, denoted as 1, 2, and 3, into an end product through intermediates 1 and 2. In this model, the final product exerts feedback control on enzyme 1. A minor pathway producing small quantities of M1 and M2 also exists. The biochemical consequences of an enzyme defect in such a reaction may lead to three major consequences: 1. Accumulation of the substrate, depending on the site of block, may be accompanied by accumulation of one or both intermediates. Moreover, an increased concentration of intermediate 2 may stimulate the minor pathway and thus 153 lead to an excess of M1and M2. Under these conditions, tissue injury may result if the precursor, the intermediates, or the products of alternative minor pathways are toxic in high concentrations. For example, in galactosemia, the deficiency of galactose-1-phosphate uridyltransferase ( Chapter 10) leads to the accumulation of galactose and consequent tissue damage. In phenylketonuria, a deficiency of phenylalanine hydroxylase ( Chapter 10) results in the accumulation of phenylalanine. Excessive accumulation of complex substrates within the lysosomes as a result of deficiency of degradative enzymes is responsible for a group of diseases generally referred to as lysosomal storage diseases. 2. An enzyme defect can lead to a metabolic block and a decreased amount of end product that may be necessary for normal function. For example, a deficiency of melanin may result from lack of tyrosinase, which is necessary for the biosynthesis of melanin from its precursor, tyrosine. This results in the clinical condition called albinism. If the end product is a feedback inhibitor of the enzymes involved in the early reactions (in Fig. 5-7 , it is shown that the product inhibits enzyme 1), the deficiency of the end product may permit overproduction of intermediates and their catabolic products, some of which may be injurious at high concentrations. A prime example of a disease with such an underlying mechanism is the Lesch-Nyhan syndrome ( Chapter 26 ). 3. Failure to inactivate a tissue-damaging substrate is best exemplified by α1 -antitrypsin (α1 -AT) deficiency. Patients who have an inherited deficiency of serum α1 -AT are unable to inactivate neutrophil elastase in their lungs. Unchecked activity of this protease leads to destruction of elastin in the walls of lung alveoli, leading eventually to pulmonary emphysema ( Chapter 15 ). TABLE 5-4 -- Biochemical and Molecular Basis of Some Mendelian Disorders Protein Type/Function Enzyme Example Molecular Lesion Disease Phenylalanine hydroxylase Splice site mutation: reduced amount Phenylketonuria Hexosaminidase Splice site mutation or frameshift mutation with stop codon: reduced amount Tay-Sachs disease Adenosine deaminase Point mutations: abnormal protein with reduced activity Severe combined immunodeficiency Enzyme Inhibitor α1 -Antitrypsin Missense mutations: impaired secretion from liver to serum Emphysema and liver disease Receptor Low-density lipoprotein receptor Deletions, point mutations: reduction of synthesis, transport to cell surface, or binding to low-density lipoprotein Familial hypercholesterolemia Vitamin D receptor Point mutations: failure of normal signaling Vitamin D-resistant rickets Transport Deletions: reduced amount α-Thalassemia Defective mRNA processing: reduced amount β-Thalassemia Point mutations: abnormal structure Sickle cell anemia Cystic fibrosis transmembrane conductance regulator Deletions and other mutations Cystic fibrosis Collagen Deletions or point mutations cause reduced amount of normal collagen or normal amounts of mutant collagen Osteogenesis imperfecta; Ehlers-Danlos syndromes Fibrillin Missense mutations Marfan syndrome Dystrophin Deletion with reduced synthesis Duchenne/Becker muscular dystrophy Spectrin, ankyrin, or protein 4.1 Heterogeneous Hereditary spherocytosis Hemostasis Factor VIII Deletions, insertions, nonsense mutations, and others: reduced synthesis or abnormal factor VIII Hemophilia A Growth Regulation Rb protein Deletions Hereditary retinoblastoma Neurofibromin Heterogeneous Neurofibromatosis type 1 Oxygen Ions Hemoglobin Structural Extracellular Cell membrane Figure 5-7 Scheme of a possible metabolic pathway in which a substrate is converted to an end product by a series of enzyme reactions. M1, M2, products of a minor pathway. TABLE 5-5 -- Classification of Ehlers-Danlos Syndromes (EDS) EDS Type * Clinical Findings Inheritance Gene Defects Classical (I/II) Skin and joint hypermobility, atrophic scars, easy bruising Autosomal dominant COL5A1, COL5A2 Hypermobility (III) Joint hypermobility, pain, dislocations Autosomal dominant Unknown Vascular (IV) Thin skin, arterial or uterine rupture, bruising, small joint hyperextensibility Autosomal dominant COL3A1 Kyphoscoliosis (VI) Hypotonia, joint laxity, congenital scoliosis, ocular fragility Autosomal recessive Lysyl-hydroxylase Arthrochalasia (VIIa,b) Severe joint hypermobility, skin changes mild, scoliosis, bruising Autosomal dominant COL1A1, COL1A2 Dermatosparaxsis (VIIc) Severe skin fragility, cutis laxa, bruising Autosomal recessive Procollagen N-peptidase *EDS were previously classified by Roman numerals. Parentheses show previous numerical equivalents. deficiency of lysyl hydroxylase results in the synthesis of collagen that lacks normal structural stability. The vascular type of EDS results from abnormalities of type III collagen. This form is genetically heterogeneous because at least three distinct types of mutations affecting the COL3A1 gene for collagen type III can give rise to this variant. Some affect the rate of synthesis of pro α1 (III) chains, others affect the secretion of type III procollagen, and still others lead to the synthesis of structurally abnormal type III collagen. Some mutant alleles behave as dominant negatives (see discussion under autosomal dominant disorders) and thus produce severe phenotypic effects. These molecular studies provide a rational basis for the pattern of transmission and clinical features that are characteristic of this variant. First, because vascular type EDS results from mutations involving a structural protein (rather than an enzyme protein), an autosomal dominant pattern of inheritance would be expected. Second, because blood vessels and intestines are known to be rich in collagen type III, an abnormality of this collagen is consistent with severe defects (e.g., spontaneous rupture) in these organs. In two forms of EDS—arthrochalasia type and dermatosparaxis type—the fundamental defect is in the conversion of type I procollagen to collagen. This step in collagen synthesis involves cleavage of noncollagen peptides at the N-terminal and C-terminal of the procollagen molecule. This is accomplished by N-terminal-specific and C-terminal-specific peptidases. The defect in the conversion of procollagen to collagen in the arthrocalasic type has been traced to mutations that affect one of the two type I collagen genes, COL1A1 and COL1A2. As a result, structurally abnormal pro α1 (I) or pro α2 (I) chains that resist cleavage of N-terminal peptides are formed. In patients with a single mutant allele, only 50% of the type I collagen chains are abnormal, but because these chains interfere with the formation of normal collagen helices, heterozygotes manifest the disease. By contrast, the related dermatosparaxis type is caused by mutations in the procollagen-N-peptidase genes, essential for the cleavage of collagens. In this case, the enzyme deficiency leads to an autosomal recessive form of inheritance. Finally, the classical type of EDS is worthy of brief mention, since molecular analysis of the variant suggests that genes other than collagen genes may be involved in the pathogenesis of EDS. In 30% to 50% of these cases, mutations in the genes for type V collagen (COL5A1 and COL5A1) have been detected. Surprisingly, despite a phenotype typical of EDS, no other collagen gene abnormalities have been found in these cases. This has led to the speculation that other proteins in the extracellular matrix, such as tenascin-X, may also be involved in regulating collagen synthesis. To summarize, the common thread in EDS is some abnormality of collagen. These disorders, however, are extremely heterogeneous. At the molecular level, a variety of defects, varying from mutations involving structural genes for collagen to those involving enzymes that are responsible for post-transcriptional modifications of mRNA, have been detected. Such molecular heterogeneity results in the expression of EDS as a clinically heterogeneous disorder with several patterns of inheritance. DISORDERS ASSOCIATED WITH DEFECTS IN RECEPTOR PROTEINS Familial Hypercholesterolemia Familial hypercholesterolemia is a "receptor disease" that is the consequence of a mutation in the gene encoding the receptor for low density lipoprotein (LDL), which is involved in the transport and metabolism of cholesterol. As a consequence of receptor abnormalities, there is a loss of feedback control and elevated levels of cholesterol that induce premature atherosclerosis, leading to a greatly increased risk of myocardial infarction.[ 19] [20] Familial hypercholesterolemia is possibly the most frequent mendelian disorder. Heterozygotes with one mutant gene, representing about 1 in 500 individuals, have from birth a twofold to threefold elevation of plasma cholesterol level, leading to tendinous xanthomas and premature atherosclerosis in adult life ( Chapter 11 ). Homozygotes, having a double dose of the mutant gene, are much more severely affected and may have fivefold to sixfold elevations in plasma cholesterol levels. These individuals develop skin xanthomas and coronary, cerebral, and peripheral vascular atherosclerosis at an early age. Myocardial infarction may develop before age 20. Large-scale studies have found that familial hypercholesterolemia is present in 3% to 6% of survivors of myocardial infarction. 157 An understanding of this disorder requires that we briefly review the normal process of cholesterol metabolism and transport. Approximately 7% of the body's cholesterol circulates in the plasma, predominantly in the form of LDL. As might be expected, the level of plasma cholesterol is influenced by its synthesis and catabolism and the liver plays a crucial role in both these processes ( Fig. 5-8 ). The first step in this complex sequence is the secretion of very-low-density lipoproteins (VLDL) by the liver into the bloodstream. VLDL particles are rich in triglycerides, although they do contain lesser amounts of cholesteryl esters. When a VLDL particle reaches the capillaries of adipose tissue or muscle, it is cleaved by lipoprotein lipase, a process that extracts most of the triglycerides. The resulting molecule, called intermediate-density lipoprotein (IDL), is reduced in triglyceride content and enriched in cholesteryl esters, but it retains two of the three apoproteins (B-100 and E) present in the parent VLDL particle (see Fig. 5-8 ). After release from the capillary endothelium, the IDL particles have one of two fates. Approximately 50% of newly formed IDL is rapidly taken up by the liver through a receptor-mediated transport. The receptor responsible for the binding of IDL to liver cell membrane recognizes both apoprotein B-100 and apoprotein E. It is called the LDL receptor, however, because it is also involved in the hepatic clearance of LDL, as described later. In the liver cells, Figure 5-8 Schematic illustration of low-density lipoprotein (LDL) metabolism and the role of the liver in its synthesis and clearance. Lipolysis of very-low-density lipoprotein (VLDL) by lipoprotein lipase in the capillaries releases triglycerides, which are then stored in fat cells and used as a source of energy in skeletal muscles. Figure 5-9 The LDL receptor pathway and regulation of cholesterol metabolism. Figure 5-10 Classification of LDL receptor mutations based on abnormal function of the mutant protein. These mutations disrupt the receptor's synthesis in the endoplasmic reticulum, transport to the Golgi complex, binding of apoprotein ligands, clustering in coated pits, and recycling in endosomes. Each class is heterogeneous at the DNA level. (Modified with permission from Hobbs HH, et al: The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 24:133–170, 1990. © 1990 by Annual Reviews.) Figure 5-11 Synthesis and intracellular transport of lysosomal enzymes. Figure 5-12 Schematic diagram illustrating the pathogenesis of lysosomal storage diseases. In the example shown, a complex substrate is normally degraded by a series of lysosomal enzymes (A, B, and C) into soluble end products. If there is a deficiency or malfunction of one of the enzymes (e.g., B), catabolism is incomplete and insoluble intermediates accumulate in the lysosomes. TABLE 5-6 -- Lysosomal Storage Diseases Disease Enzyme Deficiency Major Accumulating Metabolites Glycogenosis Type 2—Pompe disease α-1,4-Glucosidase (lysosomal glucosidase) Glycogen GM1 ganglioside β-galactosidase GM1 ganglioside, galactose-containing oligosaccharides Sphingolipidoses GM1 gangliosidosis ••Type 1—infantile, generalized ••Type 2—juvenile GM2 gangliosidosis ••Tay-Sachs disease Hexosaminidase-α subunit GM2 ganglioside ••Sandhoff disease Hexosaminidase-β subunit GM2 ganglioside, globoside ••GM2 gangliosidosis, variant AB Ganglioside activator protein GM2 ganglioside Metachromatic leukodystrophy Arylsulfatase A Sulfatide Multiple sulfatase deficiency Arylsulfatases A, B, C; steroid sulfatase; iduronate sulfatase; heparan N-sulfatase Sulfatide, steroid sulfate, heparan sulfate, dermatan sulfate Krabbe disease Galactosylceramidase Galactocerebroside Fabry disease α-Galactosidase A Ceramide trihexoside Gaucher disease Glucocerebrosidase Glucocerebroside Niemann-Pick disease: types A and B Sphingomyelinase Sphingomyelin MPS I H (Hurler) α-L-Iduronidase Dermatan sulfate, heparan sulfate MPS II (Hunter) L-Iduronosulfate sulfatase Sulfatidoses Mucopolysaccharidoses (MPS) Mucolipidoses (ML) I-cell disease (ML II) and pseudo-Hurler polydystrophy Deficiency of phosphorylating enzymes essential for the formation Mucopolysaccharide, glycolipid of mannose-6-phosphate recognition marker; acid hydrolases lacking the recognition marker cannot be targeted to the lysosomes but are secreted extracellularly Other Diseases of Complex Carbohydrates Fucosidosis α-Fucosidase Fucose-containing sphingolipids and glycoprotein fragments Mannosidosis α-Mannosidase Mannose-containing oligosaccharides Aspartylglycosaminuria Aspartylglycosamine amide hydrolase Aspartyl-2-deoxy-2-acetamido-glycosylamine Wolman disease Acid lipase Cholesterol esters, triglycerides Acid phosphate deficiency Lysosomal acid phosphatase Phosphate esters Other Lysosomal Storage Diseases of cytoplasmic inclusions can be visualized, the most prominent being whorled configurations within lysosomes composed of onion-skin layers of membranes ( Fig. 5-14B ). In time, there is progressive destruction of neurons, proliferation of microglia, and accumulation of complex lipids in phagocytes within the brain substance. A similar process occurs in the cerebellum as well as in neurons throughout the basal ganglia, brain stem, spinal cord, and dorsal root ganglia and in the neurons of the autonomic nervous system. The ganglion cells in the retina are similarly swollen with GM2 ganglioside, particularly at the margins of the macula. A cherry-red spot thus appears in the macula, representing accentuation of the normal color of the macular choroid contrasted with the pallor produced by the swollen ganglion cells in the remainder of the retina ( Chapter 29 ). This finding is characteristic of Tay-Sachs disease and other storage disorders affecting the neurons. Many alleles have been identified at the α-subunit locus, each associated with a variable degree of enzyme deficiency and hence with variable clinical manifestations. The affected infants appear normal at birth but begin to manifest signs and symptoms at about age 6 months. There is relentless motor and mental deterioration, beginning with motor incoordination, mental obtundation leading to muscular flaccidity, blindness, and increasing dementia. Sometime during the early course of the disease, the characteristic, but not pathognomonic, cherry-red spot appears in the macula of the eye grounds in almost all patients. Over the span of 1 or 2 years, a complete, pathetic vegetative state is reached, followed by death at age 2 to 3 years. Antenatal diagnosis and carrier detection are possible by enzyme assays and DNA-based analysis.[ 25] The clinical features of the two other forms of GM2 gangliosidosis (see Fig. 5-13 ), Sandhoff disease, resulting from β-subunit defect, and GM2 activator deficiency, are similar to those of Tay-Sachs disease. 162 Figure 5-13 The three-gene system required for hexosaminidase A activity and the diseases that result from defects in each of the genes. The function of the activator protein is to bind the ganglioside substrate and present it to the enzyme. (Modified from Sandhoff K, et al: The GM2 gangliosidoses. In Scriver CR, et al [eds]: The Metabolic Basis of Inherited Disease, 6th ed. New York, McGraw-Hill, 1989, p. 1824.) Figure 5-14 Ganglion cells in Tay-Sachs disease. A, Under the light microscope, a large neuron has obvious lipid vacuolation. (Courtesy of Dr. Arthur Weinberg, Department of Pathology, University of Texas Southwestern Medical Center, Dallas.) B, A portion of a neuron under the electron microscope shows prominent lysosomes with whorled configurations. Part of the nucleus is shown above. (Electron micrograph courtesy of Dr. Joe Rutledge, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-15 Niemann-Pick disease in liver. The hepatocytes and Kupffer cells have a foamy, vacuolated appearance owing to deposition of lipids. (Courtesy of Dr. Arthur Weinberg, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-16 Gaucher disease involving the bone marrow. A, Gaucher cells with abundant lipid-laden granular cytoplasm. B, Electron micrograph of Gaucher cells with elongated distended lysosomes. (Courtesy of Dr. Matthew Fries, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-17 Pathways of glycogen metabolism. Asterisks mark the enzyme deficiencies associated with glycogen storage diseases. Roman numerals indicate the type of glycogen storage disease associated with the given enzyme deficiency. Types V and VI result from deficiencies of muscle and liver phosphorylases, respectively. (Modified from Hers H, et al: Glycogen storage diseases. In Scriver CR, et al [eds]: The Metabolic Basis of Inherited Disease, 6th ed. New York, McGraw-Hill, 1989, p. 425.) Figure 5-18 Top, Simplified schema of normal glycogen metabolism in the liver and skeletal muscles. Middle, Effects of an inherited deficiency of hepatic enzymes involved in glycogen metabolism. Bottom, Consequences of a genetic deficiency in the enzymes that metabolize glycogen in skeletal muscles. Figure 5-19 Pompe disease (glycogen storage disease type II). A, Normal myocardium with abundant eosinophilic cytoplasm. B, Patient with Pompe disease (same magnification) showing the myocardial fibers full of glycogen seen as clear spaces. (Courtesy of Dr. Trace Worrell, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) TABLE 5-7 -- Principal Subgroups of Glycogenoses Clinicopathologic Category Hepatic Type Specific Type Hepatorenal—von Gierke disease (type I) Enzyme Deficiency Glucose-6-phosphatase Morphologic Changes Hepatomegaly—intracytoplasmic accumulations of glycogen and small amounts of lipid; intranuclear glycogen Renomegaly—intracytoplasmic accumulations of glycogen in cortical tubular epithelial cells Muscle phosphorylase Myopathic Type McArdle syndrome (type V) Miscellaneous Types Generalized glycogenosis Lysosomal glucosidase —Pompe disease (type II) (acid maltase) Clinical Features In untreated patients: failure to thrive, stunted growth, hepatomegaly, and renomegaly. Hypoglycemia due to failure of glucose mobilization, often leading to convulsions. Hyperlipidemia and hyperuricemia resulting from deranged glucose metabolism; many patients develop gout and skin xanthomas. Bleeding tendency due to platelet dysfunction. With treatment most survive and develop late complications, e.g., hepatic adenomas Skeletal muscle only—accumulations of glycogen predominant in subsarcolemmal location Painful cramps associated with strenuous exercise. Myoglobinuria occurs in 50% of cases. Onset in adulthood (>20 years). Muscular exercise fails to raise lactate level in venous blood. Serum creatine kinase always elevated. Compatible with normal longevity Mild hepatomegaly—ballooning of lysosomes with glycogen, creating lacy cytoplasmic pattern Massive cardiomegaly, muscle hypotonia, and cardiorespiratory failure within 2 years. A milder adult form with only skeletal muscle involvement, presenting Cardiomegaly—glycogen within sarcoplasm as well as membrane-bound with chronic myopathy Skeletal muscle—similar to changes in heart the urine if allowed to stand and undergo oxidation.[ by Garrod. 38] 39] The gene encoding homogentisic oxidase, mapped to 3q21, was cloned in 1996,[ 64 years after the initial description of the disease Morphology. The retained homogentisic acid selectively binds to collagen in connective tissues, tendons, and cartilage, imparting to these tissues a blue-black pigmentation (ochronosis) most evident in the ears, nose, and cheeks. The most serious consequences of ochronosis, however, stem from deposits of the pigment in the articular cartilages of the joints. In some obscure manner, 40 the pigmentation causes the cartilage to lose its normal resiliency and become brittle and fibrillated.[ ] Wear-and-tear erosion of this abnormal cartilage leads to denudation of the subchondral bone, and often tiny fragments of the fibrillated cartilage are driven into the underlying bone, worsening the damage. The vertebral column, particularly the intervertebral disc, is the prime site of attack, but later the knees, shoulders, and hips may be affected. The small joints of the hands and feet are usually spared. The metabolic defect is present from birth, but the degenerative arthropathy develops slowly and usually does not become clinically evident until the thirties. Although it is not lifethreatening, it may be severely crippling. The disability may be as extreme as that encountered in the severe forms of osteoarthritis ( Chapter 26 ) of the elderly, but in alkaptonuria the arthropathy occurs at a much earlier age. DISORDERS ASSOCIATED WITH DEFECTS IN PROTEINS THAT REGULATE CELL GROWTH Normal growth and differentiation of cells is regulated by two classes of genes: proto-oncogenes and tumor-suppressor genes, whose products promote or restrain cell growth ( Chapter 7 ). It is now well established that mutations in these two classes of genes are important in the pathogenesis of tumors. In the vast majority of cases, cancer-causing mutations affect somatic cells and hence are not passed in the germ line. In approximately 5% of all cancers, however, mutations transmitted through the germ line contribute to the development of cancer. Most familial cancers are inherited in an autosomal dominant fashion, but a few recessive disorders have also been described. This subject is discussed in greater detail in Chapter 7 . Here we provide an example of two common familial neoplasms. Neurofibromatosis: Types 1 and 2 Neurofibromatoses comprise two autosomal dominant disorders, affecting approximately 100,000 people in the United States. They are referred to as neurofibromatosis type 1 (previously called von Recklinghausen disease) and neurofibromatosis type 2 (previously called acoustic neurofibromatosis). Although there is some overlap in clinical features, these two entities are genetically distinct.[ 41] Neurofibromatosis type 1 is a relatively common disorder, with a frequency of almost 1 in 3000. Although approximately 50% of the patients have a definite family history consistent 169 with autosomal dominant transmission, the remainder appear to represent new mutations. In familial cases, the expressivity of the disorder is extremely variable, but the penetrance is 100%. Neurofibromatosis type 1 has three major features: (1) multiple neural tumors (neurofibromas) dispersed anywhere on or in the body; (2) numerous pigmented skin lesions, some of which are café au lait spots; and (3) pigmented iris hamartomas, also called Lisch nodules. A bewildering assortment of other abnormalities (cited later) may accompany these cardinal manifestations. Morphology. The neurofibromas arise within or are attached to nerve trunks anywhere in the skin, including the palms and soles, as well as in every conceivable internal site, including the cranial nerves. Three types of neurofibromas are found in individuals with neurofibromatosis type 1: cutaneous, subcutaneous, and plexiform. Cutaneous, or dermal, neurofibromas are soft, sessile, or pedunculated lesions that vary in number from a few to many hundreds. Subcutaneous neurofibromas grow just beneath the skin; they are firm, round masses that are often painful. The cutaneous and subcutaneous neurofibromas may be less than 1 cm in diameter; moderate-sized pedunculated lesions; or huge, multilobar pendulous masses, 20 cm or more in greatest diameter. The third variant, referred to as plexiform neurofibroma, diffusely involves subcutaneous tissue and contains numerous tortuous, thickened nerves; the overlying skin is frequently hyperpigmented. These may grow to massive proportions, causing striking enlargement of a limb or some other body part. Similar tumors may occur internally, and in general the deeply situated lesions tend to be large. Microscopically, neurofibromas reveal proliferation of all the elements in the peripheral nerve, including neurites, Schwann cells, and fibroblasts. Typically, these components are dispersed in a loose, disorderly pattern, often in a loose, myxoid stroma. Elongated, serpentine Schwann cells predominate, with their slender, spindle-shaped nuclei. The loose and disorderly architecture helps differentiate these neural tumors from schwannomas. The latter, composed entirely of Schwann cells, virtually never undergo malignant 42 transformation, whereas plexiform neurofibromas become malignant in about 5% of patients with neurofibromatosis type 1.[ ] Malignant transformation is most common in the large plexiform tumors attached to major nerve trunks of the neck or extremities. The superficial lesions, despite their size, rarely become malignant. The cutaneous pigmentations, the second major component of this syndrome, are present in more than 90% of patients. Most commonly, they appear as light brown café au lait macules, with generally smooth borders, often located over nerve trunks. They are usually round to ovoid, with their long axes parallel to the underlying cutaneous nerve. Although normal individuals may have a few café au lait spots, it is a clinical maxim that when six or more spots greater than 1.5 cm in diameter are present in an adult, the patient is likely to have neurofibromatosis type 1. Lisch nodules (pigmented hamartomas in the iris) are present in more than 94% of patients age 6 years or older. They do not produce any symptoms but are helpful in establishing the diagnosis. A wide range of associated abnormalities has been reported in these patients. Perhaps most common (seen in 30% to 50% of patients) are skeletal lesions, which take a variety of forms, including (1) erosive defects owing to contiguity of neurofibromas to bone, (2) scoliosis, (3) intraosseous cystic lesions, (4) subperiosteal bone cysts, and (5) pseudoarthrosis of the tibia. Patients with neurofibromatosis type 1 have a twofold to fourfold greater risk of developing other tumors, especially Wilms tumors, rhabdomyosarcomas, meningiomas, optic gliomas, and pheochromocytomas. Affected children are at increased risk of developing chronic myeloid leukemia. Although some patients with this condition have normal intelligence quotients (IQs), there is an unmistakable tendency for reduced intelligence. When neurofibromas arise within the gastrointestinal tract, intestinal obstruction or gastrointestinal bleeding may occur. Narrowing of a renal artery by a tumor may induce hypertension. Owing to variable expression of the gene, the range of clinical presentations is almost limitless, but ultimately the diagnosis rests on the concurrence of multiple café au lait spots and multiple skin tumors. The neurofibromatosis type 1 (NF-1) gene has been mapped to chromosome 17q11.2. It encodes a protein called neurofibromin, which down-regulates the function of the p21Ras oncoprotein (see section on oncogenes, Chapter 7 ). NF-1 therefore belongs to the family of tumor-suppressor genes. Neurofibromatosis type 2 is an autosomal dominant disorder in which patients develop a range of tumors, most commonly bilateral acoustic schwannomas and multiple meningiomas. Gliomas, typically ependymomas of the spinal cord, also occur in these patients. Many individuals with neurofibromatosis type 2 also have non-neoplastic lesions, which include nodular ingrowth of Schwann cells into the spinal cord (schwannosis), meningioangiomatosis (a proliferation of meningeal cells and blood vessels that grows into the brain), and glial hamartia (microscopic nodular collections of glial cells at abnormal locations, often in the superficial and deep layers of the cerebral cortex). Café au lait spots are present, but Lisch nodules in the iris are not found. This disorder is much less common than neurofibromatosis type 1, having a frequency of 1 in 40,000 to 50,000. The NF-2 gene, located on chromosome 22q12, is also a tumor-suppressor gene. As further discussed in Chapter 7 , the product of the NF-2 gene, called merlin, shows structural similarity to the ezrin, radixin, moesin (ERM) family of proteins. These cytoskeletal proteins interact with actin on the one hand and membrane proteins on the cell surface on the other hand. It is thought that merlin regulates contact inhibition and proliferation of Schwann cells.[ 43] Disorders with Multifactorial Inheritance As pointed out earlier, the multifactorial disorders result from the combined actions of environmental influences and two or more mutant genes having additive effects. The genetic component exerts a dosage effect—the greater the number of inherited deleterious genes, the more severe the expression of the disease. Because environmental factors significantly influence 170 the expression of these genetic disorders, the term polygenic inheritance should not be used. A number of normal phenotypic characteristics are governed by multifactorial inheritance, such as hair color, eye color, skin color, height, and intelligence. These characteristics exhibit a continuous variation in population groups, producing the standard bell-shaped curve of distribution. Environmental influences, however, significantly modify the phenotypic expression of multifactorial traits. For example, type II diabetes mellitus has many of the features of a multifactorial disorder. It is well recognized clinically that individuals often first manifest this disease after weight gain. Thus, obesity as well as other environmental influences unmasks the diabetic genetic trait. Nutritional influences may cause even monozygous twins to achieve different heights. The culturally deprived child cannot achieve his or her full intellectual capacity. The following features characterize multifactorial inheritance. These have been established for the multifactorial inheritance of congenital malformations and, in all likelihood, obtain for other multifactorial diseases.[ 44] • The risk of expressing a multifactorial disorder is conditioned by the number of mutant genes inherited. Thus, the risk is greater in siblings of patients having severe expressions of the disorder. For example, the risk of cleft lip in the siblings of an index case is 2.5% if the cleft lip is unilateral but 6% if it is bilateral. Similarly, the greater the number of affected relatives, the higher is the risk for other relatives. • The rate of recurrence of the disorder (in the range of 2% to 7%) is the same for all first-degree relatives (i.e., parents, siblings, and offspring) of the affected individual. Thus, if parents have had one affected child, the risk that the next child will be affected is between 2% and 7%. Similarly, there is the same chance that one of the parents will be affected. • The likelihood that both identical twins will be affected is significantly less than 100% but is much greater than the chance that both nonidentical twins will be affected. Experience has proven, for example, that the frequency of concordance for identical twins is in the range of 20% to 40%. • The risk of recurrence of the phenotypic abnormality in subsequent pregnancies depends on the outcome in previous pregnancies. When one child is affected, there is up to a 7% chance that the next child will be affected, but after two affected siblings, the risk rises to about 9%. • Expression of a multifactorial trait may be continuous (lack a distinct phenotype, e.g., height) or discontinuous (with a distinct phenotype, e.g., diabetes mellitus). In the latter, disease is expressed only when the combined influences of the genes and environment cross a certain threshold. In the case of diabetes, for example, the risk of phenotypic expression increases when the blood glucose levels go above a certain level. Assigning a disease to this mode of inheritance must be done with caution. It depends on many factors but first on familial clustering and the exclusion of mendelian and chromosomal modes of transmission. A range of levels of severity of a disease is suggestive of multifactorial inheritance, but, as pointed out earlier, variable expressivity and reduced penetrance of single mutant genes may also account for this TABLE 5-8 -- Multifactorial Disorders Disorder Chapter Cleft lip or cleft palate (or both) Chapter 10 Congenital heart disease Chapter 12 Coronary heart disease Chapter 12 Hypertension Chapter 11 Gout Chapter 27 Diabetes mellitus Chapter 24 Pyloric stenosis Chapter 17 phenomenon. Because of these problems, sometimes it is difficult to distinguish between mendelian and multifactorial inheritance. In contrast to the mendelian disorders, many of which are uncommon, the multifactorial group includes some of the most common ailments to which humans are heir ( Table 5-8 ). Most of these disorders are described in appropriate chapters elsewhere in this book. Normal Karyotype As is well known, human somatic cells contain 46 chromosomes; these comprise 22 homologous pairs of autosomes and two sex chromosomes, XX in the female and XY in the male. The study of chromosomes—karyotyping—is the basic tool of the cytogeneticist. The usual procedure of producing a chromosome spread is to arrest mitosis in dividing cells in metaphase by the use of mitotic spindle inhibitors (e.g., colcemid) and then to stain the chromosomes. In a metaphase spread, the individual chromosomes take the form of two chromatids connected at the centromere. A karyotype is a standard arrangement of a photographed or imaged stained metaphase spread in which chromosome pairs are arranged in order of decreasing length. A variety of staining methods that allow identification of each individual chromosome on the basis of a distinctive and reliable pattern of alternating light and dark bands along the length of the chromosome have been developed. The one most commonly employed uses a Giemsa stain and is hence called G banding. A normal male karyotype with G banding is illustrated in Figure 5-20 . With G banding, approximately 400 to 800 bands per haploid set can be detected. The resolution obtained by banding techniques can be dramatically improved by obtaining the cells in prophase. The individual chromosomes appear markedly elongated, and up to 1500 bands per karyotype may be recognized. The use of these banding techniques permits certain identification of each chromosome as well as delineation of precise breakpoints and other subtle alterations, to be described later. Before this discussion of the normal karyotype is concluded, reference must be made to commonly used cytogenetic terminology. Karyotypes are usually described using a shorthand system of notations. The following order is used: Total number of chromosomes is given first, followed by the sex chromosome complement, and finally the description of abnormalities in ascending numerical order. For example, a 171 Figure 5-20 Normal male karyotype with G banding. (Courtesy of Dr. Nancy Schneider, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-21 Details of banding pattern of the X chromosome (also called "idiogram"). Note the nomenclature of arms, regions, bands, and sub-bands. On the right side, the approximate locations of some genes that cause disease are indicated. Figure 5-22 Fluorescence in situ hybridization (FISH). Interphase nuclei of a childhood hepatic cancer (hepatoblastoma) stained with a fluorescent DNA probe that hybridizes to chromosome 20. Under ultraviolet light, each nucleus reveals three bright yellow fluorescent dots, representing three copies of chromosome 20. Normal diploid cells (not shown) have two fluorescent dots. (Courtesy of Dr. Vijay Tonk, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-23 FISH. A metaphase spread in which two fluorescent probes, one for the terminal ends of chromosome 22 and the other for the D22S75 locus, which maps to chromosome 22, have been used. The terminal ends of the two chromosomes 22 have been labeled. One of the two chromosomes does not stain with the probe for the D22S75 locus, indicating a microdeletion in this region. This deletion gives rise to the 22q11.2 deletion syndrome. (Courtesy of Dr. Nancy Schneider, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-24 Chromosome painting with a library of chromosome 22-specific DNA probes. The presence of three fluorescent chromosomes indicates that the patient has trisomy 22. (Courtesy of Dr. Charleen M. Moore, The University of Texas Health Science Center at San Antonio, TX.) Figure 5-25 Spectral karyotype. (Courtesy of Dr. Janet D. Rowley, University of Chicago Pritzker Medical School, Chicago, IL.) Figure 5-26 Types of chromosomal rearrangements. Figure 5-27 G banded karyotype of a male with trisomy 21. (Courtesy of Dr. Nancy Schneider, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-28 Clinical features and karyotypes of selected autosomal trisomies. Figure 5-29 Clinical features and karyotypes of Turner syndrome. Figure 5-30 Turner syndrome critical regions and (candidate) genes. SHOX, short homeobox gene; EIF1AX, eukaryotic initiation factor 1A; ZFX, zinc finger X (transcription factor); USP9X, homologue of Drosophila gene involved in öogenesis; DBX, dead box polypeptide 3,X, a spermatogenesis gene; UTX, ubiquitously transcribed tetratricopeptide repeat gene, X chromosome; SMCX, homologue of the Y-encoded male antigen HY; RPS4X isoform of ribosomal protein S4 involved in lymphatic development. (Courtesy of Dr. Andrew Zinn, University of Texas Southwestern Medical School, Dallas, TX.) Figure 5-31 Fragile-X, seen as discontinuity of staining. (Courtesy of Dr. Patricia Howard-Peebles, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-32 Fragile-X pedigree. Note that in the first generation all sons are normal and all females are carriers. During oogenesis in the carrier female, premutation expands to full mutation; hence in the next generation, all males who inherit the X with full mutation are affected. However, only 50% of females who inherit the full mutation are affected, and only mildly. (Courtesy of Dr. Nancy Schneider, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 5-33 A model for the action of familial mental retardation protein (FMRP) in neurons. (Adapted from Hin P, Warren ST: New insights into fragile-X syndrome: from molecules to neurobehavior. Trends Biochem Sci 28:152, 2003.) TABLE 5-9 -- Summary of Trinucleotide Repeat Disorders Disease Gene Locus Protein Repeat Normal Disease Expansions Affecting Noncoding Regions Fragile-X syndrome FMRI(FRAXA) Xq27.3 FMR-1 protein (FMRP) CGG 6–53 60–200 (pre) >230 (full) Friedreich ataxia X25 9q13-21.1 Frataxin GAA 7–34 34–80 (pre) >100 (full) Myotonic dystrophy DMPK 19q13 Myotonic dystrophy protein kinase (DMPK) CTG 5–37 50-thousands Spinobulbar muscular atrophy (Kennedy disease) AR Xq13-21 Androgen receptor (AR) CAG 9–36 38–62 Huntington disease HD 4p16.3 Huntingtin CAG 6–35 36–121 Dentatorubral-pallidoluysian atrophy (Haw River syndrome) DRPLA 12p13.31 Atrophin-1 CAG 6–35 49–88 Spinocerebellar ataxia type 1 SCA1 6p23 Ataxin-1 CAG 6–44 39–82 Spinocerebellar ataxia type 2 SCA2 12q24.1 Ataxin-2 CAG 15–31 36–63 Spinocerebellar ataxia type 3 (Machado-Joseph disease) SCA3 (MJD1) 14q32.1 Ataxin-3 CAG 12–40 55–84 Spinocerebellar ataxia type 6 SCA6 19p13 α1A -Voltage-dependent calcium channel CAG 4–18 21–33 CAG 4–35 37–306 Expansions Affecting Coding Regions subunit Spinocerebellar ataxia type 7 SCA7 3p12-13 Ataxin-7 185 Figure 5-34 Sites of expansion and the affected sequence in selected diseases caused by nucleotide repeat mutations. UTR, untranslated region. *Although not strictly a trinucleotide repeat disease, progressive myoclonus epilepsy is caused, like others in this group, by a heritable DNA expansion. The expanded segment is in the promoter region of the gene. Figure 5-35 Pedigree of Leber hereditary optic neuropathy, a disorder caused by mutation in mitochondrial DNA. Note that all progeny of an affected male are normal, but all children, male and female, of the affected female manifest disease. Figure 5-36 Diagrammatic representation of Prader-Willi and Angelman syndromes. Figure 5-37 Direct gene diagnosis: detection of coagulation factor V mutation by polymerase chain reaction (PCR) analysis. A G→A substitution in an exon destroys one of the two Mnl1 restriction sites. The mutant allele therefore gives rise to two, rather than three, fragments by PCR analysis. Figure 5-38 Diagnostic application of PCR and Southern blot analysis in fragile-X syndrome. With PCR, the differences in the size of CGG repeat between normal and premutation give rise to products of different sizes and mobility. With a full mutation, the region between the primers is too large to be amplified by conventional PCR. In Southern blot analysis the DNA is cut by enzymes that flank the CGG repeat region, and is then probed with a complementary DNA that binds to the affected part of the gene. A single small band is seen in normal males, a highermolecular-weight band in males with premutation, and a very large (usually diffuse) band in those with the full mutation. With this background, we can discuss how RFLPs can be used in gene tracking. Figure 5-39 illustrates the principle of RFLP analysis. In this example of an autosomal recessive disease, both of the parents are heterozygote carriers and the children are normal, are carriers, or are affected. In the illustrated example, the normal chromosome (A) has two restriction sites, 7.6 kb apart, whereas chromosome B, which carries the mutant gene, has a DNA sequence polymorphism resulting in the creation of an additional (third) restriction site for the same enzyme. Note that the additional restriction site has not resulted from the mutation but from a naturally occurring polymorphism. When DNA from such an individual is digested with the appropriate restriction enzyme and probed with a cloned DNA fragment that hybridizes with a stretch of sequences between 190 the restriction sites, the normal chromosome yields a 7.6 kb band, whereas the other chromosome (carrying the mutant gene) produces a smaller, 6.8 kb, band. Thus, on Southern blot analysis, two bands are noted. It is possible by this technique to distinguish family members who have inherited both normal chromosomes from those who are heterozygous or homozygous for the mutant gene. PCR followed by digestion with the appropriate restriction enzyme and gel electrophoresis can also be used to detect RFLPs if the target DNA is of the size that can be amplified by conventional PCR. • Length polymorphisms: Human DNA contains short repetitive sequences of noncoding DNA. Because the number of repeats affecting such sequences varies greatly between different individuals, the resulting length polymorphisms are quite useful for linkage analysis. These polymorphisms are often subdivided on the basis of their length into microsatellite repeats and minisatellite repeats. Microsatellites are usually less than 1 kb and are characterized by a repeat size of 2 to 6 base pairs. Minisatellite repeats, by comparison, are larger (1 to 3 kb), and the repeat motif is usually 15 to 70 base pairs. It is important to note that the number of repeats, both in microsatellites and minisatellites, is extremely variable within a given population, and hence these stretches of DNA can be used quite effectively to distinguish different chromosomes ( Fig. 5-40A ). Figure 5-40B illustrates how microsatellite polymorphisms can be used to track the inheritance of autosomal dominant polycystic kidney disease (PKD). In this case, allele C, which produces a larger PCR product than allele A or B, carries the diseaserelated gene. Hence all individuals who carry the C allele are affected. Microsatellites have assumed great importance in linkage studies and hence in the development of the human genome map. Currently, linkage to all human chromosomes can be identified by microsatellite polymorphisms.[ 86] Figure 5-39 Schematic illustration of the principles underlying restriction fragment length polymorphism analysis in the diagnosis of genetic diseases. Figure 5-40 Schematic diagram of DNA polymorphisms resulting from a variable number of CA repeats. The three alleles produce PCR products of different sizes, thus identifying their origins from specific chromosomes. In the example depicted, allele C is linked to a mutation responsible for autosomal dominant polycystic kidney disease (PKD). Application of this to detect progeny carrying the disease gene is illustrated in one hypothetical pedigree. References 1. Rimoin DL, et al: Nature and frequency of genetic disease. In Rimoin DL, et al (eds): Emery and Rimoin's Principles and Practice of Medical Genetics, 3rd ed. New York, Churchill Livingstone, 1997, p 32. 2. Guttmacher AE, Collins FS: Genomic medicine—a primer. N Engl J Med 347:1512, 2002. 2A. Rammensee H-G: Protein surgery. Science 427:203, 2004. 3. Joos L, Eryuksel E, Brutsche MH: Functional genomics and gene microarrays—the use in research and clinical medicine. Swiss Med Wkly 133:31, 2003. 4. 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N Engl J Med 349:562, 2003. 86. Koreth J, et al: Microsatellites and PCR genomic analysis. J Pathol 178:239, 1996. 193 Chapter 6 - Diseases of Immunity Abul K. Abbas MD 194 General Features of the Immune System Although vital to survival, the immune system is similar to the proverbial two-edged sword. On the one hand, immunodeficiency states render humans easy prey to infections and possibly tumors; on the other hand, a hyperactive immune system may cause fatal disease, as in the case of an overwhelming allergic reaction to the sting of a bee. In yet another series of derangements, the immune system may lose its normal capacity to distinguish self from non-self, resulting in immune reactions against one's own tissues and cells (autoimmunity). This chapter considers diseases caused by too little immunity as well as those resulting from too much immunologic reactivity. We also consider amyloidosis, a disease in which an abnormal protein, derived in some cases from fragments of immunoglobulins, is deposited in tissues. First, we review some advances in the understanding of innate and adaptive immunity and lymphocyte biology, then give a brief description of the histocompatibility genes because their products are relevant to several immunologically mediated diseases and to the rejection of transplants. INNATE AND ADAPTIVE IMMUNITY The physiologic function of the immune system is to protect individuals from infectious pathogens. The mechanisms that are responsible for this protection fall into two broad categories ( Fig. 6-1 ). Innate immunity (also called natural, or native, immunity) refers to defense mechanisms that are present even before infection and have evolved to specifically recognize microbes and protect multicellular organisms against infections. Adaptive immunity (also called acquired, or specific, immunity) consists of mechanisms that are stimulated by (adapt to) microbes and are capable of also recognizing nonmicrobial substances, called antigens. Innate immunity is the first line of defense, because it is always ready Figure 6-1 Innate and adaptive immunity. The principal mechanisms of innate immunity and adaptive immunity are shown. Box 6-1. Toll-like Receptors The Toll-like receptors (TLRs) are membrane proteins that recognize a variety of microbe-derived molecules and stimulate innate immune responses against the microbes. The first protein to be identified in this family was the Drosophila Toll protein, which is involved in establishing the dorsal-ventral axis during embryogenesis of the fly, as well as mediating antimicrobial responses. Ten different mammalian TLRs have been identified based on sequence homology to Drosophila Toll, and they are named TLR1-10. All these receptors contain leucine-rich repeats flanked by characteristic cysteine-rich motifs in their extracellular regions, and a conserved signaling domain in their cytoplasmic region that is also found in the cytoplasmic tails of the IL-1 and IL-18 receptors and is called the Toll/IL-1 receptor (TIR) domain. The TLRs are expressed on many different cell types that participate in innate immune responses, including macrophages, dendritic cells, neutrophils, NK cells, mucosal epithelial cells, and endothelial cells. Mammalian TLRs are involved in responses to widely divergent types of molecules that are commonly expressed by microbial but not mammalian cells (see Figure ). Some of the microbial products that stimulate TLRs include Gram-negative bacterial lipopolysaccharide (LPS), Gram-positive bacterial peptidoglycan, bacterial lipoproteins, the bacterial flagellar protein flagellin, heat shock protein 60, unmethylated CpG DNA motifs (found in many bacteria), and double-stranded RNA (found in RNA viruses). The specificity of TLRs for microbial products is dependent on associations between different TLRs and non-TLR adapter molecules. For instance, LPS first binds to soluble LPS-binding protein (LBP) in the blood or extracellular fluid, and this complex serves to facilitate LPS binding to CD14, which exists as both a soluble plasma protein and a glycophosphatidylinositol-linked membrane protein on most cells. Once LPS binds to CD14, LBP dissociates, and the LPS-CD14 complex physically associates with TLR4. An additional extracellular accessory protein, called MD2, also binds to the complex with CD14. LPS, CD14, and MD2 are all required for efficient LPS-induced signaling, but it is not yet clear if direct physical interaction of LPS with TLR4 is necessary. Signaling by TLRs results in the activation of transcription factors, notably NF-κB (see Figure). Ligand binding to the TLR at the cell surface leads to recruitment of cytoplasmic signaling molecules, the first of which is the adapter protein MyD88. A kinase called IL-1 receptor associated kinase (IRAK) is recruited into the signaling complex. IRAK undergoes autophosphorylation, dissociates from MyD88, and activates another signaling molecule, called TNF-receptor (TNF-R) associated factor-6 (TRAF-6). TRAF-6 then activates the I-κB kinase cascade, leading to activation of the NF-κB transcription factor. In some cell types certain TLRs also engage other signaling pathways, such as the MAP kinase cascade, leading to activation of the AP-1 transcription factor. Some TLRs may use adapter proteins other than MyD88. The relative importance of these various pathways of TLR signaling, and the way the "choice" of pathways is made, are not well understood. The genes that are expressed in response to TLR signaling encode proteins important in many different components of innate immune responses. These include inflammatory cytokines (TNF, IL-1, and IL-12), endothelial adhesion molecules (E-selectin), and proteins involved in microbial killing mechanisms (inducible nitric oxide synthase). The particular genes expressed will depend on the responding cell type. Figure 6- A, Different TLRs are involved in responses to different microbial products. B, Signaling by a prototypic TLR, TLR4, in response to bacterial LPS. An adapter protein links the TLR to a kinase, which activates transcription factors such as NF-κB and AP-1. TIR, Toll/IL-1 receptor domain. 196 of innate immunity, providing protection against inhaled microbes. The adaptive immune system consists of lymphocytes and their products, including antibodies. The receptors of lymphocytes are much more diverse than those of the innate immune system, but lymphocytes are not inherently specific for microbes, and they are capable of recognizing a vast array of foreign substances. In the remainder of this introductory section we focus on lymphocytes and the reactions of the adaptive immune system. CELLS AND TISSUES OF THE IMMUNE SYSTEM There are two main types of adaptive immunity—cell-mediated (or cellular) immunity, which is responsible for defense against intracellular microbes, and humoral immunity, which protects against extracellular microbes and their toxins ( Fig. 6-2 ). Cellular immunity is mediated by T (thymus-derived) lymphocytes, and humoral immunity is mediated by B (bone marrow-derived) lymphocytes and their secreted products, Figure 6-2 Humoral and cell-mediated immunity. Figure 6-3 Histology of a lymph node. A, The organization of the lymph node, with an outer cortex containing follicles and an inner medulla. B, The location of B cells (stained green, using the immunofluorescence technique) and T cells (stained red) in a lymph node. C, A germinal center. Figure 6-4 The T-cell receptor (TCR) complex. A, Schematic illustration of TCRα and TCRβ chains linked to the CD3 complex. B, Recognition of MHC-associated peptide displayed on an antigen-presenting cell (top) by the TCR. Note that the TCR-associated ζ chains and CD3 complex deliver signals (signal 1) upon antigen recognition, and CD28 delivers signals (signal 2) upon recognition of costimulators (B7 molecules). Figure 6-5 Structure of antibodies and the B-cell antigen receptor. A, The B-cell receptor complex composed of membrane IgM (or IgD, not shown) and the associated signaling proteins Igα and Igβ. CD21 is a receptor for a complement component that also promotes B-cell activation. B, Crystal structure of a secreted IgG molecule, showing the arrangement of the variable (V) and constant (C) regions of the heavy (H) and light (L) chains. (Courtesy of Dr. Alex McPherson, University of California, Irvine, CA.) Figure 6-6 The morphology and functions of dendritic cells (DC). A, The morphology of cultured dendritic cells. (Courtesy of Dr. Y-J. Liu, M. D. Anderson Cancer Center, Houston.) B, The location of dendritic cells (Langerhans cells) in the epidermis. (Courtesy of Dr. Y-J. Liu, M. D. Anderson Cancer Center, Houston.) C, The role of dendritic cells in capturing microbial antigens from epithelia and transporting them to regional lymph nodes. Figure 6-7 A highly activated natural killer cell with abundant cytoplasmic granules. (Courtesy of Dr. Noelle Williams, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-8 Schematic representation of NK-cell receptors and killing. NK cells express activating and inhibitory receptors; some examples of each are indicated. Normal cells are not killed because inhibitory signals from normal MHC class I molecules override activating signals. In tumor cells or virus-infected cells, there is increased expression of ligands for activating receptors, and reduced expression or alteration of MHC molecules, which interrupts the inhibitory signals, allowing activation of NK cells and lysis of target cells. KIR, killer cell Ig-like recepors. Figure 6-9 The HLA complex and the structure of HLA molecules. A, The location of genes in the HLA complex is shown. The sizes and distances between genes are not to scale. B, Schematic diagrams and crystal structures of class I and class II HLA molecules. (Crystal structures are courtesy of Dr. P. Bjorkman, California Institute of Technology, Pasadena, CA.) Figure 6-10 Antigen processing and recognition. The sequence of events in the processing of a cytoplasmic protein antigen and its display by class I MHC molecules are shown at the top. The recognition of this MHC-displayed peptide by a CD8+ T cell is shown at the bottom. TABLE 6-1 -- Association of HLA with Disease Disease HLA Allele Relative Risk Ankylosing spondylitis B27 90 Postgonococcal arthritis B27 14 Acute anterior uveitis B27 14 Rheumatoid arthritis DR4 •4 Chronic active hepatitis DR3 13 Primary Sjögren syndrome DR3 •9 Type-1 diabetes DR3 •5 DR4 •6 DR3/DR4 20 MECHANISMS OF HYPERSENSITIVITY REACTIONS Humans live in an environment teeming with substances capable of producing immunologic responses. Contact with antigen leads not only to induction of a protective immune response, but also to reactions that can be damaging to tissues. Exogenous antigens occur in dust, pollens, foods, drugs, microbiologic agents, chemicals, and many blood products used in clinical practice. The immune responses that may result from such exogenous antigens take a variety of forms, ranging from annoying but trivial discomforts, such as itching of the skin, to potentially fatal diseases, such as bronchial asthma. The various reactions produced are called hypersensitivity reactions, and tissue injury in these reactions may be caused by humoral or cell-mediated immune mechanisms. Injurious immune reactions may be evoked not only by exogenous environmental antigens, but also by endogenous tissue antigens. Some of these immune reactions are triggered by homologous antigens that differ among individuals with different genetic backgrounds. Transfusion reactions and graft rejection are examples of immunologic disorders evoked by homologous antigens. Another category of disorders, those incited by self-, or autologous, antigens, constitutes the important group of autoimmune diseases (discussed later). These diseases arise because of the emergence of immune responses against self-antigens. Hypersensitivity diseases can be classified on the basis of the immunologic mechanism that mediates the disease ( Table 6-2 ). This classification is of value in distinguishing the manner in which the immune response ultimately causes tissue injury and disease, and the accompanying pathologic alterations. Prototypes of each of these immune mechanisms are presented in the subsequent sections. • In immediate hypersensitivity (type I hypersensitivity), the immune response releases vasoactive and spasmogenic substances that act on vessels and smooth muscle and proinflammatory cytokines that recruit inflammatory cells. • In antibody-mediated disorders (type II hypersensitivity), secreted antibodies participate directly in injury to cells by promoting their phagocytosis or lysis and injury to tissues by inducing inflammation. Antibodies may also interfere with cellular functions and cause disease without tissue injury. • In immune complex-mediated disorders (type III hypersensitivity), antibodies bind antigens and then induce inflammation directly or by activating complement. The leukocytes that are recruited (neutrophils and monocytes) produce tissue damage by release of lysosomal enzymes and generation of toxic free radicals. • In cell-mediated immune disorders (type IV hypersensitivity), sensitized T lymphocytes are the cause of the cellular and tissue injury. Most hypersensitivity diseases show a genetic predisposition. Modern methods of mapping disease-associated susceptibility 206 TABLE 6-2 -- Mechanisms of Immunologically Mediated Diseases Type Prototype Disorder Immune Mechanisms Pathologic Lesions Immediate (type I) hypersensitivity Anaphylaxis; allergies; bronchial asthma (atopic forms) Production of IgE antibody → immediate release of vasoactive amines and other mediators from mast cells; recruitment of inflammatory cells (late-phase reaction) Vascular dilation, edema, smooth muscle contraction, mucus production, inflammation Antibody-mediated (type II) hypersensitivity Autoimmune hemolytic anemia; Goodpasture syndrome Production of IgG, IgM → binds to antigen on target cell or tissue → phagocytosis or lysis of target cell by activated complement or Fc receptors; recruitment of leukocytes Cell lysis; inflammation Immune complex-mediated (type III) hypersensitivity Systemic lupus erythematosus; some forms of glomerulonephritis; serum sickness; Arthus reaction Deposition of antigen-antibody complexes → complement activation → recruitment of leukocytes by complement products and Fc receptors → release of enzymes and other toxic molecules Necrotizing vasculitis (fibrinoid necrosis); inflammation Cell-mediated (type IV) hypersensitivity Contact dermatitis; multiple sclerosis; type I, diabetes; transplant rejection; tuberculosis Activated T lymphocytes → i) release of cytokines and macrophage activation; ii) T cell-mediated cytotoxicity Perivascular cellular infiltrates; edema; cell destruction; granuloma formation genes are revealing the complex nature of these genetic influences. Many susceptibility loci have been identified in different diseases. Among the genes known to be associated with hypersensitivity diseases are MHC genes, but many non-MHC genes also play a role. Immediate (Type I) Hypersensitivity Immediate, or type I, hypersensitivity is a rapidly developing immunologic reaction occurring within minutes after the combination of an antigen with antibody bound to mast cells in 20 21 individuals previously sensitized to the antigen.[ ] [ ] These reactions are often called allergy, and the antigens that elicit them are allergens. Immediate hypersensitivity may occur as a systemic disorder or as a local reaction. The systemic reaction usually follows injection of an antigen to which the host has become sensitized. Often within minutes, a state of shock is produced, which is sometimes fatal. The nature of local reactions varies depending on the portal of entry of the allergen and may take the form of localized cutaneous swellings (skin allergy, hives), nasal and conjunctival discharge (allergic rhinitis and conjunctivitis), hay fever, bronchial asthma, or allergic gastroenteritis (food allergy). Many local type I hypersensitivity reactions have two well-defined phases ( Fig. 6-11 ). The immediate, or initial, response is characterized by vasodilation, vascular leakage, and depending on the location, smooth muscle spasm or glandular secretions. These changes usually become evident within 5 to 30 minutes after exposure to an allergen and tend to subside in 60 minutes. In many instances (e.g., allergic rhinitis and bronchial asthma), a second, late-phase reaction sets in 2 to 24 hours later without additional exposure to antigen and may last for several days. This latephase reaction is characterized by infiltration of tissues with eosinophils, neutrophils, basophils, monocytes, and CD4+ T cells as well as tissue destruction, typically in the form of mucosal epithelial cell damage. Because mast cells are central to the development of immediate hypersensitivity, we first review some of their salient characteristics and then discuss the immune mechanisms that underlie this form of hypersensitivity.[ 22] Mast cells are bone Figure 6-11 Immediate hypersensitivity. A, Kinetics of the immediate and late-phase reactions. The immediate vascular and smooth muscle reaction to allergen develops within minutes after challenge (allergen exposure in a previously sensitized individual), and the late-phase reaction develops 2 to 24 hours later. B, C, Morphology: The immediate reaction (B) is characterized by vasodilation, congestion, and edema, and the late phase reaction (C) is characterized by an inflammatory infiltrate rich in eosinophils, neutrophils, and T cells. (Courtesy of Dr. Daniel Friend, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-12 Pathogenesis of immediate (type I) hypersensitivity reaction. The late-phase reaction is dominated by leukocyte infiltration and tissue injury. TH 2, T-helper type 2 CD4 cells. Figure 6-13 Activation of mast cells in immediate hypersensitivity and release of their mediators. ECF, eosinophil chemotactic factor; NCF, neutrophil chemotactic factor; PAF, plateletactivating factor. TABLE 6-3 -- Summary of the Action of Mast Cell Mediators in Immediate (Type I) Hypersensitivity Action Vasodilation, increased vascular permeability Mediator Histamine PAF Leukotrienes C4 , D4 , E4 Neutral proteases that activate complement and kinins Prostaglandin D2 Smooth muscle spasm Leukotrienes C4 , D4 , E4 Histamine Prostaglandins PAF Cellular infiltration Cytokines, e.g., TNF Leukotriene B4 Eosinophil and neutrophil chemotactic factors (not defined biochemically) PAF PAF, platelet-activating factor; TNF, tumor necrosis factor. produce leukotriene C4 and PAF and directly activate mast cells to release mediators. Thus, the recruited cells amplify and sustain the inflammatory response without additional exposure to the triggering antigen. It is now believed that this late-phase inflammatory response is a major cause of symptoms in some type I hypersensitivity disorders, such as allergic asthma. Therefore, treatment of these diseases requires the use of broad-spectrum anti-inflammatory drugs, such as steroids. A final point that should be mentioned in this general discussion of immediate hypersensitivity is that susceptibility to these reactions is genetically determined. The term atopy refers to a predisposition to develop localized immediate hypersensitivity reactions to a variety of inhaled and ingested allergens. Atopic individuals tend to have higher serum IgE levels, and more IL-4-producing TH 2 cells, compared with the general population. A positive family history of allergy is found in 50% of atopic individuals. The basis of familial predisposition is not clear, 27 but studies in patients with asthma reveal linkage to several gene loci.[ ] Candidate genes have been mapped to 5q31, where genes for the cytokines IL-3, IL-4, IL-5, IL-9, IL-13, and GMCSF are located, consistent with the idea that these cytokines are involved in the reactions. Linkage has also been noted to 6p, close to the HLA complex, suggesting that the inheritance of certain HLA alleles permits reactivity to certain allergens. Another asthma-associated locus is on chromosome 11q13, the location of the gene encoding the β chain of the high-affinity IgE receptor, but many studies have failed to establish a linkage of atopy with the FcepsilonRI β chain chain or even this chromosomal region. To summarize, immediate (type I) hypersensitivity is a complex disorder resulting from an IgE-mediated triggering of mast cells and subsequent accumulation of inflammatory cells at sites of antigen deposition. These events are regulated in large part by the induction of TH 2-type helper T cells that promote synthesis of IgE and accumulation of inflammatory cells, particularly eosinophils. The clinical features result from release of mast-cell mediators as well as the accumulation of an eosinophilrich inflammatory exudate. With this consideration of the basic mechanisms of type I hypersensitivity, we turn to some conditions that are important examples of IgE-mediated disease. Systemic Anaphylaxis Systemic anaphylaxis is characterized by vascular shock, widespread edema, and difficulty in breathing. In humans, systemic anaphylaxis may occur after administration of foreign 28 proteins (e.g., antisera), hormones, enzymes, polysaccharides, and drugs (such as the antibiotic penicillin).[ ] The severity of the disorder varies with the level of sensitization. Extremely small doses of antigen may trigger anaphylaxis, for example, the tiny amounts used in ordinary skin testing for various forms of allergies. Within minutes after exposure, itching, hives, and skin erythema appear, followed shortly thereafter by a striking contraction of respiratory bronchioles and respiratory distress. Laryngeal edema results in hoarseness. Vomiting, abdominal cramps, diarrhea, and laryngeal obstruction follow, and the patient may go into shock and even die within the hour. The risk of anaphylaxis must be borne in mind when certain therapeutic agents are administered. Although patients at risk can generally be identified by a previous history of some form of allergy, the absence of such 210 a history does not preclude the possibility of an anaphylactic reaction. Local Immediate Hypersensitivity Reactions Local immediate hypersensitivity, or allergic, reactions are exemplified by so-called atopic allergy. About 10% of the population suffers from allergies involving localized reactions to common environmental allergens, such as pollen, animal dander, house dust, foods, and the like. Specific diseases include urticaria, angioedema, allergic rhinitis (hay fever), and some forms of asthma, all discussed elsewhere in this book. The familial predisposition to the development of this type of allergy has been mentioned earlier. Antibody-Mediated (Type II) Hypersensitivity Type II hypersensitivity is mediated by antibodies directed toward antigens present on cell surfaces or extracellular matrix. The antigenic determinants may be intrinsic to the cell membrane or matrix, or they may take the form of an exogenous antigen, such as a drug metabolite, that is adsorbed on a cell surface or matrix. In either case, the hypersensitivity reaction results from the binding of antibodies to normal or altered cell-surface antigens. Three different antibody-dependent mechanisms involved in this type of reaction are depicted in Figure 614 and described next. Most of these reactions involve the effector mechanisms that are used by antibodies, namely the complement system and phagocytes. Opsonization and Complement- and Fc Receptor-Mediated Phagocytosis The depletion of cells targeted by antibodies is, to a large extent, because the cells are coated (opsonized) with molecules that make them attractive for phagocytes. When antibodies are deposited on the surfaces of cells, they may activate the complement system (if the antibodies are of the IgM or IgG class). Complement activation generates byproducts, mainly C3b and C4b, which are deposited on the surfaces of the cells and recognized by phagocytes that express receptors for these proteins. In addition, cells opsonized by IgG antibodies are recognized by phagocyte Fc receptors, which are specific for the Fc portions of some IgG subclasses. The net result is the phagocytosis of the opsonized cells and their destruction ( Fig. 6-14A ). Complement activation on cells also leads to the formation of the membrane attack complex, which disrupts membrane integrity by "drilling holes" through the lipid bilayer, thereby causing osmotic lysis of the cells. Antibody-mediated destruction of cells may occur by another process called antibody-dependent cellular cytotoxicity (ADCC). This form of antibody-mediated cell injury does not involve fixation of complement but instead requires the cooperation of leukocytes. Cells that are coated with low concentrations of IgG antibody are killed by a variety of effector cells, which bind to the target by their receptors for the Fc fragment of IgG, and cell lysis proceeds without phagocytosis. ADCC may be mediated by monocytes, neutrophils, eosinophils, and NK cells. Although, in most instances, IgG antibodies are involved in ADCC, in certain cases (e.g., eosinophil-mediated cytotoxicity against parasites), IgE antibodies are used. The role of ADCC in hypersensitivity diseases is uncertain. Clinically, antibody-mediated cell destruction and phagocytosis occur in the following situations: (1) transfusion reactions, in which cells from an incompatible donor react with and are opsonized by preformed antibody in the host; (2) erythroblastosis fetalis, in which there is an antigenic difference between the mother and the fetus, and antibodies (of the IgG class) from the mother cross the placenta and cause destruction of fetal red cells; (3) autoimmune hemolytic anemia, agranulocytosis, and thrombocytopenia, in which individuals produce antibodies to their own blood cells, which are then destroyed; and (4) certain drug reactions, in which antibodies are produced that react with the drug, which may be attached to the surface of erythrocytes or other cells. Complement- and Fc Receptor-Mediated Inflammation When antibodies deposit in extracellular tissues, such as basement membranes and matrix, the resultant injury is because of inflammation and not because of phagocytosis or lysis of cells. The deposited antibodies activate complement, generating byproducts, such as C5a (and to a lesser extent C4a and C3a), that recruit neutrophils and monocytes. The same cells also bind to the deposited antibodies via their Fc receptors. The leukocytes are activated, they release injurious substances, such as enzymes and reactive oxygen intermediates, and the result is damage to the tissues ( Fig. 6-14B ). It was once thought that complement was the major mediator of antibody-induced inflammation, but knockout mice lacking Fc receptors also show striking reduction in these reactions. It is now believed that inflammation in antibody-mediated (and immune complex-mediated) diseases is because of both complement and Fc receptordependent reactions.[ 29] Antibody-mediated inflammation is the mechanism responsible for tissue injury in some forms of glomerulonephritis, vascular rejection in organ grafts, and other diseases ( Table 6-4 ). As we shall discuss in more detail below, the same reaction is involved in immune complex-mediated diseases. Antibody-Mediated Cellular Dysfunction In some cases, antibodies directed against cell-surface receptors impair or dysregulate function without causing cell injury or inflammation. For example, in myasthenia gravis, antibodies reactive with acetylcholine receptors in the motor end-plates of skeletal muscles impair neuromuscular transmission and therefore cause muscle weakness ( Fig. 6-14C ). In pemphigus vulgaris, antibodies against desmosomes disrupt intercellular junctions in epidermis, leading to the formation of skin vesicles. The converse (i.e., antibody-mediated stimulation of cell function) is noted in Graves disease. In this disorder, antibodies against the thyroid-stimulating hormone receptor on thyroid epithelial cells stimulate the cells, resulting in hyperthyroidism. Immune Complex-Mediated (Type III) Hypersensitivity Antigen-antibody complexes produce tissue damage mainly by eliciting inflammation at the sites of deposition. The toxic 211 Figure 6-14 Schematic illustration of the three major mechanisms of antibody-mediated injury. A, Opsonization of cells by antibodies and complement components and ingestion by phagocytes. B, Inflammation induced by antibody binding to Fc receptors of leukocytes and by complement breakdown products. C, Antireceptor antibodies disturb the normal function of receptors. In these examples, antibodies against the thyroid stimulating hormone (TSH) receptor activate thyroid cells in Graves disease, and acetylcholine (ACh) receptor antibodies impair neuromuscular transmission in myasthenia gravis. TABLE 6-4 -- Examples of Antibody-Mediated Diseases (Type II Hypersensitivity) Disease Target Antigen Mechanisms of Disease Clinicopathologic Manifestations Autoimmune hemolytic anemia Erythrocyte membrane proteins (Rh blood group antigens, I antigen) Opsonization and phagocytosis of erythrocytes Hemolysis, anemia Autoimmune thrombocytopenic purpura Platelet membrane proteins (gpllb:Illa intergrin) Opsonization and phagocytosis of platelets Bleeding Pemphigus vulgaris Proteins in intercellular junctions of epidermal cells (epidermal cadherin) Antibody-mediated activation of proteases, disruption of intercellular adhesions Skin vesicles (bullae) Vasculitis caused by ANCA Neutrophil granule proteins, presumably released from activated neutrophils Neutrophil degranulation and inflammation Vasculitis Goodpasture syndrome Noncollagenous protein in basement membranes of kidney glomeruli and lung alveoli Complement- and Fc receptor-mediated inflammation Nephritis, lung hemorrhage Acute rheumatic fever Streptococcal cell wall antigen; antibody cross-reacts with myocardial antigen Inflammation, macrophage activation Myocarditis, arthritis Myasthenia gravis Acetylcholine receptor Antibody inhibits acetylcholine binding, downmodulates receptors Muscle weakness, paralysis Graves disease (hyperthyroidism) TSH receptor Antibody-mediated stimulation of TSH receptors Hyperthyroidism Insulin-resistant diabetes Insulin receptor Antibody inhibits binding of insulin Hyperglycemia, ketoacidosis Pernicious anemia Intrinsic factor of gastric parietal cells Neutralization of intrinsic factor, decreased absorption of vitamin B12 Abnormal erythropoiesis, anemia ANCA, antineutrophil cytoplasmic antibodies; TSH, thyroid-stimulating hormone. From Abbas AK, Lichtman H: Cellular and Molecular Immunology. 5th edition. WB Saunders Company, Philadelphia, 2003. Examples of immune complex disorders and the antigens involved are listed in Table 6-5 . Immune complex-mediated diseases can be generalized, if immune complexes are formed in the circulation and are deposited in many organs, or localized to particular organs, such as the kidney (glomerulonephritis), joints (arthritis), or the small blood vessels of the skin if the complexes are formed and deposited locally. These two patterns are considered separately. Systemic Immune Complex Disease Acute serum sickness is the prototype of a systemic immune complex disease; it was at one time a frequent sequela to the administration of large amounts of foreign serum (e.g., immune serum from horses used for passive immunization.) The TABLE 6-5 -- Examples of Immune Complex-Mediated Diseases Disease Antigen Involved Clinicopathologic Manifestations Systemic lupus erythematosus DNA, nucleoproteins, others Nephritis, arthritis, vasculitis Polyarteritis nodosa Hepatitis B virus surface antigen (in some cases) Vasculitis Poststreptococcal glomerulonephritis Streptococcal cell wall antigen(s); may be "planted" in glomerular basement membrane Nephritis Acute glomerulonephritis Bacterial antigens (Treponema); parasite antigens (malaria, schistosomes); tumor antigens Nephritis Reactive arthritis Bacterial antigens (Yersinia) Acute arthritis Arthus reaction Various foreign proteins Cutaneous vasculitis Serum sickness Various proteins, e.g., foreign serum (anti-thymocyte globulin) Arthritis, vasculitis, nephritis occurrence of diseases caused by immune complexes was suspected in the early 1900s by a physician named Clemens von Pirquet. Patients with diphtheria infection were being treated with serum from horses immunized with the diphtheria toxin. Von Pirquet noted that some of these patients developed arthritis, skin rash, and fever, and the symptoms appeared more rapidly with repeated injection of the serum. Von Pirquet concluded that the treated patients made antibodies to horse serum proteins, these antibodies formed complexes with the injected proteins, and the disease was due to the antibodies or immune complexes. He called this disease "serum disease"; it is now known as serum sickness. In modern times the disease is infrequent, but it is an informative model that has taught us a great deal about systemic immune complex disorders. 213 For the sake of discussion, the pathogenesis of systemic immune complex disease can be divided into three phases: (1) formation of antigen-antibody complexes in the circulation; (2) deposition of the immune complexes in various tissues, thus initiating; and (3) an inflammatory reaction at the sites of immune complex deposition ( Fig. 6-15 ). The first phase is initiated by the introduction of antigen, usually a protein, and its interaction with immunocompetent cells, resulting in the formation of antibodies approximately a week after the injection of the protein. These antibodies are secreted into the blood, where they react with the antigen still present in the circulation to form antigen-antibody complexes. In the second phase, the circulating antigen-antibody complexes are deposited in various tissues. The factors that determine whether immune complex formation will lead to tissue deposition and disease are not fully understood, but two possible influences are the size of the immune complexes and the functional status of the mononuclear phagocyte system: • Large complexes formed in great antibody excess are rapidly removed from the circulation by the mononuclear phagocyte system and are therefore relatively harmless. The most pathogenic complexes are of small or intermediate size (formed in slight antigen excess), which bind less avidly to phagocytic cells and therefore circulate longer. • Because the mononuclear phagocyte system normally filters out the circulating immune complexes, its overload or intrinsic dysfunction increases the probability of persistence of immune complexes in circulation and tissue deposition. In addition, several other factors, such as charge of the immune complexes (anionic versus cationic), valency of the antigen, avidity of the antibody, affinity of the antigen to various tissue components, three-dimensional (lattice) structure of the complexes, and hemodynamic factors, influence the tissue deposition of complexes. Because most of these influences have been investigated with reference to deposition of immune complexes in the glomeruli, they are discussed further in Chapter 20 . In addition to the renal glomeruli, the favored sites of immune complex deposition are joints, skin, heart, serosal surfaces, and small blood vessels. For complexes to leave the microcirculation and deposit in the vessel wall, an increase in vascular permeability must occur. This is believed to occur when immune complexes bind to inflammatory cells through their Fc or C3b receptors and trigger release of vasoactive mediators as well as permeability-enhancing cytokines. Mast cells may also be involved in this phase of the reaction. Once complexes are deposited in the tissues, they initiate an acute inflammatory reaction (third phase). During this phase (approximately 10 days after antigen administration), clinical features such as fever, urticaria, arthralgias, lymph node enlargement, and proteinuria appear. Wherever complexes deposit, the tissue damage is similar. Two mechanisms are believed to cause inflammation at the sites of deposition ( Fig. 6-16 ): (1) activation of the complement cascade, and (2) activation of neutrophils and macrophages through their Fc receptors. As discussed in Chapter 2 , complement activation promotes inflammation mainly by production of chemotactic factors, which direct the migration of polymorphonuclear leukocytes and monocytes Figure 6-15 Schematic illustration of the three sequential phases in the induction of systemic immune complex-mediated disease (type III hypersensitivity). Figure 6-16 Pathogenesis of immune complex-mediated tissue injury. The morphologic consequences are depicted as boxed areas. Figure 6-17 Immune complex vasculitis. The necrotic vessel wall is replaced by smudgy, pink "fibrinoid" material. (Courtesy of Dr. Trace Worrell, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-18 Mechanisms of T cell-mediated (type IV) hypersensitivity reactions. A, In delayed type hypersensitivity reactions, CD4+ T cells (and sometimes CD8+ cells) respond to tissue antigens by secreting cytokines that stimulate inflammation and activate phagocytes, leading to tissue injury. B, In some diseases, CD8+ cytolytic T lymphocytes (CTLs) directly kill tissue cells. APC, antigenpresenting cell. TABLE 6-6 -- Examples of T Cell-Mediated (Type IV) Hypersensitivity Disease Specificity of Pathogenic T Cells Clinicopathologic Manifestations Type 1 diabetes mellitus Antigens of pancreatic islet β cells (insulin, glutamic acid decarboxylase, others) Insulitis (chronic inflammation in islets), destruction of β cells; diabetes Multiple sclerosis Protein antigens in central nervous system myelin (myelin basic protein, proteolipid protein) Demyelination in CNS with perivascular inflammation; paralysis, ocular lesions Rheumatoid arthritis Unknown antigen in joint synovium (type II collagen?); role of antibodies? Chronic arthritis with inflammation, destruction of articular cartilage and bone Peripheral neuropathy; GuillainBarré syndrome? Protein antigens of peripheral nerve myelin Neuritis, paralysis and inflammation ( Fig. 6-17 ). Thrombi are formed in the vessels, resulting in local ischemic injury. Cell-Mediated (Type IV) Hypersensitivity The cell-mediated type of hypersensitivity is initiated by antigen-activated (sensitized) T lymphocytes. It includes the delayed type hypersensitivity reactions mediated by CD4+ T cells, and direct cell cytotoxicity mediated by CD8+ T cells ( Fig. 6-18 ). It is the principal pattern of immunologic response not only to a variety of intracellular microbiologic agents, such as Mycobacterium tuberculosis, but also to many viruses, fungi, protozoa, and parasites. So-called contact skin sensitivity to chemical agents and graft rejection are other instances of cellmediated reactions. In addition, many autoimmune diseases are now known to be caused by T cell-mediated reactions ( Table 6-6 ). The two forms of T cell-mediated hypersensitivity are described next. 216 Figure 6-19 Delayed hypersensitivity in the skin. A, Perivascular infiltration by T cells and mononuclear phagocytes. B, Immunoperoxidase staining reveals a predominantly perivascular cellular infiltrate that marks positively with anti-CD4 antibodies. (Courtesy of Dr. Louis Picker, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-20 A section of a lymph node shows several granulomas, each made up of an aggregate of epithelioid cells and surrounded by lymphocytes. The granuloma in the center shows several multinucleate giant cells. (Courtesy of Dr. Trace Worrell, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-21 Schematic illustration of the events that give rise to the formation of granulomas in cell-mediated (type IV) hypersensitivity reactions. Note the role played by T cell-derived cytokines. Figure 6-22 Contact dermatitis showing an epidermal blister (vesicle) with dermal and epidermal mononuclear infiltrates. (Courtesy of Dr. Louis Picker, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-23 Schematic representation of the events that lead to the destruction of histoincompatible grafts. In the direct pathway, donor class I and class II antigens on antigen-presenting cells in the graft (along with B7 molecules, not shown) are recognized by CD8+ cytotoxic T cells and CD4+ helper T cells, respectively, of the host. CD4+ cells proliferate and produce cytokines that induce tissue damage by a local delayed hypersensitivity reaction and stimulate B cells and CD8+ T cells. CD8+ T cells responding to graft antigens differentiate into cytotoxic T lymphocytes that kill graft cells. In the indirect pathway, graft antigens are displayed by host APCs and activate CD4+ cells, which damage the graft by a local delayed hypersensitivity reaction. The example shown is of a kidney allograft. Figure 6-24 Acute cellular rejection of a renal allograft. A, An intense mononuclear cell infiltrate occupies the space between the tubules. B, T cells (stained brown by the immunoperoxidase technique) are abundant in the interstitium and infiltrating a tubule. (Courtesy of Dr. Robert Colvin, Department of Pathology, Massachusetts General Hospital, Boston, MA.) Figure 6-25 Antibody-mediated damage to the blood vessel in a renal allograft. The blood vessel is markedly thickened, and the lumen is obstructed by proliferating fibroblasts and foamy macrophages. (Courtesy of Dr. Ihsan Housini, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-26 Chronic rejection in a kidney allograft. A, Changes in the kidney in chronic rejection. B, Graft arteriosclerosis. The vascular lumen is replaced by an accumulation of smooth muscle cells and connective tissue in the vessel intima. (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA.) TABLE 6-7 -- Autoimmune Diseases Organ-Specific Systemic Hashimoto thyroiditis Systemic lupus erythematosus Autoimmune hemolytic anemia Rheumatoid arthritis Autoimmune atrophic gastritis of pernicious anemia Sjögren syndrome Multiple sclerosis Reiter syndrome Autoimmune orchitis Inflammatory myopathies Goodpasture syndrome Systemic sclerosis (scleroderma) Autoimmune thrombocytopenia Polyarteritis nodosa Insulin-dependent diabetes mellitus Myasthenia gravis Graves disease Primary biliary cirrhosis * * * * Autoimmune (chronic active) hepatitis Ulcerative colitis * * *The evidence supporting an autoimmune basis of these disorders is not strong. autoimmunity are type I diabetes mellitus, in which the autoreactive T cells and antibodies are specific for β cells of the pancreatic islets, and multiple sclerosis, in which autoreactive T cells react against central nervous system myelin. An example of systemic autoimmune disease is SLE, in which a diversity of antibodies directed against DNA, platelets, red cells, and protein-phospholipid complexes result in widespread lesions throughout the body. In the middle of the spectrum falls Goodpasture syndrome, in which antibodies to basement membranes of lung and kidney induce lesions in these organs. It is obvious that autoimmunity results from the loss of self-tolerance, and the question arises as to how this happens. Before we look for answers to this question, we review the mechanisms of immunologic tolerance to self-antigens. Immunologic Tolerance Immunologic tolerance is a state in which the individual is incapable of developing an immune response to a specific antigen. Self-tolerance refers to lack of responsiveness to an individual's own antigens, and it underlies our ability to live in harmony with our cells and tissues. Several mechanisms, albeit not well understood, have been postulated to explain the tolerant state. They can be broadly classified into two groups: central tolerance and peripheral tolerance.[ 37] [38] [39] [40] Each of these is considered briefly. Central Tolerance. This refers to death (deletion) of self-reactive T- and B-lymphocyte clones during their maturation in the central lymphoid organs (the thymus for T cells and the bone marrow for B cells). Deletion of developing intrathymic T cells has been extensively investigated. Experiments with transgenic mice provide abundant evidence that T lymphocytes that bear receptors for selfantigens undergo apoptosis within the thymus during the process of T-cell maturation. It 224 is proposed that many autologous protein antigens, including antigens thought to be restricted to peripheral tissues, are processed and presented by thymic antigen-presenting cells in association with self-MHC molecules.[ 37] A protein called AIRE (autoimmune regulator) is thought to stimulate expression of many "peripheral" self-antigens in the thymus and is thus critical for deletion of immature self-reactive T cells.[ polyendocrinopathy ( Chapter 24 ). The 38] Mutations in the AIRE gene (either spontaneous in humans or created in knockout mice) are the cause of an autoimmune Figure 6-27 Schematic illustration of the mechanisms involved in central and peripheral tolerance. The principal mechanisms of tolerance in CD4+ T cells are shown. APC, antigenpresenting cell. Figure 6-28 Pathogenesis of autoimmunity. Autoimmunity results from multiple factors, including susceptibility genes that may interfere with self-tolerance and environmental triggers (inflammation, other inflammatory stimuli) that promote lymphocyte entry into tissues, activation of lymphocytes, and tissue injury. Figure 6-29 Role of infections in autoimmunity. Infections may promote activation of self-reactive lymphocytes by inducing the expression of costimulators (A), or microbial antigens may mimic self-antigens and activate self-reactive lymphocytes as a cross-reaction (B). TABLE 6-8 -- 1997 Revised Criteria for Classification of Systemic Lupus Erythematosus (Not Available) Data from Tan EM, et al: The revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271, 1982; and Hochberg, MC: Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 40:1725, 1997. mechanisms that maintain self-tolerance. Antibodies have been identified against an array of nuclear and cytoplasmic components of the cell that are neither organ nor species specific. In addition, a third group of antibodies is directed against cell-surface antigens of blood cells. Apart from their value in the diagnosis and management of patients with SLE, these antibodies are of major pathogenetic significance, as, for example, in the immune complex-mediated glomerulonephritis so typical of this disease.[ 56 56] ANAs are directed against several nuclear antigens and can be grouped into four categories:[ ] (1) antibodies to DNA, (2) antibodies to histones, (3) antibodies to nonhistone proteins bound to RNA, and (4) antibodies to nucleolar antigens. Table 6-9 lists several ANAs and their association with SLE as well as with other autoimmune diseases to be discussed later. Several techniques are used to detect ANAs. Clinically the most commonly used method is indirect immunofluorescence, which detects a variety of nuclear antigens, including DNA, RNA, and proteins (collectively called generic ANAs). The pattern of nuclear fluorescence suggests the type of antibody present in the patient's serum. Four basic patterns are recognized: • Homogeneous or diffuse nuclear staining usually reflects antibodies to chromatin, histones and, occasionally, double-stranded DNA. • Rim or peripheral staining patterns are most commonly indicative of antibodies to double-stranded DNA. • Speckled pattern refers to the presence of uniform or variable-sized speckles. This is one of the most commonly observed patterns of fluorescence and therefore the least specific. It reflects the presence of antibodies to non-DNA nuclear constituents. Examples include Sm antigen, ribonucleoprotein, and SS-A and SS-B reactive antigens ( Table 6-9 ). • Nucleolar pattern refers to the presence of a few discrete spots of fluorescence within the nucleus and represents antibodies to nucleolar RNA. This pattern is reported most often in patients with systemic sclerosis. The fluorescence patterns are not absolutely specific for the type of antibody, and because many autoantibodies may be present, combinations of patterns are frequent. The immunofluorescence test for ANA is positive in virtually every patient with SLE; hence this test is sensitive, but it is not specific because patients with other autoimmune diseases also frequently score positive (see Table 6-9 ). Furthermore, approximately 5% to 15% of normal individuals have low titers of these antibodies. The incidence increases with age. Detection of antibodies to specific nuclear antigens requires specialized techniques. Of the numerous nuclear antigen-antibody systems,[ 6-9 . Antibodies to double-stranded DNA and the so-called Smith (Sm) antigen are virtually diagnostic of SLE. 57] some that are clinically useful are listed in Table There is some, albeit imperfect, correlation between the presence or absence of certain ANAs and clinical manifestations. For example, high titers of double-stranded DNA antibodies are 56] usually associated with active renal disease. Conversely the risk of nephritis is low if anti-SS-B antibodies are present.[ 229 TABLE 6-9 -- Antinuclear Antibodies in Various Autoimmune Diseases Disease, % Positive Nature of Antigen Antibody System Many nuclear antigens (DNA, RNA, proteins) Generic ANA (indirect IF) Native DNA Anti-double-stranded DNA Histones Antihistone SLE Drug-Induced LE Systemic Sclerosis— Diffuse Limited Scleroderma— CREST Sjögren Syndrome Inflammatory Myopathies >95 >95 70–90 70–90 50–80 40–60 <5 <5 <5 <5 <5 <5 <5 <5 <5 50–70 Core proteins of small nuclear ribonucleoprotein particles (Smith antigen) Anti-Sm Ribonucleoprotein (U1RNP) Nuclear RNP RNP <5 <5 <5 <5 <5 30–40 <5 15 10 <5 <5 SS-A(Ro) 30–50 <5 <5 <5 10 RNP SS-B(La) 10–15 <5 <5 <5 <5 DNA topoisomerase I Scl-70 <5 <5 Centromeric proteins Anticentromere <5 <5 22–36 Histidyl-t-RNA synthetase Jo-1 <5 <5 <5 10–18 <5 <5 <5 <5 <5 <5 Boxed entries indicate high correlation. SLE, systemic lupus erythematosus; LE, lupus erythematosus; ANA, antinuclear antibodies; RNP, ribonucleoprotein. In addition to ANAs, lupus patients have a host of other autoantibodies. Some are directed against elements of the blood, such as red cells, platelets, and lymphocytes; others are directed 58 against proteins complexed to phospholipids. In recent years, there has been much interest in these so-called antiphospholipid antibodies. [ ] They are present in 40% to 50% of lupus patients. Although initially believed to be directed against anionic phospholipids, they are actually directed against epitopes of plasma proteins that are revealed when the proteins are complexed to phospholipids. A variety of protein substrates have been implicated, including prothrombin, annexin V, β2 -glycoprotein I, protein S, and protein C.[ 59] Antibodies against the phospholipid-β2 -glycoprotein complex also bind to cardiolipin antigen, used in syphilis serology, and therefore lupus patients may have a false-positive test result for syphilis. Some of these antibodies interfere with in vitro clotting tests, such as partial thromboplastin time. Therefore, these antibodies are sometimes referred to as lupus anticoagulant. Despite having a 60 circulating anticoagulant that delays clotting in vitro, these patients have complications associated with a hypercoagulable state.[ ] They have venous and arterial thromboses, which may be associated with recurrent spontaneous miscarriages and focal cerebral or ocular ischemia. This constellation of clinical features, in association with lupus, is referred to as the secondary antiphospholipid antibody syndrome. The pathogenesis of thrombosis in these patients is unknown; possible mechanisms are discussed in Chapter 4 . Some patients develop these autoantibodies and the clinical syndrome without associated SLE. They are said to have the primary antiphospholipid syndrome ( Chapter 4 ). Given the presence of all these autoantibodies, we still know little about the mechanism of their emergence. Three converging lines of investigation hold center stage today: genetic predisposition, some nongenetic (environmental) factors, and a fundamental abnormality in the immune system. Genetic Factors. SLE is a complex genetic trait with contribution from MHC and multiple non-MHC genes. Many lines of evidence support a genetic predisposition. [ 51] [61] • Family members of patients have an increased risk of developing SLE. Up to 20% of clinically unaffected first-degree relatives of SLE patients reveal autoantibodies and other immunoregulatory abnormalities. • There is a higher rate of concordance (>20%) in monozygotic twins when compared with dizygotic twins (1% to 3%). Monozygotic twins who are discordant for SLE have 62 similar patterns and titers of autoantibodies.[ ] These data suggest that the genetic makeup regulates the formation of autoantibodies, but the expression of the disease (i.e., tissue injury) is influenced by non-genetic (possibly environmental) factors. • Studies of HLA associations further support the concept that MHC genes regulate production of specific autoantibodies, rather than conferring a generalized predisposition to SLE. Specific alleles of the HLA-DQ locus have been linked to the production of anti-double-stranded DNA, anti-Sm, and antiphospholipid antibodies. • Some lupus patients (approximately 6%) have inherited deficiencies of early complement components, such as C2, C4, or C1q. Lack of complement may impair removal of circulating immune complexes by the mononuclear phagocyte system, thus favoring tissue deposition. Knockout mice lacking C4 or certain complement receptors are also prone to develop lupus-like autoimmunity. Various mechanisms have been invoked, including failure to clear immune complexes and loss of B-cell self-tolerance. It has also been proposed that deficiency of C1q results in failure of phagocytic 230 63 clearance of apoptotic cells.[ ]Such cells are produced normally, and if they are not cleared their nuclear components may elicit immune responses. • In animal models of SLE, several non-MHC susceptibility loci have been identified. The best-known animal model is the (NZBxNZW)F1 mouse strain. In different versions of this strain, up to 20 loci are believed to be associated with the disease. [ 51] Environmental Factors. There are many indications that, in addition to genetic factors, several environmental or non-genetic factors must be involved in the pathogenesis of SLE. The clearest example comes from 64 the observation that drugs such as hydralazine, procainamide, and D-penicillamine can induce an SLE-like response in humans.[ ] Exposure to ultraviolet light is another environmental factor that exacerbates the disease in many individuals. How ultraviolet light acts is not entirely clear, but it is suspected of modulating the immune response. For example, it induces keratinocytes to produce IL-1, a factor known to influence the immune response. In addition, UV irradiation may induce apoptosis in cells, and alter the DNA in such a way that it becomes 65 immunogenic. [ ] Sex hormones seem to exert an important influence on the occurrence and manifestations of SLE. During the reproductive years, the frequency of SLE is 10 times greater in women than in men, and exacerbation has been noted during normal menses and pregnancy. Immunologic Factors. With all the immunologic findings in SLE patients, there can be little doubt that some fundamental derangement of the immune system is involved in the pathogenesis of SLE. Although a variety of immunologic abnormalities affecting both T cells and B cells have been detected in patients with SLE, it has been difficult to relate any one of them to the causation of this disease. For years, it had been thought that an intrinsic B-cell hyperactivity is fundamental to the pathogenesis of SLE. Polyclonal B-cell activation can be readily demonstrated in patients with SLE and in murine models of this disease. Molecular analyses of anti-double-stranded DNA antibodies, however, strongly suggest that pathogenic autoantibodies are not derived from polyclonally activated B cells. Instead, it appears that the production of tissue-damaging antibodies is driven by self-antigens and results from an antigen-specific helper T cell-dependent B66 66A cell response with many characteristics of responses to foreign antigens.[ ] These observations have shifted the onus of driving the autoimmune response squarely on helper T cells.[ ] Based on these findings, a model for the pathogenesis of SLE has been proposed ( Fig. 6-30 ). Other contributing factors include defective clearance of apoptotic cells, mentioned above, and dysregulation of cytokines, notably interferons.[ 61] SLE is a heterogeneous disease, however, and as mentioned earlier, the production of different autoantibodies is regulated by 67] distinct genetic factors. Hence, there may well be distinct immunoregulatory disturbances in patients with different genetic backgrounds and autoantibody profiles.[ Regardless of the exact sequence by which autoantibodies are formed, they are clearly the mediators of tissue injury. Most of the visceral lesions are mediated by immune complexes (type III hypersensitivity). DNA-anti-DNA complexes can be detected in the glomeruli and small blood vessels. Low levels of serum complement and granular deposits of complement Figure 6-30 Model for the pathogenesis of systemic lupus erythematosus. (Modified from Kotzin BL: Systemic lupus erythematosus. Cell 65:303, 1996. Copyright 1996, Cell Press.) TABLE 6-10 -- Clinical and Pathologic Manifestations of Systemic Lupus Erythematosus Clinical Manifestation Prevalence in Patients, % Hematologic 100 Arthritis •90 Skin •85 Fever •83 Fatigue •81 Weight loss •63 Renal •50 Central nervous system •50 Pleuritis •46 Myalgia •33 Pericarditis •25 Gastrointestinal •21 Paynaud phenomenon •20 Ocular •15 Peripheral neuropathy •14 lesions result from the deposition of immune complexes and are found in the blood vessels, kidneys, connective tissue, and skin. 68] An acute necrotizing vasculitis involving small arteries and arterioles may be present in any tissue.[ stages, vessels undergo fibrous thickening with luminal narrowing. The arteritis is characterized by fibrinoid deposits in the vessel walls. In chronic Kidney. The kidney is a frequent target of injury in SLE. The principal mechanism of injury is immune complex deposition in renal structures, including glomeruli, tubular and peritubular capillary basement membranes, and larger blood vessels. Other forms of injury may include a thrombotic process involving the glomerular capillaries and extraglomerular vasculature, thought to be caused by antiphospholipid antibodies. 69 A morphologic classification of the patterns of immune complex-mediated glomerular injury in SLE has proven to be clinically useful.[ ] There are several versions of the World Health Organization (WHO) classification of lupus nephritis, but in all, five patterns are recognized: (1) minimal or no detectable abnormalities (class I), which is rare, seen in renal biopsies from less than 5% of SLE patients; (2) mesangial lupus glomerulonephritis (class II); (3) focal proliferative glomerulonephritis (class III); (4) diffuse proliferative glomerulonephritis (class IV); and (5) membranous glomerulonephritis (class V). None of these patterns is specific for lupus. Mesangial lupus glomerulonephritis is characterized by mesangial cell proliferation and lack of involvement of glomerular capillary walls. It is seen in 10% to 25% of patients, most of whom have minimal clinical manifestations, such as mild hematuria or transient proteinuria. There is a slight to moderate increase in the intercapillary mesangial matrix as well as in the number of mesangial cells. Despite the mild histologic changes, granular mesangial deposits of immunoglobulin and complement are always present. Such deposits presumably reflect the earliest change because filtered immune complexes accumulate primarily in the mesangium. The other changes to be described are usually superimposed on the mesangial changes. Focal proliferative glomerulonephritis is seen in 20% to 35% of patients. It is a focal lesion, affecting fewer than 50% of the glomeruli and generally only portions of each glomerulus. Typically, one or two tufts in an otherwise normal glomerulus exhibit swelling and proliferation of endothelial and mesangial cells, infiltration with neutrophils, and sometimes fibrinoid deposits and intracapillary thrombi ( Fig. 6-31 ). Occasionally, affected glomeruli exhibit global injury. Focal lesions are associated with hematuria and proteinuria. In some patients, the nephritis progresses to diffuse proliferative disease. Diffuse proliferative glomerulonephritis is the most serious of the renal lesions in SLE, occurring in 35% to 60% of patients who undergo biopsy. Anatomic changes are dominated by proliferation of endothelial, mesangial and, sometimes, epithelial cells ( Fig. 6-32 ), producing in some cases epithelial crescents that fill the Bowman space ( Chapter 20 ). The presence of fibrinoid necrosis, crescents, prominent infiltration by leukocytes, cell death as indicated by apoptotic bodies, and hyaline thrombi indicates active disease. Most or all glomeruli are involved in both kidneys, and the entire glomerulus is frequently affected. Patients with diffuse lesions are usually overtly symptomatic, showing microscopic or gross hematuria as well as proteinuria that is severe enough to cause the nephrotic syndrome in more than 50% of patients. Hypertension and mild to severe renal insufficiency are also common. Membranous glomerulonephritis is a designation given to glomerular disease in which the principal histologic change consists of widespread thickening of the capillary walls. The lesions are similar to those encountered in idiopathic membranous glomerulonephritis, described more fully in Chapter 20 . This type of lesion is seen in 10% to 15% of patients with Figure 6-31 Lupus nephritis. There are two focal necrotizing lesions in the glomerulus (arrowheads). (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-32 Lupus nephritis, diffuse proliferative type. Note the marked increase in cellularity throughout the glomerulus. (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-33 Immunofluorescence micrograph stained with fluorescent anti-IgG from a patient with diffuse proliferative lupus nephritis. One complete glomerulus and part of another one are seen. Note the mesangial and capillary wall deposits of IgG. (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-34 Electron micrograph of a renal glomerular capillary loop from a patient with systemic lupus erythematosus nephritis. Subendothelial dense deposits correspond to "wire loops" seen by light microscopy. Deposits are also present in the mesangium. (Courtesy of Dr. Jean Olson, Department of Pathology, University of California San Francisco, San Francisco, CA.) Figure 6-35 Lupus nephritis showing a glomerulus with several "wire loop" lesions representing extensive subendothelial deposits of immune complexes. (Periodic acid-Schiff [PAS] stain.) (Courtesy of Dr. Helmut Rennke, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-36 Systemic lupus erythematosus involving the skin. A, An H&E-stained section shows liquefactive degeneration of the basal layer of the epidermis and edema at the dermoepidermal junction. (Courtesy of Dr. Jag Bhawan, Boston University School of Medicine, Boston, MA.) B, An immunofluorescence micrograph stained for IgG reveals deposits of immunoglobulin along the dermal-epidermal junction. (Courtesy of Dr. Richard Sontheimer, Department of Dermatology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-37 Libman-Sacks endocarditis of the mitral valve in lupus erythematosus. The vegetations attached to the margin of the thickened valve leaflet are indicated by arrows. (Courtesy of Dr. Fred Schoen, Department of Pathology, Brigham and Women's Hospital, Boston, MA.) Figure 6-38 Sjögren syndrome. A, Enlargement of the salivary gland. (Courtesy of Dr. Richard Sontheimer, Department of Dermatology, University of Texas Southwestern Medical School, Dallas, TX.) B, Intense lymphocytic and plasma cell infiltration with ductal epithelial hyperplasia in a salivary gland. (Courtesy of Dr. Dennis Burns, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 6-39 Schematic illustration of the possible mechanisms leading to systemic sclerosis. Figure 6-40 Systemic sclerosis. A, Normal skin. B, Skin biopsy from a patient with systemic sclerosis. Note the extensive deposition of dense collagen in the dermis with virtual absence of appendages (e.g. hair follicles) and foci of inflammation (arrow). Figure 6-41 Advanced systemic sclerosis. The extensive subcutaneous fibrosis has virtually immobilized the fingers, creating a clawlike flexion deformity. Loss of blood supply has led to cutaneous ulcerations. (Courtesy of Dr. Richard Sontheimer, Department of Dermatology, University of Texas Southwestern Medical School, Dallas, TX.) TABLE 6-11 -- Examples of Infections in Immunodeficiencies Pathogen Type T-Cell-Defect B-Cell Defect Bacteria Bacterial sepsis Streptococci, staphylococci, Haemophilus Viruses Cytomegalovirus, Epstein-Barr virus, severe varicella, chronic infections with respiratory and intestinal viruses Enteroviral encephalitis Fungi and parasites Candida, Pneumocystis carinii Severe intestinal giardiasis Granulocyte Defect Staphylococci, Pseudomonas Candida, Nocardia, Aspergillus Complement Defect Neisserial infections, other pyogenic bacterial infections Special features Aggressive disease with opportunistic pathogens, failure to clear infections Recurrent sinopulmonary infections, sepsis, chronic meningitis From Puck JM: Primary immunodeficiency diseases. JAMA 278:1835, 1997. Copyright 1997, American Medical Association. of T cells are often indistinguishable clinically from combined deficiencies of T and B cells. Although originally thought to be quite rare, some forms, such as IgA deficiency, are common, and collectively they are a significant health problem, especially in children. Most primary immunodeficiencies manifest themselves in infancy, between 6 months and 2 years of life, and they are detected because the affected infants are susceptible to recurrent infections. The nature of infecting organisms depends to some extent on the nature of the underlying defect, as summarized in Table 6-11 . Detailed classification of the primary immunodeficiencies according to the suggested cellular defect may be found in the WHO report on immunodeficiency. [92] Defects of phagocytes were discussed in Chapter 2 . Here we present selected examples of other immunodeficiencies. We begin with isolated defects in B cells, followed by a discussion of combined immunodeficiencies and defects in complement proteins. Finally, Wiskott-Aldrich syndrome, a complex disorder affecting lymphocytes as well as platelets, is presented. With rapid advances in genetic analyses, in the past ten years the mutations responsible for many primary immunodeficiencies have been identified.[ 93] X-Linked Agammaglobulinemia of Bruton 94 X-linked agammaglobulinemia is one of the more common forms of primary immunodeficiency.[ ] It is characterized by the failure of B-cell precursors (pro-B cells and pre-B cells) to mature into B cells. During normal B-cell maturation in the bone marrow, the immunoglobulin heavy-chain genes are rearranged first, followed by rearrangement of the light chain genes. In X-linked agammaglobulinemia, B-cell maturation stops after the rearrangement of heavy chain genes. Because light chains are not produced, the complete immunoglobulin molecule (which contains heavy and light chains) cannot be assembled and transported to the cell membrane. Free heavy chains can be found in the cytoplasm. This block in differentiation is due to 95 mutations in a cytoplasmic tyrosine kinase, called B-cell tyrosine kinase (Btk).[ ] Btk is a protein tyrosine kinase associated with the antigen receptor complex of pre-B and mature B cells. It is needed to transduce signals from the antigen receptor that are critical for driving maturation. When it is mutated, the pre-B cell receptor cannot deliver signals, and maturation stops at this stage. The BTK gene maps to the long arm of the X chromosome at Xq21.22. 241 Figure 6-42 Scheme of lymphocyte development and sites of block in primary immunodeficiency diseases. The affected genes are indicated in parentheses for some of the disorders. ADA, adenosine deaminase; CD40L, CD40 ligand; SCID, severe combined immunodeficiency. Figure 6-43 Schematic illustration of an HIV-1 virion. The viral particle is covered by a lipid bilayer that is derived from the host cell. Figure 6-44 HIV proviral genome. Several viral genes and their corresponding functions are illustrated. The genes outlined in red are unique to HIV; others are shared by all retroviruses. Figure 6-45 Pathogenesis of HIV-1 infection. Initially, HIV-1 infects T cells and macrophages directly or is carried to these cells by Langerhans cells. Viral replication in the regional lymph nodes leads to viremia and widespread seeding of lymphoid tissue. The viremia is controlled by the host immune response (not shown), and the patient then enters a phase of clinical latency. During this phase, viral replication in both T cells and macrophages continues unabated, but there is some immune containment of virus (not illustrated). There continues a gradual erosion of CD4+ cells by productive infection (or other mechanisms, not shown). Ultimately, CD4+ cell numbers decline, and the patient develops clinical symptoms of full-blown AIDS. Macrophages are also parasitized by the virus early; they are not lysed by HIV-1, and they may transport the virus to tissues, particularly the brain. Figure 6-46 Mechanism of HIV entry into host cells. Interactions with CD4 and CCR5 coreceptor are illustrated. (Adapted with permission from Wain-Hobson S: HIV. One on one meets two. Nature 384:117, 1996. Copyright 1996, Macmillam Magazines Limited.) Figure 6-47 The life cycle of HIV. The steps from viral entry to production of infectious virions are illustrated. Figure 6-48 Mechanisms of CD4 cell loss in HIV infection. Figure 6-49 HIV infection showing the formation of giant cells in the brain. (Courtesy of Dr. Dennis Burns, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) TABLE 6-12 -- Major Abnormalities of Immune Function in AIDS Lymphopenia Predominantly due to selective loss of the CD4+ helper-inducer T-cell subset; inversion of CD4:CD8 ratio Decreased T-Cell Function In Vivo Preferential loss of memory T cells Susceptibility to opportunistic infections Susceptibility to neoplasms Decreased delayed-type hypersensitivity Altered T-Cell Function In Vitro Decreased proliferative response to mitogens, alloantigens, and soluble antigens Decreased specific cytotoxicity Decreased helper function for pokeweed mitogen-induced B-cell immunoglobulin production Decreased IL-2 and TFN-γ production Polyclonal B-Cell Activation Hypergammaglobulinemia and circulating immune complexes Inability to mount de novo antibody response to a new antigen or vaccine Refractoriness to the normal signals for B-cell activation in vitro Altered Monocyte or Macrophage Functions Decreased chemotaxis and phagocytosis Decreased HLA class II antigen expression Diminished capacity to present antigen to T cells Increased spontaneous secretion of IL-1, TNF, IL-6 HIV infection of macrophages has three important implications. First, monocytes and macrophages represent a veritable virus factory and reservoir, whose output remains largely protected from host defenses. Second, macrophages provide a safe vehicle for HIV to be transported to various parts of the body, including the nervous system. Third, in late stages of HIV infection, when the CD4+ T-cell numbers decline greatly, macrophages may be an important site of continued viral replication.[ 135] In contrast to tissue macrophages, the number of monocytes in circulation infected by HIV is low, yet there are unexplained functional defects that have important consequences for host defense. These defects include impaired microbicidal activity, decreased chemotaxis, decreased secretion of IL-1, inappropriate secretion of TNF, and, most important, poor capacity to present antigens to T cells. Studies have documented that, in addition to macrophages, two types of dendritic cells are also important targets for the initiation and maintenance of HIV infection: mucosal and follicular dendritic cells. It is thought that mucosal dendritic cells are infected by the virus and transport it to regional lymph 253 nodes, where CD4+ T cells are infected.[ 136] Dendritic cells also express a lectin-like receptor that specifically binds HIV and displays it in an intact, infectious form to T cells, thus 137 cells.[ ] 138 promoting infection of the T Follicular dendritic cells in the germinal centers of lymph nodes are, similar to macrophages, important reservoirs of HIV.[ ] Although some follicular dendritic cells may be susceptible to HIV infection, most virus particles are found on the surface of their dendritic processes. Follicular dendritic cells have receptors for the Fc portion of immunoglobulins, and hence they trap HIV virions coated with anti-HIV antibodies. The antibody-coated virions localized to follicular dendritic cells retain the ability to infect CD4+ T cells as they traverse the intricate meshwork formed by the dendritic processes of the follicular dendritic cells. To summarize, CD4+ T cells, macrophages, and follicular dendritic cells contained in the lymphoid tissues are the major sites of HIV infection and persistence. Although much attention has been focused on T cells, macrophages, and dendritic cells because they can be infected by HIV, patients with AIDS also display profound abnormalities of Bcell function. Paradoxically, these patients have hypergammaglobulinemia and circulating immune complexes owing to polyclonal B-cell activation. This may result from multiple interacting factors: reactivation of or reinfection with cytomegalovirus and EBV, both of which are polyclonal B-cell activators, can occur; gp41 itself can promote B-cell growth and differentiation; and HIV-infected macrophages produce increased amounts of IL-6, which stimulates proliferation of B cells. Despite the presence of spontaneously activated B cells, patients with AIDS are unable to mount antibody responses to new antigens. This could be due, in part, to lack of T-cell help, but antibody responses against T-independent antigens are also suppressed, and hence there may be other defects in B cells as well. Impaired humoral immunity renders these patients prey to disseminated infections caused by encapsulated bacteria, such as S. pneumoniae and H. influenzae, both of which require antibodies for effective opsonization and clearance. Pathogenesis of Central Nervous System Involvement. 139 140 The pathogenesis of neurologic manifestations deserves special mention because, in addition to the lymphoid system, the nervous system is a major target of HIV infection.[ ] [ ] Macrophages and microglia, cells in the central nervous system that belong to the monocyte and macrophage lineage, are the predominant cell types in the brain that are infected with HIV. It is widely believed that HIV is carried into the brain by infected monocytes. In keeping with this, the HIV isolates from the brain are almost exclusively M-tropic. The mechanism of HIVinduced damage of the brain, however, remains obscure. Because neurons are not infected by HIV, and the extent of neuropathologic changes is often less than might be expected from the severity of neurologic symptoms, most workers believe that neurologic deficit is caused indirectly by viral products and by soluble factors produced by infected microglia. Included among the soluble factors are the usual culprits, such as IL-1, TNF, and IL-6. In addition, nitric oxide induced in neuronal cells by gp41 has been implicated. Direct damage of neurons by soluble HIV gp120 has also been postulated. According to some investigators, these diverse soluble neurotoxins act by triggering excessive entry of Ca2+ into the neurons through their action on glutamate-activated ion channels that regulate intracellular calcium. Natural History of HIV Infection The course of HIV infection can be best understood in terms of an interplay between HIV and the immune system. Three phases reflecting the dynamics of virus-host interaction can be 141 recognized: (1) an acute retroviral syndrome; (2) a middle, chronic phase; and (3) full-blown AIDS (see Fig. 6-45 ; also Fig. 6-50 ). [ ] We first present the cardinal features of the phases of HIV infection and their associated clinical syndromes then recount the sequential virologic and immunologic findings during the course of HIV infection. 142 The acute retroviral syndrome represents the initial or primary response of an immunocompetent adult to HIV infection. [ ] It is characterized initially by a high level of virus production, viremia, and widespread seeding of the lymphoid tissues. The initial infection, however, is readily controlled by the development of an antiviral immune response. It is estimated that 40% to 90% of individuals who acquire a primary infection develop the viral syndrome 3 to 6 weeks after infection, and this resolves spontaneously in 2 to 4 weeks. Clinically, this phase is associated with a self-limited acute illness with nonspecific symptoms, including sore throat, myalgias, fever, rash, weight loss, and fatigue, resembling a flulike syndrome. Other clinical features, such as rash, cervical adenopathy, diarrhea, and vomiting, may also occur. The middle chronic phase represents a stage of relative containment of the virus, associated with a period of clinical latency. The immune system is largely intact, but there is continuous HIV replication, predominantly in the lymphoid tissues, which may last for several years. Patients are either asymptomatic or develop persistent generalized lymphadenopathy. In addition, many patients have minor opportunistic infections, such as thrush and herpes zoster. Thrombocytopenia may also be noted ( Chapter 13 ). Persistent lymphadenopathy with significant constitutional symptoms (fever, rash, fatigue) reflects the onset of immune system decompensation, escalation of viral replication, and onset of the crisis phase. The final phase is progression to AIDS. It is characterized by a breakdown of host defense, a dramatic increase in plasma virus, and clinical disease. Typically the patient presents with longlasting fever (>1 month), fatigue, weight loss, and diarrhea. After a variable period, serious opportunistic infections, secondary neoplasms, or clinical neurologic disease (grouped under the rubric AIDS indicator diseases, discussed below) supervene, and the patient is said to have developed AIDS. In the absence of treatment, most but not all patients with HIV infection progress to AIDS after a chronic phase lasting from 7 to 10 years. Exceptions to this typical course are exemplified by long-term nonprogressors and by rapid progressors. Nonprogressors are defined as untreated HIV-1-infected individuals who remain asymptomatic for 10 years or more, with stable CD4 + counts and low levels of plasma viremia. In rapid progressors, the middle, chronic phase is telescoped to 2 to 3 years after primary infection. The possible basis for these variant outcomes is discussed later. With this overview of the phases of HIV disease, we can consider some details of host-parasite relationships during the course of a typical HIV infection. The initial entry of the virus may be through a mucosal surface, as in sexual intercourse (via rectal or cervical mucosa) or via blood exposure (e.g., after intravenous drug use). From the mucosal portal, the virus is carried to the regional lymph nodes by dendritic cells. 254 Figure 6-50 Typical course of HIV infection. A, During the early period after primary infection, there is widespread dissemination of virus and a sharp decrease in the number of CD4+ T cells in peripheral blood. An immune response to HIV ensues, with a decrease in viremia followed by a prolonged period of clinical latency. During this period, viral replication continues. The CD4+ T-cell count gradually decreases during the following years, until it reaches a critical level below which there is a substantial risk of opportunistic diseases. (Redrawn from Fauci AS, Lane HC: Human immunodeficiency virus disease: AIDS and related conditions. In Fauci AS, et al (eds): Harrison's Principles of Internal Medicine, 14th ed. New York, McGraw-Hill, 1997, p 1791.) B, Immune response to HIV infection. A cytolytic T lymphocyte (CTL) response to HIV is detectable by 2 to 3 weeks after the initial infection and peaks by 9 to 12 weeks. Marked expansion of virus-specific CD8+ T cell clones occurs during this time, and up to 10% of a patient's CTLs may be HIV specific at 12 weeks. The humoral immune response to HIV peaks at about 12 weeks. TABLE 6-13 -- CDC Classification Categories of HIV Infection CD4+ T-Cell Categories 1. ≥500/µL 2. 200–499/µL 3. ≤200/µL A. Asymptomatic, acute (primary) HIV, or persistent generalized lymphadenopathy A1 A2 A3 B. Symptomatic, not A or C conditions B1 B2 B3 Clinical Categories C. AIDS indicator conditions: including constitutional disease, neurologic disease, or secondary infection or neoplasm Data from CDC. Centers for Disease Control and Prevention: 1993 revised classification system and expanded surveillance definition for AIDS among adolescents and adults. MMWR 41 (RR-17): 1, 1992. CD4+ cell count and the development of AIDS, there is extensive turnover of the virus. In other words, HIV infection lacks a phase of true microbiologic latency, that is, a phase during which all the HIV is in the form of proviral DNA, and no cell is productively infected. Before this discussion of the virus-host relationships is ended, some comments on those patients who are considered long-term nonprogressors are in order. Individuals in this group remain asymptomatic for long periods of time (10 years or more), have low levels of viremia, and have stable CD4+ cell counts. People with such an uncommon clinical course have attracted great attention in the hope that studying them may shed light on host and viral factors that influence disease progression. Studies to date suggest that this group is heterogeneous with respect to the factors that influence the course of the disease. In a small subset of nonprogressors, the infecting HIV had deletions or mutations in the nef gene, suggesting that Nef proteins are critical to disease progression. In most cases, the viral isolates do not show any qualitative abnormalities. In all cases, there is evidence of a vigorous anti-HIV immune response, but the immune correlates of protection are still unknown. Some of these patients have high levels of HIV-specific CD8+ cells, and these levels are maintained over the course of infection. It is not clear whether the robust CD8+ cell response is the cause or consequence of the slow progression. Further studies, it is hoped, will provide the answers to this and other questions critical to disease progression. Clinical Features of AIDS The clinical manifestations of HIV infection can be readily surmised from the foregoing discussion. They range from a mild acute illness to severe disease. Because the salient clinical features of the acute early and chronic middle phases of HIV infection were described earlier, here we summarize the clinical manifestations of the terminal phase, AIDS. At the outset it should be pointed out that the clinical manifestations and opportunistic infections associated with HIV infection may differ in different parts of the world. Typically, HIV-infected individuals in Africa show a more rapid progression of the disease and a shorter survival time than in other geographic areas. Importantly, the clinical course of the disease has been greatly modified by new anti-retroviral therapies, and many complications that were once devestating are now infrequent. In the United States, the typical adult patient with AIDS presents with fever, weight loss, diarrhea, generalized lymphadenopathy, multiple opportunistic infections, neurologic disease and, in many cases, secondary neoplasms. The infections and neoplasms listed in Table 6-14 are included in the surveillance definition of AIDS.[ 149] Opportunistic infections account for the majority of deaths in patients with AIDS. The actual frequency of infections varies in different regions of the world, and has been greatly reduced by the advent of HAART.[ 150] A brief summary of selected opportunistic infections is provided here. Extensive reviews on the subject are available.[ 151] [152] Approximately 15% to 30% of HIV-infected people develop pneumonia caused by the opportunistic fungus P. carinii (representing reactivation of a prior latent infection), despite prophylaxis. Prior to HAART, this infection was the presenting feature in about 20% of cases, but the incidence is much less in patients who respond to HAART. The risk of developing this infection is extremely high in individuals with fewer than 200 CD4+ cells/µL. Even in these patients there has been a substantial decline in the incidence of this infection because of effective prophylaxis. An increasing number of patients present with an opportunistic infection other than P. carinii pneumonia. Among the most common pathogens are Candida, cytomegalovirus, TABLE 6-14 -- AIDS-Defining Opportunistic Infections and Neoplasms Found in Patients with HIV Infection INFECTIONS Protozoal and Helminthic Infections Cryptosporidiosis or isosporidiosis (enteritis) Pneumocytosis (pneumonia or disseminated infection) Toxoplasmosis (pneumonia or CNS infection) Fungal Infections Candidiasis (esophageal, tracheal, or pulmonary) Cryptococcosis (CNS infection) Coccidioidomycosis (disseminated) Histoplasmosis (disseminated) Bacterial Infections Mycobacteriosis (atypical, e.g., M. avium-intracellulare, disseminated or extrapulmonary; M. tuberculosis, pulmonary or extrapulmonary) Nocardiosis (pneumonia, meningitis, disseminated) Salmonella infections, disseminated Viral Infections Cytomegalovirus (pulmonary, intestinal, retinitis, or CNS infections) Herpes simplex virus (localized or disseminated) Varicella-zoster virus (localized or disseminated) Progressive multifocal leukoencephalopathy) NEOPLASMS Kaposi sarcoma B-cell non-Hodgkin lymphomas Primary lymphoma of the brain Invasive cancer of uterine cervix CNS, central nervous system. 256 atypical and typical mycobacteria, Cryptococcus neoformans, Toxoplasma gondii, Cryptosporidium, herpes simplex virus, papovaviruses, and Histoplasma capsulatum. Candidiasis is the most common fungal infection in patients with AIDS. Candida infection of the oral cavity (thrush) and esophagus are the two most common clinical manifestations of candidiasis in HIV-infected patients. In asymptomatic HIV-infected individuals, oral candidiasis is a sign of immunologic decompensation, and it often heralds the transition to AIDS. Invasive candidiasis is not common in patients with AIDS, and it usually occurs when there is drug-induced neutropenia or use of indwelling catheters. Cytomegalovirus may cause disseminated disease, although, more commonly, it affects the eye and gastrointestinal tract. Chorioretinitis was seen in approximately 25% of patients pre-HAART, but this has decreased by over 50% after the intiation of HAART. Cytomegalovirus retinitis occurs almost exclusively in patients with CD4+ cell counts below 50/µl. Gastrointestinal disease, seen in 5% to 10% of cases, manifests as esophagitis and colitis, the latter associated with multiple mucosal ulcerations. Disseminated bacterial infection with atypical mycobacteria (mainly M. avium153 intracellulare) also occurs late, in the setting of severe immunosuppression. Coincident with the AIDS epidemic, the incidence of tuberculosis has risen dramatically. [ ] Worldwide, almost a third of all deaths in AIDS patients are attributable to tuberculosis; in the United states, about 5% of patients with AIDS develop active tuberculosis. Patients with AIDS have reactivation of latent pulmonary disease as well as outbreaks of primary infection. In contrast to infection with atypical mycobacteria, M. tuberculosis manifests itself early in the course of AIDS. As with tuberculosis in other settings, the infection may be confined to lungs or may involve multiple organs. The pattern of expression depends on the degree of immunosuppression; dissemination is more common in patients with very low CD4+ cell counts. Most worrisome are reports indicating that a growing number of isolates are resistant to multiple drugs. Cryptococcosis occurs in about 10% of AIDS patients. Among fungal infections that prey on HIV-infected individuals, it is second only to candidiasis. As in other settings with immunosuppression, meningitis is the major clinical manifestation of cryptococcosis. In contrast to Cryptococcus, T. gondii, another frequent invader of the central nervous system in AIDS, causes encephalitis and is responsible for 50% of all mass lesions in the central nervous system. JC virus, a human papovavirus, is another important cause of central nervous system infections in HIV-infected patients. It causes progressive multifocal leukoencephalopathy ( Chapter 28 ). Herpes simplex virus infection is manifested by mucocutaneous ulcerations involving the mouth, esophagus, external genitalia, and perianal region. Persistent diarrhea, so common in patients with AIDS, is often caused by infections with protozoans such as Cryptosporidium, Isospora belli, or microsporidia. These patients have chronic, profuse, watery diarrhea with massive fluid loss. Diarrhea may also result from infection with enteric bacteria, such as Salmonella and Shigella, as well as M. avium-intracellulare. Depressed humoral immunity renders AIDS patients susceptible to severe, recurrent bacterial pneumonias. 154 155 Patients with AIDS have a high incidence of certain tumors, especially Kaposi sarcoma (KS), non-Hodgkin B-cell lymphoma, cervical cancer in women, and anal cancer in men.[ ] [ ] It is estimated that 25% to 40% of HIV-infected individuals will eventually develop a malignancy. A common feature of these tumors is that they are all believed to be caused by oncogenic DNA viruses, that is, Kaposi sarcoma herpesvirus (Kaposi sarcoma), EBV (B-cell lymphoma), human papillomavirus (cervical and anal carcinoma). The increased risk of malignancy is thus mainly a consequence of increased susceptibility to infections by these viruses and decreased immunity against the tumors. KS, a vascular tumor that is otherwise rare in the United States, is the most common neoplasm in patients with AIDS. The morphology of KS and its occurrence in patients not infected with HIV are discussed in Chapter 11 . At the onset of the AIDS epidemic, up to 30% of infected homosexual or bisexual men had KS, but in recent years, with use of HAART there has been a marked decline in its incidence, from 15 cases per 1000 person years to less than 5 cases. The lesions of KS are characterized by the proliferation of spindle-shaped cells that express markers of both endothelial (vascular or lymphatic) and smooth muscle lineages. There is also a profusion of slit-like vascular spaces, suggesting that the lesions may arise from primitive mesenchymal precursors of vascular channels. In addition, KS lesions display chronic inflammatory cell infiltrates. There is still some debate about whether the lesions represent an exuberant hyperplasia or a malignant neoplasm, but the weight of evidence favors the former. For instance, spindle cells in many KS lesions are polyclonal or oligoclonal, although more advanced lesions occasionally show monoclonality.[ 156] Moreover, spindle cells in many KS [157] lesions are diploid, dependent on growth factors for their proliferation, and do not form tumors in immunodeficient mice. When KS cells are implanted subcutaneously in such mice, they transiently induce slit-like new blood vessels and inflammatory infiltrates in the surrounding tissue; these elements recall features of human KS, but interestingly are of murine origin. When the human KS cells involute, these elements also regress. These observations suggest that KS pathogenesis involves a complex web of paracrine signaling interactions among different types of cells, no one of which is fully autonomous. One popular view envisions that spindle cells produce pro-inflammatory and angiogenic factors, recruiting the inflammatory 157] and neovascular components of the lesion, while the latter components supply signals that aid in spindle cell survival or growth[ ( Fig. 6-51 ). But what initiates this cycle of events? Clues to this came from the observation that not all HIV patients are at equal risk for KS development. AIDS-related KS is twenty times more frequent in individuals who acquire HIV by sexual routes compared to those who acquire it parenterally. This observation suggested that a sexually transmitted agent other than HIV might 158 be implicated in KS etiology and prompted a search for new viruses in KS. This search yielded a novel herpesvirus, aptly labeled KS herpesvirus (KSHV), or human herpesvirus 8.[ ] Epidemiologic studies strongly link KSHV to KS development. Infection is uncommon in the general population and strikingly increased in prevalence in groups in which KS is common. In individual patients, KSHV infection precedes KS development and is highly correlated with increased KS risk. KSHV DNA is found in virtually all KS lesions, including those that occur in HIV-negative populations. In the lesions, KSHV is strikingly localized to the spindle cells, which display predominantly latent infection.[ 159] Thus, 257 Figure 6-51 Proposed role of HIV, KSHV (HHV8), and cytokines in the pathogenesis of Kaposi sarcoma. Cytokines are produced by the mesenchymal cells infected by KSHV, or by HIVinfected CD4+ cells. B cells may also be infected by KSHV; their role in the disease is unclear. Figure 6-52 Amyloidosis. A, A section of the liver stained with Congo red reveals pink-red deposits of amyloid in the walls of blood vessels and along sinusoids. B, Note the yellow-green birefringence of the deposits when observed by polarizing microscope. (Courtesy of Dr. Trace Worrell and Sandy Hinton, Department of Pathology, University of Texas Southwestern Medical School, Dallas TX.) Figure 6-53 Structure of an amyloid fibril, depicting the β-pleated sheet structure and binding sites for the Congo red dye, which is used for diagnosis of amyloidosis. (Modified from Glenner GG: Amyloid deposit and amyloidosis. The β-fibrilloses. N Engl J Med 52:148, 1980. By permission of The New England Journal of Medicine.) TABLE 6-15 -- Classification of Amyloidosis Clinicopathologic Category Associated Diseases Major Fibril Protein Chemically Related Precursor Protein Systemic (Generalized) Amyloidosis Immunocyte dyscrasias with amyloidosis (primary amyloidosis) Multiple myeloma and other monoclonal Bcell proliferations AL Immunoglobulin light chains, chiefly λ type Reactive systemic amyloidosis (secondary amyloidosis) Chronic inflammatory conditions AA SAA Hemodialysis-associated amyloidosis Chronic renal failure Aβ2 m β2 -microglobulin Familial Mediterranean fever — AA SAA Familial amyloidotic neuropathies (several types) — ATTR Transthyretin Systemic senile amyloidosis — ATTR Transthyretin Alzheimer disease Aβ APP Hereditary amyloidosis Localized Amyloidosis Senile cerebral Endocrine ••Medullary carcinoma of thyroid — A Cal Calcitonin ••Islet of Langerhans Type II diabetes AIAPP Islet amyloid peptide Isolated atrial amyloidosis — AANF Atrial natriuretic factor Prion diseases Various prion diseases of the CNS Misfolded prion protein (PrPSC ) Normal prion protein PrP In addition, other minor components are always present in amyloid. These include serum amyloid P component, proteoglycans, and highly sulfated glycosaminoglycans. Serum amyloid P protein may contribute to amyloid deposition by stabilizing the fibrils and decreasing their clearance. Classification of Amyloidosis. According to devoted "amyloidologists," who congregate every few years to discuss their favorite protein, amyloid should be classified based on its constituent chemical fibrils into 170 categories such as AL, AA, and ATTR and not based on clinical syndromes.[ ] Because a given biochemical form of amyloid (e.g., AA) may be associated with amyloid deposition in diverse clinical settings, we follow a combined biochemical-clinical classification for our discussion ( Table 6-15 ). Amyloid may be systemic (generalized), involving several organ systems, or it may be localized, when deposits are limited to a single organ, such as the heart. As should become evident, several different biochemical forms of amyloid are encompassed by such segregation. On clinical grounds, the systemic, or generalized, pattern is subclassified into primary amyloidosis, when associated with some immunocyte dyscrasia, or secondary amyloidosis, when it occurs as a complication of an underlying chronic inflammatory or tissue destructive process. Hereditary or familial amyloidosis constitutes a separate, albeit heterogeneous group, with several distinctive patterns of organ involvement. Immunocyte Dyscrasias with Amyloidosis (Primary Amyloidosis). Amyloid in this category is usually systemic in distribution 261 and is of the AL type. With approximately 1275 to 3200 new cases every year in the United States, this is the most common form of amyloidosis. In many of these cases, the patients have some form of plasma cell dyscrasia. Best defined is the occurrence of systemic amyloidosis in 5% to 15% of patients with multiple myeloma, a plasma-cell tumor characterized by multiple osteolytic lesions throughout the skeletal system ( Chapter 14 ). The malignant B cells characteristically synthesize abnormal amounts of a single specific immunoglobulin (monoclonal gammopathy), producing an M (myeloma) protein spike on serum electrophoresis. In addition to the synthesis of whole immunoglobulin molecules, only the light chains (referred to as Bence Jones protein) of either the λ or the κ variety may be elaborated and found in the serum. By virtue of the small molecular size of the Bence Jones protein, it is frequently excreted in the urine. The amyloid deposits contain the same light chain protein. Almost all the patients with myeloma who develop amyloidosis have Bence Jones proteins in the serum or urine, or both, but a great majority of myeloma patients who have free light chains do not develop amyloidosis. Clearly, therefore, the presence of Bence Jones proteins, although necessary, is by itself not enough to produce amyloidosis. We discuss later the other factors, such as the type of light chain produced (amyloidogenic potential) and the subsequent handling (possibly degradation) that may have a bearing on whether Bence Jones proteins are deposited as amyloid. The great majority of patients with AL amyloid do not have classic multiple myeloma or any other overt B-cell neoplasm; such cases have been traditionally classified as primary amyloidosis because their clinical features derive from the effects of amyloid deposition without any other associated disease. In virtually all such cases, however, monoclonal immunoglobulins or free light chains, or both, can be found in the serum or urine. Most of these patients also have a modest increase in the number of plasma cells in the bone marrow, which presumably secrete the precursors of AL protein. Clearly, these patients have an underlying B-cell dyscrasia in which production of an abnormal protein, rather than production of tumor masses, is the predominant manifestation. Recent studies have revealed chromosomal translocations in many of these patients, suggesting the presence of neoplastic clones.[ Whether most of these clones would evolve into myeloma if the patients lived long enough can only be a matter for speculation. 171] Reactive Systemic Amyloidosis. The amyloid deposits in this pattern are systemic in distribution and are composed of AA protein. This category was previously referred to as secondary amyloidosis because it is secondary to an associated inflammatory condition. The feature common to most of the conditions associated with reactive systemic amyloidosis is protracted breakdown of cells resulting from a wide variety of infectious and noninfectious chronic inflammatory conditions. At one time, tuberculosis, bronchiectasis, and chronic osteomyelitis were the most important underlying conditions, but with the advent of effective antimicrobial chemotherapy, the importance of these conditions has diminished. More commonly now, reactive systemic amyloidosis complicates rheumatoid arthritis, other connective tissue disorders such as ankylosing spondylitis, and inflammatory bowel disease, particularly Crohn disease and ulcerative colitis. Among these, the most frequent associated condition is rheumatoid arthritis. Amyloidosis is reported to occur in approximately 3% of patients with rheumatoid arthritis and is clinically significant in one half of those affected. Heroine abusers who inject the drug subcutaneously also have a high occurrence rate of generalized AA amyloidosis. The chronic skin infections associated with "skin-popping" of narcotics seem to be responsible for amyloidosis in this group of patients. Reactive systemic amyloidosis may also occur in association with nonimmunocyte-derived tumors, the two most common being renal cell carcinoma and Hodgkin disease. Hemodialysis-Associated Amyloidosis. Patients on long-term hemodialysis for renal failure develop amyloidosis owing to deposition of β2 -microglobulin. This protein is present in high concentrations in the serum of patients with renal disease and is retained in circulation because it cannot be filtered through the cuprophane dialysis membranes. In some series, as many as 60% to 80% of the patients on longterm dialysis developed amyloid deposits in the synovium, joints, and tendon sheaths. Heredofamilial Amyloidosis. A variety of familial forms of amyloidosis have been described. Most of them are rare and occur in limited geographic areas. The most common and best studied is an autosomal recessive 172 condition called familial Mediterranean fever.[ ] This is a febrile disorder of unknown cause characterized by attacks of fever accompanied by inflammation of serosal surfaces, including peritoneum, pleura, and synovial membrane. This disorder is encountered largely in individuals of Armenian, Sephardic Jewish, and Arabic origins. It is associated with widespread tissue involvement indistinguishable from reactive systemic amyloidosis. The amyloid fibril proteins are made up of AA proteins, suggesting that this form of amyloidosis is related to the recurrent bouts of inflammation that characterize this disease. The gene for familial Mediterranean fever has been cloned, and its product is called pyrin (for its relation to fever). Although its exact function is not known, it has been suggested that pyrin is responsible for regulating acute inflammation, presumably by inhibiting the function of neutrophils.[ of this mutation to the disease is not understood. 173] The relationship In contrast to familial Mediterranean fever, a group of autosomal dominant familial disorders is characterized by deposition of amyloid predominantly in the nerves—peripheral and autonomic. These familial amyloidotic polyneuropathies have been described in different parts of the world. As mentioned previously, in all of these genetic disorders, the fibrils are made up of mutant transthyretins (ATTR). Localized Amyloidosis. Sometimes, amyloid deposits are limited to a single organ or tissue without involvement of any other site in the body. The deposits may produce grossly detectable nodular masses or be evident only on microscopic examination. Nodular (tumor-forming) deposits of amyloid are most often encountered in the lung, larynx, skin, urinary bladder, tongue, and the region about the eye. Frequently, there are infiltrates of lymphocytes and plasma cells in the periphery of these amyloid masses, raising the question of whether the mononuclear infiltrate is a response to the deposition of amyloid or instead is responsible for it. At least in some cases, the amyloid consists of AL protein and may therefore represent a localized form of immunocyte-derived amyloid. Endocrine Amyloid. Microscopic deposits of localized amyloid may be found in certain endocrine tumors, such as 262 medullary carcinoma of the thyroid gland, islet tumors of the pancreas, pheochromocytomas, and undifferentiated carcinomas of the stomach, and in the islets of Langerhans in patients with type II diabetes mellitus. In these settings, the amyloidogenic proteins seem to be derived either from polypeptide hormones (e.g., medullary carcinoma) or from unique proteins (e.g., islet amyloid polypeptide). Amyloid of Aging. 174 Several well-documented forms of amyloid deposition occur with aging.[ ] Senile systemic amyloidosis refers to the systemic deposition of amyloid in elderly patients (usually in their seventies and eighties). Because of the dominant involvement and related dysfunction of the heart, this form was previously called senile cardiac amyloidosis. Those who are symptomatic present with a restrictive cardiomyopathy and arrhythmias. The amyloid in this form is composed of the normal TTR molecule. In addition to the sporadic senile systemic amyloidosis, another form, affecting predominantly the heart, that results from the deposition of a mutant form of TTR has also been recognized. Approximately 4% of the black population in the United States is a carrier of the mutant allele, and cardiomyopathy has been identified in both homozygous and heterozygous patients. The precise prevalance of patients with this mutation who develop clinically manifest cardiac disease is not known. Pathogenesis. Amyloidosis results from abnormal folding of proteins, which are deposited as fibrils in extracellular tissues and disrupt normal function. Misfolded proteins are often unstable and selfassociate, ultimately leading to the formation of oligomers and fibrils that are deposited in tissues. The reason diverse conditions are associated with amyloidosis may be that each of these conditions results in excessive production of proteins that are prone to misfolding. The proteins that form amyloid fall into two general categories: (1) normal proteins that have an inherent tendency to fold improperly, associate and form fibrils, and do so when they are produced in Figure 6-54 Proposed schema of the pathogenesis of the major forms of amyloid fibrils. Figure 6-55 Amyloidosis of the kidney. The glomerular architecture is almost totally obliterated by the massive accumulation of amyloid. Figure 6-56 Cardiac amyloidosis. The atrophic myocardial fibers are separated by structureless, pink-staining amyloid (arrows). References 1. Janeway CA, Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 20:197, 2002. 2. Takeda K, Kaisho T, Akira S: Toll-like receptors. Ann Rev Immunol 21:335, 2003. 3. Cyster JG: Chemokines and cell migration in secondary lymphoid organs. Science 286:2098, 1999. 4. Mebius RE: Organogenesis of lymphoid tissues. Nat Rev Immurol 3:292, 2003. 5. Davis MM, et al: Ligand recognition by alpha beta T cell receptors. 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Gilmore JD, et al: Amyloid load and clinical outcome in AA amyloidosis in relation to circulating concentration of serum amyloid A protein. Lancet 358:24, 2001. 268 269 Chapter 7 - Neoplasia 270 1 2 In the year 2000, there were 10 million new cases of cancer and 6 million cancer deaths worldwide.[ ] [ ] In the United States each year, almost 1.5 million individuals learn for the first time that they have some type of cancer. Not included in these figures are more than 1 million new cases of the most common types of nonpigmented skin cancers and incipient, noninvasive cancers. Not only these noninvasive lesions but many invasive tumors as well can be cured. Nonetheless, according to American Cancer Society estimates, cancer caused 3 approximately 556,000 deaths in 2003, corresponding to 1500 cancer deaths per day, accounting for about 23% of all deaths in the United States.[ ] Some good news, however, has 4 emerged: cancer mortality for both men and women in the United States declined during the last decade of the 20th century.[ ] Thus, there has been progress, but the problem is still overwhelming. The discussion that follows deals with both benign tumors and cancers; the latter receive more attention. The focus is on the basic morphologic and biologic properties of tumors and on the present understanding of the molecular basis of carcinogenesis. We also discuss the interactions of the tumor with the host and the host response to tumors. Although the discussion of therapy is beyond the scope of this chapter, there are now dramatic improvements in therapeutic responses and 5-year survival rates with many forms of malignancy, notably the leukemias and lymphomas. A greater proportion of cancers is being cured or arrested today than ever before. Definitions Neoplasia literally means the process of "new growth," and a new growth is called a neoplasm. The term tumor was originally applied to the swelling caused by inflammation. Neoplasms also may induce swellings, but by long precedent, the non-neoplastic usage of tumor has passed into limbo; thus, the term is now equated with neoplasm. Oncology (Greek oncos = tumor) is the study of tumors or neoplasms. Cancer is the common term for all malignant tumors. Although the ancient origins of this term are somewhat uncertain, it probably derives from the Latin for crab, cancer—presumably because a cancer "adheres to any part that it seizes upon in an obstinate manner like the crab." 5 Although all physicians know what they mean when they use the term neoplasm, it has been surprisingly difficult to develop an accurate definition. The eminent British oncologist Willis[ ] has come closest: "A neoplasm is an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which evoked the change." We know that the persistence of tumors, even after the inciting stimulus is gone, results from heritable genetic alterations that are passed down to the progeny of the tumor cells. These genetic changes allow excessive and unregulated proliferation that becomes autonomous (independent of physiologic growth stimuli), although tumors generally remain dependent on the host for their nutrition and blood supply. As we shall discuss later, the entire population of cells within a tumor arises from a single cell that has incurred genetic change, and hence tumors are said to be clonal. Nomenclature All tumors, benign and malignant, have two basic components: (1) proliferating neoplastic cells that constitute their parenchyma and (2) supportive stroma made up of connective tissue and blood vessels. Although parenchymal cells represent the proliferating "cutting edge" of neoplasms and so determine their behavior and pathologic consequences, the growth and evolution of neoplasms are critically dependent on their stroma. An adequate stromal blood supply is requisite, and the stromal connective tissue provides the framework for the parenchyma. In addition, there is cross-talk between tumor cells and stromal cells that appears to directly influence the growth of tumors. In some tumors, the stromal support is scant and so the neoplasm is soft and fleshy. Sometimes the parenchymal cells stimulate the formation of an abundant collagenous stroma, referred to as desmoplasia. Some tumors—for example, some cancers of the female breast—are stony hard or scirrhous. The nomenclature of tumors is, however, based on the parenchymal component. Benign Tumors. In general, benign tumors are designated by attaching the suffix -oma to the cell of origin. Tumors of mesenchymal cells generally follow this rule. For example, a benign tumor arising from fibroblastic cells is called a fibroma, a cartilaginous tumor is a chondroma, and a tumor of osteoblasts is an osteoma. In contrast, nomenclature of benign epithelial tumors is more complex. They are variously classified, some based on their cells of origin, others on microscopic architecture, and still others on their macroscopic patterns. Adenoma is the term applied to a benign epithelial neoplasm that forms glandular patterns as well as to tumors derived from glands but not necessarily reproducing glandular patterns. On this basis, a benign epithelial neoplasm that arises from renal tubular cells growing in the form of numerous tightly clustered small glands would be termed an adenoma, as would a heterogeneous mass of adrenal cortical cells growing in no distinctive pattern. Benign epithelial neoplasms producing microscopically or macroscopically visible finger-like or warty projections from epithelial surfaces are referred to as papillomas ( Fig. 7-1 ). Those that form large cystic masses, as in the ovary, are referred to as cystadenomas. Figure 7-1 Papilloma of the colon with finger-like projections into the lumen. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-2 Colonic polyp. A, This benign glandular tumor (adenoma) is projecting into the colonic lumen and is attached to the mucosa by a distinct stalk. B, Gross appearance of several colonic polyps. Figure 7-3 This mixed tumor of the parotid gland contains epithelial cells forming ducts and myxoid stroma that resembles cartilage. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-4 A, Gross appearance of an opened cystic teratoma of the ovary. Note the presence of hair, sebaceous material, and tooth. B, A microscopic view of a similar tumor shows skin, sebaceous glands, fat cells, and a tract of neural tissue (arrow). TABLE 7-1 -- Nomenclature of Tumors Tissue of Origin Benign Malignant Composed of One Parenchymal Cell Type Tumors of mesenchymal origin ••Connective tissue and derivatives Fibroma Fibrosarcoma Lipoma Liposarcoma Chondroma Chondrosarcoma Osteoma Osteogenic sarcoma ••Blood vessels Hemangioma Angiosarcoma ••Lymph vessels Lymphangioma Lymphangiosarcoma Endothelial and related tissues ••Synovium Synovial sarcoma ••Mesothelium Mesothelioma ••Brain coverings Blood cells and related cells Meningioma Invasive meningioma ••Hematopoietic cells Leukemias ••Lymphoid tissue Lymphomas Muscle ••Smooth Leiomyoma Leiomyosarcoma ••Striated Rhabdomyoma Rhabdomyosarcoma Squamous cell papilloma Squamous cell or epidermoid carcinoma Tumors of epithelial origin ••Stratified squamous ••Basal cells of skin or adnexa ••Epithelial lining of glands or ducts Basal cell carcinoma Adenoma Adenocarcinoma Papilloma Papillary carcinomas Cystadenoma Cystadenocarcinoma ••Respiratory passages Bronchial adenoma Bronchogenic carcinoma ••Renal epithelium Renal tubular adenoma Renal cell carcinoma ••Liver cells Liver cell adenoma Hepatocellular carcinoma ••Urinary tract epithelium (transitional) Transitional cell papilloma Transitional cell carcinoma ••Placental epithelium Hydatidiform mole Choriocarcinoma ••Testicular epithelium (germ cells) Seminoma Embryonal carcinoma Tumors of melanocytes Nevus Malignant melanoma Pleomorphic adenoma (mixed tumor of salivary origin) Malignant mixed tumor of salivary gland origin More Than One Neoplastic Cell Type—Mixed Tumors, Usually Derived from One Germ Cell Layer Salivary glands Renal anlage Wilms tumor More Than One Neoplastic Cell Type Derived from More Than One Germ Cell Layer—Teratogenous Totipotential cells in gonads or in embryonic rests Mature teratoma, dermoid cyst Immature teratoma, teratocarcinoma have primitive-appearing, unspecialized cells. In general, benign tumors are well differentiated ( Fig. 7-6 ). The neoplastic cell in a benign smooth muscle tumor—a leiomyoma—so closely resembles the normal cell that it may be impossible to recognize it as a tumor by microscopic examination of individual cells. Only the massing of these cells into a nodule discloses the neoplastic nature of the lesion. One may get so close to the tree that one loses sight of the forest. Malignant neoplasms, in contrast, range from well differentiated to undifferentiated. Malignant neoplasms composed of undifferentiated cells are said to be anaplastic. Lack of differentiation, or anaplasia, is considered a hallmark of malignant transformation. Anaplasia literally means "to form backward," implying a reversion from a high level of differentiation to a lower level. There is substantial evidence, however, that most cancers do not represent "reverse differentiation" of mature normal cells but, in fact, arise from stem cells that are present in all specialized tissues. The well-differentiated cancer ( Fig. 7-7 ) evolves from maturation or specialization of undifferentiated cells as they proliferate, whereas the undifferentiated malignant tumor derives from proliferation without complete maturation of the transformed cells. Lack of differentiation, or anaplasia, is marked by a number of morphologic changes. • Pleomorphism. Both the cells and the nuclei characteristically display pleomorphism—variation in size and shape ( Fig. 7-8 ). Cells may be found that are many times larger than their neighbors, and other cells may be extremely small and primitive appearing. 274 • Abnormal nuclear morphology. Characteristically the nuclei contain an abundance of DNA and are extremely dark staining (hyperchromatic). The nuclei are disproportionately large for the cell, and the nucleus-to-cytoplasm ratio may approach 1:1 instead of the normal 1:4 or 1:6. The nuclear shape is very variable, and the chromatin is often coarsely clumped and distributed along the nuclear membrane. Large nucleoli are usually present in these nuclei. • Mitoses. As compared with benign tumors and some well-differentiated malignant neoplasms, undifferentiated tumors usually possess large numbers of mitoses, reflecting the higher proliferative activity of the parenchymal cells. The presence of mitoses, however, does not necessarily indicate that a tumor is malignant or that the tissue is neoplastic. Many normal tissues exhibiting rapid turnover, such as bone marrow, have numerous mitoses, and non-neoplastic proliferations such as hyperplasias contain many cells in mitosis. More important as a morphologic feature of malignant neoplasia are atypical, bizarre mitotic figures, sometimes producing tripolar, quadripolar, or multipolar spindles ( Fig. 7-9 ). • Loss of polarity. In addition to the cytologic abnormalities, the orientation of anaplastic cells is markedly disturbed (i.e., they lose normal polarity). Sheets or large masses of tumor cells grow in an anarchic, disorganized fashion. • Other changes. Another feature of anaplasia is the formation of tumor giant cells, some possessing only a single huge polymorphic nucleus and others having two or more nuclei. These giant cells are not to be confused with inflammatory Langhans or foreign body giant cells, which are derived from macrophages and contain many small, normal-appearing nuclei. In the cancer giant cell, the nuclei are hyperchromatic and large in relation to the cell. Although growing tumor cells obviously require a blood 275 supply, often the vascular stroma is scant, and in many anaplastic tumors, large central areas undergo ischemic necrosis. Figure 7-5 Leiomyoma of the uterus. This benign, well-differentiated tumor contains interlacing bundles of neoplastic smooth muscle cells that are virtually identical in appearance to normal smooth muscle cells in the myometrium. Figure 7-6 Benign tumor (adenoma) of the thyroid. Note the normal-looking (well-differentiated), colloid-filled thyroid follicles. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-7 Malignant tumor (adenocarcinoma) of the colon. Note that compared with the well-formed and normal-looking glands characteristic of a benign tumor (see Fig. 7-6 ), the cancerous glands are irregular in shape and size and do not resemble the normal colonic glands. This tumor is considered differentiated because gland formation can be seen. The malignant glands have invaded the muscular layer of the colon. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-8 Anaplastic tumor of the skeletal muscle (rhabdomyosarcoma). Note the marked cellular and nuclear pleomorphism, hyperchromatic nuclei, and tumor giant cells. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-9 Anaplastic tumor showing cellular and nuclear variation in size and shape. The prominent cell in the center field has an abnormal tripolar spindle. Figure 7-10 Well-differentiated squamous cell carcinoma of the skin. The tumor cells are strikingly similar to normal squamous epithelial cells, with intercellular bridges and nests of keratin pearls (arrow). (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-11 A, Carcinoma in situ. This low-power view shows that the entire thickness of the epithelium is replaced by atypical dysplastic cells. There is no orderly differentiation of squamous cells. The basement membrane is intact and there is no tumor in the subepithelial stroma. B, A high-power view of another region shows failure of normal differentiation, marked nuclear and cellular pleomorphism, and numerous mitotic figures extending toward the surface. The basement membrane (below) is not seen in this section. Figure 7-12 Biology of tumor growth. The left panel depicts minimal estimates of tumor cell doublings that precede the formation of a clinically detectable tumor mass. It is evident that by the time a solid tumor is detected, it has already completed a major portion of its life cycle as measured by cell doublings. The right panel illustrates clonal evolution of tumors and generation of tumor cell heterogeneity. New subclones arise from the descendants of the original transformed cell, and with progressive growth the tumor mass becomes enriched for those variants that are more adept at evading host defenses and are likely to be more aggressive. (Adapted from Tannock IF: Biology of tumor growth. Hosp Pract 18:81, 1983.) Figure 7-13 Schematic representation of tumor growth. As the cell population expands, a progressively higher percentage of tumor cells leaves the replicative pool by reversion to G0 , differentiation, and death. Figure 7-14 Fibroadenoma of the breast. The tan-colored, encapsulated small tumor is sharply demarcated from the whiter breast tissue. Figure 7-15 Microscopic view of fibroadenoma of the breast seen in Figure 7-14 . The fibrous capsule (right) delimits the tumor from the surrounding tissue. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-16 Cut section of an invasive ductal carcinoma of the breast. The lesion is retracted, infiltrating the surrounding breast substance, and would be stony hard on palpation. Figure 7-17 The microscopic view of the breast carcinoma seen in Figure 7-16 illustrates the invasion of breast stroma and fat by nests and cords of tumor cells (compare with fibroadenoma shown in Fig. 7-15 ). The absence of a well-defined capsule should be noted. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-18 Colon carcinoma invading pericolonic adipose tissue. (Courtesy of Dr. Melissa Upton, University of Washington, Seattle, WA.) Figure 7-19 Axillary lymph node with metastatic breast carcinoma. The subcapsular sinus (top) is distended with tumor cells. Nests of tumor cells have also invaded the subcapsular cortex. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallas, TX.) Figure 7-20 A liver studded with metastatic cancer. Figure 7-21 Microscopic view of liver metastasis. A pancreatic adenocarcinoma has formed a metastatic nodule in the liver. (Courtesy of Dr. Trace Worrell, University of Texas Southwestern Medical School, Dallax, TX.) Figure 7-22 Comparison between a benign tumor of the myometrium (leiomyoma) and a malignant tumor of similar origin (leiomyosarcoma). TABLE 7-2 -- Comparisons Between Benign and Malignant Tumors Characteristics Benign Malignant Differentiation/anaplasia Well differentiated; structure may be typical of tissue of origin Some lack of differentiation with anaplasia; structure is often atypical Rate of growth Usually progressive and slow; may come to a standstill or regress; mitotic figures are rare and normal Erratic and may be slow to rapid; mitotic figures may be numerous and abnormal Local invasion Usually cohesive and expansile well-demarcated masses that do not invade or infiltrate surrounding normal tissues Locally invasive, infiltrating the surrounding normal tissues; sometimes may be seemingly cohesive and expansile Metastasis Absent Frequently present; the larger and more undifferentiated the primary, the more likely are metastases reasons discussed later, they do not indicate the inevitable development of metastases. The distinguishing features of benign and malignant tumors discussed in this overview are summarized in Table 7-2 and Figure 7-22 . With this background on the structure and behavior of neoplasms, we now discuss the origin of tumors, starting with insights gained from the epidemiology of cancer and followed by the molecular basis of carcinogenesis. Epidemiology Because cancer is a disorder of cell growth and behavior, its ultimate cause has to be defined at the cellular and subcellular levels. Study of cancer patterns in populations, however, 282 can contribute substantially to knowledge about the origins of cancer. For example, the concept that chemicals can cause cancer arose from the astute observations of Sir Percival Pott, who related the increased incidence of scrotal cancer in chimney sweeps to chronic exposure to soot. Thus, major insights into the cause of cancer can be obtained by epidemiologic studies that relate particular environmental, hereditary, and cultural influences to the occurrence of malignant neoplasms. In addition, certain diseases associated with an increased risk of developing cancer can provide insights into the pathogenesis of malignancy. Therefore, in the following discussion, we first summarize the overall incidence of cancer to provide an insight into the magnitude of the cancer problem, and then review a number of factors relating to both the patient and the environment that influence predisposition to cancer. CANCER INCIDENCE In some measure, an individual's likelihood of developing a cancer is expressed by national incidence and mortality rates. For example, residents of the United States have about a one in 3 five chance of dying of cancer. There were, it is estimated, about 556,000 deaths from cancer in 2003, representing 23% of all mortality,[ ] a frequency surpassed only by deaths caused by cardiovascular diseases. These data do not include an additional 1 million, for the most part readily curable, non-melanoma cancers of the skin and 100,000 cases of carcinoma in situ, largely of the uterine cervix but also of the breast. The major organ sites affected and the estimated frequency of cancer deaths are shown in Figure 7-23 . The most common tumors in men are prostate, lung, and colorectal cancers. In women, cancers of the breast, lung, and colon and rectum are the most frequent. Cancers of the lung, female breast, prostate, and colon/rectum constitute more than 50% of cancer diagnoses and cancer deaths in the U.S. population.[ 15] Figure 7-23 Cancer incidence and mortality by site and sex. Excludes basal cell and squamous cell skin cancers and in situ carcinomas, except urinary bladder. (Adapted from Jemal A, et al: Cancer statistics, 2003. CA Cancer J Clin 53:5, 2003.) Figure 7-24 Age-adjusted cancer death rates for selected sites in the United States, adjusted for the 2000 U.S. population. (Adapted from Jemal A, et al: Cancer statistics, 2003. CA Cancer J Clin 53:5, 2003.) Figure 7-25 The change in incidence of various cancers with migration from Japan to the United States provides evidence that the occurrence of cancers is related to components of the environment that differ in the two countries. The incidence of each kind of cancer is expressed as the ratio of the death rate in the population being considered to that in a hypothetical population of California whites with the same age distribution; the death rates for whites are thus defined as 1. The death rates among immigrants and immigrants' sons tend consistently toward California norms. (From Cairns J: The cancer problem. In Readings from Scientific American—Cancer Biology. New York, WH Freeman, 1986, p. 13.) TABLE 7-3 -- Occupational Cancers Agents or Groups of Agents Human Cancer Site for Which Reasonable Evidence Is Available Typical Use or Occurrence Arsenic and arsenic compounds Lung, skin, hemangiosarcoma Byproduct of metal smelting. Component of alloys, electrical and semiconductor devices, medications and herbicides, fungicides, and animal dips Asbestos Lung, mesothelioma; gastrointestinal tract (esophagus, stomach, large intestine) Formerly used for many applications because of fire, heat, and friction resistance; still found in existing construction as well as fire-resistant textiles, friction materials (i.e., brake linings), underlayment and roofing papers, and floor tiles Benzene Leukemia, Hodgkin lymphoma Principal component of light oil. Although use as solvent is discouraged, many applications exist in printing and lithography, paint, rubber, dry cleaning, adhesives and coatings, and detergents. Formerly widely used as solvent and fumigant Beryllium and beryllium compounds Lung Missile fuel and space vehicles. Hardener for lightweight metal alloys, particularly in aerospace applications and nuclear reactors Cadmium and cadmium compounds Prostate Uses include yellow pigments and phosphors. Found in solders. Used in batteries and as alloy and in mental platings and coatings Chromium compounds Lung Component of metal alloys, paints, pigments, and preservatives Ethylene oxide Leukemia Ripening agent for fruits and nuts. Used in rocket propellant and chemical synthesis, in fumigants for foodstuffs and textiles, and in sterilants for hospital equipment Nickel compounds Nose, lung Nickel plating. Component of ferrous alloys, ceramics, and batteries. Byproduct of stainless steel arc welding Radon and its decay products Lung From decay of minerals containing uranium. Can be serious hazard in quarries and underground mines Vinyl chloride Angiosarcoma, liver Refrigerant. Monomer for vinyl polymers. Adhesive for plastics. Formerly inert aerosol propellant in pressurized containers Modified from Stellman JM, Stellman SD: Cancer and workplace. CA Cancer J Clin 46:70, 1996. of the colon and in virtually 100% of cases are fated to develop a carcinoma of the colon by age 50. Other autosomal dominant cancer syndromes are the Li-Fraumeni syndrome resulting from germ line mutations of the p53 gene, multiple endocrine neoplasia types 1 and 2 (MEN-1 and MEN-2), and hereditary nonpolyposis colon cancer (HNPCC), a condition caused by inactivation of a mismatch repair gene (also listed below among repair defects). There are several features that characterize inherited cancer syndromes: • In each syndrome, tumors involve specific sites and tissues, although they may involve more than one site. For example, in MEN-2, caused by a mutation of the RET protooncogene, thyroid, parathyroid, and adrenals are involved. There is no increase in predisposition to cancers in general. • Tumors within this group are often associated with a specific marker phenotype. For example, there may be multiple benign tumors in the affected tissue, as occurs in familial polyposis of the colon and in MEN. Sometimes, there are abnormalities in tissue that are not the target of transformation (e.g., Lisch nodules and café-au-lait spots in neurofibromatosis type 1; see Chapter 5 ). • As in other autosomal dominant conditions, both incomplete penetrance and variable expressivity occur. Defective DNA Repair Syndromes. Besides the dominantly inherited precancerous conditions, a group of cancerpredisposing conditions is collectively characterized by defects in DNA repair and resultant DNA instability. These conditions generally have an autosomal recessive pattern of inheritance. Included in this group are xeroderma pigmentosum, ataxiatelangectasia, and Bloom syndrome, all rare diseases characterized by genetic instability resulting from defects in DNA repair genes. Also included here is hereditary nonpolypoid colon cancer (HNPCC), an autosomal dominant 24 condition caused by inactivation of a DNA mismatch repair gene.[ ] HNPCC is the most common cancer predisposition syndrome, increasing the susceptibility to cancer in the colon and also in some other organs such as the small intestine, endometrium, and ovary ( Chapter 17 ). Familial Cancers. Besides the inherited syndromes of cancer susceptibility, cancer may occur at higher frequency in certain families without a clearly defined pattern of transmission. Virtually all the common types of cancers that occur sporadically have also been reported to occur in familial forms. Examples include carcinomas of colon, breast, ovary, and 286 TABLE 7-4 -- Reported Deaths for the Five Leading Cancer Types for Males by Age, US, 2000 All Ages Under Age 20 All cancers Age 20–39 All cancers All cancers 286,082 Lung and bronchus 90,415 * 31,078 307 28,484 105 14,238 Non-Hodgkin lymphoma 104 Non-Hodgkin lymphoma 11,812 79 14,428 2345 7821 Urinary Bladder 8179 Non-Hodgkin lymphoma 2308 15,630 Colon and rectum Pancreas Liver 431 15,420 2929 444 16,626 Prostate Prostate Esophagus Colon and rectum 57,470 4801 481 70,883 Lung and bronchus Colon and rectum Pancreas Non-Hodgkin lymphoma 158,990 15,827 611 All cancers Lung and bronchus Colon and rectum Lung and bronchus Endocrine system 50,069 626 Age 80+ All cancers Lung and bronchus Leukemia Bones and joints Pancreas * 430 Brain and ONS Colon and rectum 4832 Brain and ONS Age 60–70 All cancers 1298 Leukemia Prostate Age 40–59 3222 Leukemia 6107 3187 "All Cancers" excludes in situ carcinomas except urinary bladder. Source: US Mortality Public Use Data Tape, 2000, National Center for Health Statistics, Centers for Disease Control and Prevention, Hyattsville, MD 2002. *ONS = other nervous system. brain, as well as melanomas. Features that characterize familial cancers include early age at onset, tumors arising in two or more close relatives of the index case, and sometimes, multiple or bilateral tumors. Familial cancers are not associated with specific marker phenotypes. For example, in contrast to the familial adenomatous polyp syndrome, familial colonic cancers do not arise in pre-existing benign polyps. The transmission pattern of familial cancers is not clear. In general, siblings have a relative risk between two and three (two to three times greater than unrelated individuals). Segregation analyses of large families usually show that predisposition to the tumors is dominant, but multifactorial inheritance cannot be easily ruled out. It is likely that familial susceptibility to cancer may depend on multiple low-penetrance alleles, each contributing to only a small increase in the risk of tumor development. It has been estimated that 10% to 20% of patients with breast or ovarian cancer have a first- or second-degree relative with one of these tumors. Although two breast cancer susceptibility genes, named BRCA1 and BRCA2, have been identified, mutation of these genes occurs in no more than 3% of breast cancers. Thus, mutations in BRCA1 and BRCA2 cannot account for the large 25 proportion of familial breast cancers.[ ] Changes in other genes, probably in low-penetrance susceptibility alleles, appear to be necessary for the development of these tumors. A similar situation occurs in familial melanomas, in which a mutation of the p16INK4a tumor suppressor gene has been identified. However, mutation in this gene accounts for only about 20% of familial melanoma kindreds, suggesting that other factors are involved in the familial predisposition.[ 26] Interactions Between Genetic and Non-Genetic Factors. What can be said about the influence of heredity on the majority of malignant neoplasms? It could be argued that they are largely of environmental origin, but lack of family history does not preclude an inherited component. It is generally difficult to sort out the hereditary and acquired basis of a tumor because these factors often interact closely. The interaction between genetic and non-genetic factors is particularly complex when tumor development depends on the action of multiple contributory genes. Even in tumors with a well-defined inherited component, the risk of developing the tumor can be greatly influenced by non-genetic factors. For instance, breast cancer risk in female carriers of BRCA-1 or BRCA-2 mutations is almost 27 28 three-fold higher for women born after 1940, compared to the risks for women born before that year.[ ] [ ] Furthermore, the genotype can significantly influence the likelihood of developing environmentally induced cancers. Inherited variations (polymorphisms) of enzymes that metabolize procarcinogens to their active carcinogenic forms (see "Initiation of Carcinogenesis") can influence the susceptibility to cancer. Of interest in this regard are genes that encode the cytochrome P-450 enzymes. As discussed later under "Chemical Carcinogenesis," polymorphism at one of the P-450 loci confers inherited susceptibility to lung cancers in cigarette smokers. More such correlations are likely to be found. NONHEREDITARY PREDISPOSING CONDITIONS The only certain way of avoiding cancer is not to be born; to live is to incur the risk. The risk is greater than average, however, under many circumstances, as is evident from the predisposing influences discussed earlier. Certain clinical conditions are also important. Because cell replication is involved in neoplastic transformation, regenerative, hyperplastic, and dysplastic proliferations are fertile soil for the origin of a malignant tumor. There is a well-defined association between certain forms of endometrial hyperplasia and endometrial carcinoma and between cervical dysplasia and cervical carcinoma ( Chapter 22 ). The bronchial mucosal metaplasia and dysplasia of habitual cigarette smokers are ominous 287 TABLE 7-5 -- Reported Deaths for the Five Leading Cancer Types for Females by Age, US, 2000 All Ages Under Age 20 All cancers All cancers Lung and bronchus 973 Leukemia 5617 302 Brain and ONS * 41,872 Endocrine system Breast Uterine cervix Leukemia Lung and bronchus Lung and bronchus 80,697 Lung and bronchus 39,311 Breast 10,613 Colon and rectum All cancers 131,871 11,937 538 Age 80+ All cancers 47,850 1444 238 Age 60–70 All cancers Breast 65,016 Breast Age 40–59 All cancers 267,009 Colon and rectum Age 20–39 14,693 Colon and rectum 17,842 Colon and rectum 12,379 Breast 28,950 Pancreas 82 Bones and joints 15,094 Ovary 452 Lung and bronchus 76 Soft tissue 14,060 3619 Ovary Pancreas 397 Brain and ONS * 69 12,612 3033 Pancreas Pancreas 7825 Ovary 354 10,648 1871 5319 Non-Hodgkin lymphoma 7217 4039 "All Cancers" excludes in situ carcinomas except urinary bladder. Source: US Mortality Public Use Data Tape, 2000, National Center for Health Statistics, Centers for Disease Control and Prevention, Hyattsville, MD, 2002. *ONS = other nervous system. antecedents of bronchogenic carcinoma. About 80% of hepatocellular carcinomas arise in cirrhotic livers, which are characterized by active parenchymal regeneration ( Chapter 18 ). Chronic Inflammation and Cancer. In 1863 Virchow proposed that cancer develops at sites of chronic inflammation and the potential relationships between cancer and inflammation have been studied since then.[ exemplified by the increased risk of cancer development in patients affected by a TABLE 7-6 -- Inherited Predisposition to Cancer Inherited Cancer Syndromes (Autosomal Dominant) Gene Inherited Predisposition ••RB Retinoblastoma ••p53 Li-Fraumeni syndrome (various tumors) ••p16INK4A Melanoma ••APC Familial adenomatous polyposis/colon cancer ••NF1, NF2 Neurofibromatosis 1 and 2 ••BRCA1, BRCA2 Breast and ovarian tumors ••MEN1, RET Multiple endocrine neoplasia 1 and 2 ••MSH2, MLH1, MSH6 Hereditary nonpolyposis colon cancer ••PATCH Nevoid basal cell carcinoma syndrome Familial Cancers Familial clustering of cases, but role of inherited predisposition not clear for each individual 29] This is ••Breast cancer ••Ovarian cancer ••Pancreatic cancer Inherited Autosomal Recessive Syndromes of Defective DNA Repair ••Xeroderma pigmentosum ••Ataxia-telangiectasia ••Bloom syndrome ••Fanconi anemia variety of chronic inflammatory diseases of the gastrointestinal tract. These include ulcerative colitis, Crohn disease, Helicobacter pylori gastritis, viral hepatitis, and chronic pancreatitis. 30 The precise mechanisms that link inflammation and cancer development have not been established.[ ] Chronic inflammatory reactions may result in the production of cytokines, which stimulate the growth of transformed cells. In some cases, chronic inflammation may increase the pool of tissue stem cells, which become subject to the effect of mutagens. Interestingly, chronic inflammation may also directly promote genomic instability in cells through the production of reactive oxygen species (ROS), thus predisposing to malignant transformation. Whatever the precise mechanism, such a link may have practical implications. For instance, expression of the enzyme cyclooxygenase-2 (COX-2), which converts arachidonic acid into prostaglandins ( Chapter 2 ), is induced by inflammatory stimuli and is increased in colon cancers and other tumors.[ active and promising area of research.[ 31] The development of COX-2 inhibitors for cancer treatment is an 32] Precancerous Conditions. Certain non-neoplastic disorders—the chronic atrophic gastritis of pernicious anemia, solar keratosis of the skin, chronic ulcerative colitis, and leukoplakia of the oral cavity, vulva, and penis—have such a well-defined association with cancer that they have been termed precancerous conditions. This designation is somewhat unfortunate because in the great majority of these lesions no malignant neoplasm emerges. Nonetheless, the term persists because it calls attention to the increased risk. Certain forms of benign neoplasia also constitute precancerous conditions. The villous adenoma of the colon, as it increases in size, develops cancerous change in up to 50% of cases. It might be asked: Is there not a risk with all benign neoplasms? Although some risk may be inherent, a large cumulative experience indicates that most benign neoplasms do not become cancerous. Nonetheless, numerous examples could be offered of cancers arising, albeit rarely, in benign tumors; for example, a leiomyosarcoma beginning in a leiomyoma, and carcinoma appearing in longstanding pleomorphic adenomas. Generalization is impossible 288 because each type of benign neoplasm is associated with a particular level of risk ranging from virtually never to frequently. Only follow-up studies of large series of each neoplasm can establish the level of risk, and always the question remains: Did the cancer arise from a non-malignant cell in the benign tumor or did the benign tumor contain, from the outset, a silent or indolent malignant focus? Molecular Basis of Cancer The literature on the molecular basis of cancer continues to proliferate at such a rapid pace that it is easy to get lost in the growing forest of information. We list some fundamental principles before delving into the details of the molecular basis of cancer. • Nonlethal genetic damage lies at the heart of carcinogenesis. Such genetic damage (or mutation) may be acquired by the action of environmental agents, such as chemicals, radiation, or viruses, or it may be inherited in the germ line. The term "environmental," used in this context, involves any acquired defect caused by exogenous agents or endogenous products of cell metabolism. Not all mutations, however, are "environmentally" induced. Some may be spontaneous and stochastic. • A tumor is formed by the clonal expansion of a single precursor cell that has incurred the genetic damage (i.e., tumors are monoclonal). Clonality of tumors can be assessed in women who are heterozygous for polymorphic X-linked markers, such as the enzymes glucose-6-phosphate dehydrogenase (G6PD), iduronate-2-sulfatase and phosphoglycerate kinase. The principle underlying such an analysis is illustrated in Figure 7-26 . The most commonly used method to determine tumor clonality involves the analysis of methylation 33 patterns adjacent to the highly polymorphic locus of the human androgen receptor gene (HUMARA).[ ] The frequency of HUMARA polymorphism in the general population is more than 90%, so it is easy to establish clonality by showing that all the cells in a tumor express the same allele. For tumors with a specific translocation, such as in myeloid leukemias, the presence of the translocation can be used to assess clonality. Immunoglobulin receptor and T-cell receptor gene rearrangements serve as markers of clonality in Band T-cell lymphomas, respectively. • Four classes of normal regulatory genes—the growth-promoting protooncogenes, the growth-inhibiting tumor suppressor genes, genes that regulate programmed cell death (apoptosis), and genes involved in DNA repair—are the principal targets of genetic damage. Mutant alleles of protooncogenes are considered dominant because they transform cells despite the presence of a normal counterpart. In contrast, both normal alleles of the tumor suppressor genes must be damaged for transformation to occur, so this family of genes is sometimes referred to as recessive oncogenes. However, there are exceptions to this rule, and some tumor suppressor genes lose their suppressor activity when a single 34 allele is lost or inactivated.[ ] This loss of function of a recessive gene caused by damage of a single allele is called haploinsufficiency. Genes that regulate apoptosis may be dominant, as are protooncogenes, or they may behave as tumor suppressor genes. • DNA repair genes affect cell proliferation or survival indirectly by influencing the ability of the organism to repair nonlethal damage in other genes, including protooncogenes, tumor suppressor genes, and genes that regulate apoptosis. A disability in the DNA repair genes can predispose to mutations in the genome and hence to neoplastic 35 transformation. Such propensity to mutations is called a mutator phenotype. [ ] With some exceptions, both alleles of DNA repair genes must be inactivated to induce such genomic instability; in this sense, DNA repair genes may also be considered as tumor suppressor genes. • Carcinogenesis is a multistep process at both the phenotypic and the genetic levels. A malignant neoplasm has several phenotypic attributes, such as excessive growth, local invasiveness, and the ability to form distant metastases. These characteristics are acquired in a stepwise fashion, a phenomenon called tumor progression. At the molecular level, progression results from accumulation of genetic 289 lesions that in some instances are favored by defects in DNA repair. Figure 7-26 Diagram depicting the use of X-linked isoenzyme cell markers as evidence of the monoclonality of neoplasms. Because of random X inactivation, all females are mosaics with two cell populations (with G6PD isoenzyme A or B in this case). When neoplasms that arise in women who are heterozygous for X-linked markers are analyzed, they are made up of cells that contain the active maternal (XA ) or the paternal (XB ) X chromosome but not both. Figure 7-27 Flow chart depicting a simplified scheme of the molecular basis of cancer. Figure 7-28 Expression of cyclin-cyclin-dependent kinase (CDK) complexes during the cell cycle. The phases of the cycle are indicated inside the arrows. (Modified from Pollard TD, Earnshaw WC: Cell Biology. Philadelphia, WB Saunders, 2002.) Figure 7-29 Schematic illustration of the role of cyclins, CDKs, and cyclin-dependent kinase inhibitors in regulating the G1 /S cell-cycle transition. External signals activate multiple signal transduction pathways, including those involving the MYC and RAS genes, which lead to synthesis and stabilization of cyclin D (there are several D cyclins, but, for simplification, we refer to them as "cyclin D"). Cyclin D binds to CDK4, forming a complex with enzymatic activity (cyclin D can also bind to CDK6, which appears to have a similar role as CDK4). The cyclin D-CDK4 complex phosphorylates RB, located in the E2F/DP1/RB complex in the nucleus, activating the transcriptional activity of E2F (E2F is a family of transcription factors, which we refer to as "E2F"), which leads to transcription of cyclin E, cyclin A and other proteins needed for the cell to go through the late G1 restriction point. The cell cycle can be blocked by the Cip/Kip inhibitors p21 and p27 (red boxes) and the INK4A/ARF inhibitors p16INK4A and p14ARF (green boxes). Cell-cycle arrest in response to DNA damage and other cellular stresses is mediated through p53. The levels of p53 are under negative regulation by MDM2, through a feedback loop that is inhibited by p14ARF. Figure 7-30 Mechanism of cell-cycle regulation by RB. In a resting cell, RB is a component of the E2F/DP1/RB complex, which represses gene transcription through the recruitment of histone deacetylase, an enzyme that alters the conformation of chromatin, making it more compact. Phosphorylation of RB by cyclin D-CDK4 removes histone deacetylase from chromatin, allowing the activation of E2F transcriptional activity (RB can also be phosphorylated by cyclin E-CDK2). E2F-mediated transcription of cyclins E and A, and of genes required for DNA replication, permit the passage through the G1 restriction point. (Adapted from Pollard TD, Earnshaw WC: Cell Biology. Philadelphia, WB Saunders, 2002, p. 689.) TABLE 7-7 -- Main Cell-Cycle Components and Their Inhibitors Cell-Cycle Component Main Function Cyclin-Dependent Kinases • CDK4 Forms a complex with cyclin D. The complex phosphorylates RB, allowing the cell to progress through the G1 restriction point. • CDK2 Forms a complex with cyclin E in late G1 , which is involved in the G1 /S transition. Forms a complex with cyclin A at the S phase that facilitates the G2 /M transition. Forms a complex with cyclin B, which acts on the G2 /M transition. • CDK1 Inhibitors • Cip/Kip family: p21, p27 Block the cell cycle by binding to cyclin-CDK complexes. p21 is induced by the tumor suppressor p53. p27 responds to growth suppressors such as transforming growth factor-β. • 1NK4/ARF family: p16INK4A, p14ARF p16INK4a binds to cyclin D-CDK4 and promotes the inhibitory effects of RB. p14ARF increases p53 levels by inhibiting MDM2 activity. Checkpoint Components • p53 Tumor suppressor altered in the majority of cancers; causes cell-cycle arrest and apoptosis. Acts mainly through p21 to cause cell-cycle arrest. Causes apoptosis by inducing the transcription of pro-apoptotic genes such as BAX. Levels of p53 are negatively regulated by MDM2 through a feedback loop. p53 is required for the G1 /S checkpoint and is a main component of the G2 /M checkpoint. • Ataxia-telangiectasia mutated (ATM) Activated by mechanisms that sense double stranded DNA breaks. Transmits signals to arrest the cell cycle after DNA damage. Acts through p53 in the G1 /S checkpoint. At the G2 /M checkpoint, it acts both through p53-dependent mechanisms and through the inactivation of CDC25 phosphatase, which disrupts the cyclin B-CDK1 complex. Component of a network of genes that include BRCA1 and BRCA2, which link DNA damage with cell-cycle arrest and apoptosis. three components, p21, p27, and p57, which bind to and inactivate the complexes formed between cyclins and CDKs. Transcriptional activation of p21 is under the control of p53, a tumor suppressor gene that is mutated in a large proportion of human cancers. The main role of p53 in the cell cycle is one of surveillance, triggering checkpoint controls that slow down or stop cell-cycle progression of damaged cells, or causes apoptosis. The human INK4a/ARF locus (a notation for "inhibitor of kinase 4/alternative reading frame") encodes two proteins, p16INK4a and p14ARF, which block the cell cycle and act as tumor suppressors. p16INK4a competes with cyclin D for binding to CDK4 and inhibits the ability of the cyclin D-CDK4 complex to phosphorylate RB, thus causing cell-cycle arrest at late G1 . It is frequently mutated or inactivated by hypermethylation (discussed later) in human cancers. The INK4a locus encodes a second gene product, p14ARF (p19ARF in mice), which acts on p53. p14ARF arises from an alternative reading of the INK4a gene, providing for an "economical" way to utilize 42] gene-coding sequences.[ degradation. Although both p16INK4a and p14ARF block the cell cycle, their targets are different; p16INK4a acts on cyclin D-CDK4, whereas p14ARF prevents p53 Cell-Cycle Checkpoints. The cell cycle has its own internal controls, called checkpoints. There are two main checkpoints, one at the G1 /S transition and another at G2 /M.[ 43] [44] The S phase is the point of no return in the cell cycle, and before a cell makes the final commitment to replicate, the G1 /S checkpoint checks for DNA damage. If DNA damage is present, the DNA repair machinery and mechanisms that arrest the cell cycle are put in motion. The delay in cell-cycle progression provides the time needed for DNA repair; if the damage is not repairable, apoptotic pathways are activated to kill the cell. Thus, the G1 /S checkpoint prevents the replication of cells that have defects in DNA, which would be perpetuated as mutations or chromosomal breaks in the progeny of the cell. DNA damaged after its replication can still be repaired as long as the chromatids have not separated. The G2 /M checkpoint monitors the completion of DNA replication and checks whether the cell can safely initiate mitosis and separate sister chromatids. This checkpoint is particularly important in cells exposed to ionizing radiation. Cells damaged by ionizing radiation activate the G2 /M checkpoint and arrest in G2 ; defects in this checkpoint give rise to chromosomal abnormalities. To function properly, cell-cycle checkpoints require sensors of DNA damage, signal transducers, and effector molecules. [ 44] The sensors and transducers of DNA damage appear to be similar for the G1 /S and G2 /M checkpoints. They include, as sensors, proteins of the RAD family and ataxia telangiectasia mutated (ATM) and as transducers, the CHK kinase families. The checkpoint effector molecules differ, depending on the cell-cycle stage at which they act. In the G1 /S checkpoint, cell-cycle arrest is mostly mediated through p53, which induces the cell-cycle inhibitor p21. Arrest of the cell cycle by the G2 /M checkpoint involves both p53-dependent and independent mechanisms. Defect in cell-cycle checkpoint components is a major cause of genetic instability in cancer cells. With this background on the cell cycle and its control, we now proceed to discuss the genes that determine the malignant phenotype. This discussion will take place in the context of the seven fundamental changes in cell physiology (listed earlier) that are the hallmarks of malignant cells. SELF-SUFFICIENCY IN GROWTH SIGNALS: ONCOGENES Genes that promote autonomous cell growth in cancer cells are called oncogenes, and their normal cellular counterparts are called protooncogenes. Protooncogenes are physiologic regulators of cell proliferation and differentiation; oncogenes are characterized by the ability to promote cell growth in the 293 absence of normal mitogenic signals. Their products, called oncoproteins, resemble the normal products of protooncogenes with the exception that oncoproteins are devoid of important regulatory elements. Their production in the transformed cells becomes constitutive, that is, not dependent on growth factors or other external signals. To aid in the understanding of the nature and functions of oncoproteins, and their role in cancer, it is necessary to briefly mention the sequential steps that characterize normal cell proliferation. Under physiologic conditions, cell proliferation can be readily resolved into the following steps: • The binding of a growth factor to its specific receptor generally located on the cell membrane • Transient and limited activation of the growth factor receptor, which, in turn, activates several signal-transducing proteins on the inner leaflet of the plasma membrane • Transmission of the transduced signal across the cytosol to the nucleus via second messengers or by signal transduction molecules that directly activate transcription • Induction and activation of nuclear regulatory factors that initiate DNA transcription • Entry and progression of the cell into the cell cycle, ultimately resulting in cell division With this background, we can readily identify the strategies used by cancer cells to acquire self-sufficiency in growth signals. They can be grouped on the basis of their role in growth factor-mediated signal transduction cascades and cell-cycle regulation. We start with a description of oncogenes and their protein products, and how these were discovered. Protooncogenes, Oncogenes, and Oncoproteins As often happens in science, the discovery of protooncogenes was not straightforward. These cellular genes were first discovered in their mutated or "oncogenic" forms as "passengers" within the genome of acute transforming retroviruses by the 1989 Nobel laureates Harold Varmus and Michael Bishop. These retroviruses cause rapid induction of tumors in animals and can also transform animal cells in vitro. Molecular dissection of their genomes revealed the presence of unique transforming sequences (viral oncogenes [v-onc]) not found in the genomes of nontransforming retroviruses. Most surprisingly, molecular hybridization revealed that the v-onc sequences were almost identical to sequences found in normal cellular DNA. From this evolved the concept that during evolution, cellular oncogenes were transduced (captured) by the virus through a chance recombination with the DNA of a (normal) host cell that had been infected by the virus. Because they were discovered initially as viral genes, these protooncogenes were named after their viral homologues. Each v-onc is designated by a three-letter word that relates the oncogene to the virus from which it was isolated. Thus, the v-onc contained in feline sarcoma virus is referred to as v-FES, whereas the oncogene in simian sarcoma virus is called v-SIS. The corresponding protooncogenes are referred to as FES and SIS, dropping the prefix. The viral oncogenes are not present in several cancer-causing RNA viruses. One such example is a group of so-called slow transforming viruses that cause leukemias in rodents after a long latent period. The mechanism by which they cause neoplastic transformation implicates protooncogenes. Molecular dissection of the cells transformed by these leukemia viruses revealed that the proviral DNA is always integrated (inserted) near a protooncogene. One consequence of proviral insertion near a protooncogene is to induce a structural change in the cellular gene, thus converting it into a cellular oncogene (c-onc, or onc). This mode of protooncogene activation is called insertional mutagenesis. Alternatively, strong retroviral promoters inserted in the vicinity of the protooncogenes lead to dysregulated expression of the cellular gene. Although the study of transforming animal retroviruses provided the first glimpse of protooncogenes, these investigations did not explain the origin of human tumors, which (with rare exceptions) are not caused by infection with retroviruses. Hence the question was raised: Do nonviral tumors contain oncogenic DNA sequences? The answer was provided by experiments involving DNA-mediated gene transfer (DNA transfection). When DNA extracted from several different human tumors was transfected into mouse fibroblast cell lines in vitro, the recipient cells acquired some properties of neoplastic cells. The conclusion from such experiments was inescapable: DNA of spontaneously arising cancers contains oncogenic sequences, or oncogenes. One of the first oncogenic sequences detected in cancers was a mutated form of the RAS protooncogene. This protooncogene is the forbear of v-oncs contained in Harvey (H) and Kirsten (K) sarcoma viruses. A large number of protooncogenes have been identified during the past 20 years, most of which do not have a viral counterpart. Protooncogenes have multiple roles, participating in cellular functions related to growth and proliferation. Proteins encoded by protooncogenes may function as growth factor ligands and receptors, signal transducers, transcription factors, and cell-cycle components ( Fig. 7-31 ). Oncoproteins encoded by oncogenes generally serve similar functions as their normal counterparts ( Table 7-8 ). However, because they are constitutively expressed, oncoproteins endow the cell with self-sufficiency in growth.[ 45] To summarize, protooncogenes may be converted into cellular oncogenes (c-oncs) that are involved in tumor development. Two questions follow: (1) What are the functions of oncogene products, the oncoproteins? (2) How do the normally "civilized" protooncogenes turn into "enemies within"? These issues are discussed below. Growth Factors. Many cancer cells develop growth self-sufficiency by acquiring the ability to synthesize the same growth factors to which they are responsive. The protooncogene SIS, which encodes the β chain of platelet-derived growth factor (PDGF), is overproduced in many tumors, especially low-grade astrocytomas and osteosarcomas. Furthermore, it appears that the same tumors also express receptors for PDGF and are hence responsive to autocrine stimulation. Although an autocrine loop is considered to be an important element in the pathogenesis of several tumors, in most instances the growth factor gene itself is not altered or mutated. More commonly, products of other oncogenes such as RAS (that lie along many signal transduction pathways) cause overexpression of growth factor genes, thus forcing the cells to secrete large amounts of growth factors, such as transforming growth factor-α (TGF-α). This growth factor is related to epidermal growth factor (EGF) and induces proliferation by binding to the EGF receptor. TGF-α is often 294 Figure 7-31 Subcellular localization and functions of major classes of cancer-associated genes. The protooncogenes are colored red, cancer suppressor genes blue, DNA repair genes green, and genes that regulate apoptosis purple. TABLE 7-8 -- Selected Oncogenes, Their Mode of Activation, and Associated Human Tumors Category Protooncogene Mode of Activation Associated Human Tumor Growth Factors PDGF-β chain SIS Overexpression Astrocytome Osteosarcoma Fibroblast growth factors HST-1 Overexpression Stomach cancer INT-2 Amplification Bladder cancer Breast cancer Melanoma TGFα TGFα Overexpression Astrocytomas Hepatocellular carcinomas HGF HGF Overexpression Thyroid cancer ERB-B1 (ECFR) Overexpression Squamous cell carcinomas of lung, gliomas ERB-B2 Amplification Breast and ovarian cancers CSF-1 receptor FMS Point mutation Leukemia Receptor for neurotrophic factors RET Point mutation Multiple endocrine neoplasia 2A and B, familial medullary thyroid carcinomas PDGF receptor PDGF-R Overexpression Gliomas Receptor for stem cell (steel) factor KIT Point mutation Gastrointestinal stromal tumors and other soft tissue tumors K-RAS Point mutation Colon, lung, and pancreatic tumors H-RAS Point mutation Bladder and kidney tumors N-RAS Point mutation Melanomas, hematologic malignancies ABL Translocation Chronic myeloid leukemia Growth Factor Receptors EGF-receptor family Proteins Involved in Signal Transduction GTP-binding Nonreceptor tyrosine kinase Acute lymphoblastic leukemia RAS signal transduction BRAF Point mutation Melanomas WNT signal transduction β-catenin Point mutation Hepatoblastomas, hepatocellular carcinoma Overexpression Nuclear Regulatory Proteins Transcriptional activators C-MYC Translocation Burkitt lymphoma N-MYC Amplification Neuroblastoma, small cell carcinoma of lung L-MYC Amplification Small cell carcinoma of lung CYCLIN D Translocation Mantle cell lymphoma Amplification Breast and esophageal cancers CYCLIN E Overexpression Breast cancer CDK4 Amplification or point mutation Glioblastoma, melanoma, sarcoma Cell-Cycle Regulators Cyclins Cyclin-dependent kinase domain alter the substrate specificity of the tyrosine kinase and lead to thyroid and adrenal tumors but no involvement of the parathyroid. Complete loss of RET function results in Hirschsprung disease ( Chapter 17 ), in which there is lack of development of intestinal nerve plexuses. In all these familial conditions, the affected individuals inherit the RET mutation in the germ line. Sporadic medullary carcinomas of the thyroid are associated with somatic rearrangements of the RET gene, generally similar to those found in MEN 2B.[ 46] [47] Oncogenic conversions by mutations and rearrangements have been found in other growth factor receptor genes. Point mutations that activate c-FMS, the gene encoding the colonystimulating factor 1 (CSF-1) receptor, have been detected in myeloid leukemias. In certain chronic myelomonocytic leukemias with the t(12;9) translocation, the entire cytoplasmic domain of the PDGF receptor is fused with a segment of the ETS family transcription factor, resulting in permanent dimerization of the PDGF receptor. Far more common than mutations of these protooncogenes is overexpression of normal forms of growth factor receptors. In sporadic papillary thyroid carcinomas, c-MET is overexpressed 48 in almost every case.[ ] In these tumors, increased expression of c-MET is not caused by gene mutation but results from enhanced transcription of the gene. In some tumors, increased receptor expression results from gene amplification, but in many cases, the molecular basis of 296 increased receptor expression is not fully known. Two members of the EGF receptor family are most commonly involved. The normal form of ERB B1, the EGF receptor gene, usually referred to as EGFR, is overexpressed in up to 80% of squamous cell carcinomas of the lung, in 50% or more of high-grade astrocytomas called glioblastomas ( Chapter 28 ), in 80% to 49 50 100% of head and neck tumors, and less commonly, in carcinomas of the urinary bladder and the gastrointestinal tract.[ ] [ ] In contrast, the ERB B2 gene (also called HER 2/Neu), the second member of the EGF receptor family, is amplified in approximately 25% of breast cancers and in human adenocarcinomas arising within the ovary, lung, stomach, and salivary glands.[ 51] Because the molecular alteration in ERB B2 is specific for the cancer cells, new therapeutic agents consisting of monoclonal antibodies against ERB B2 have been developed and are currently in use clinically.[ 49] [51] This type of therapy, directed to a specific alteration in the cancer cell, is called targeted therapy. [ 52] Another example of very successful targeted 53 tract.[ ] cancer therapy is the blockage of receptor tyrosine kinase activity of c-KIT in stromal tumors of the gastrointestinal In these tumors, a mutation in c-KIT, the gene encoding the receptor for stem cell factor (also known as steel factor), constitutively activates the receptor tyrosine kinase, independent of ligand binding. Signal-Transducing Proteins. Several examples of oncoproteins that mimic the function of normal cytoplasmic signal-transducing proteins have been found. Most such proteins are strategically located on the inner leaflet of the plasma membrane, where they receive signals from outside the cell (e.g., by activation of growth factor receptors) and transmit them to the cell's nucleus. Biochemically, the signal-transducing proteins are heterogeneous. The best and most well studied example of a signal-transducing oncoprotein is the RAS family of guanine triphosphate (GTP)-binding proteins (G proteins). The RAS Oncogene. The RAS proteins were discovered as products of viral oncogenes. Point mutation of RAS family genes is the single most common abnormality of dominant oncogenes in human tumors. 54 Approximately 15% to 20% of all human tumors contain mutated versions of RAS proteins.[ ] Several distinct mutations of RAS have been identified in cancer cells, all of which dramatically reduce the GTPase activity of the RAS proteins. The mutations generally involve codons 12, 59, or 61 of HRAS, KRAS, and NRAS. The frequency of such mutations varies with different tumors, but in some types it is very high. For example, 90% of pancreatic adenocarcinomas and cholangiocarcinomas contain a RAS point mutation, as do about 50% of 55 56 57 colon, endometrial, and thyroid cancers and 30% of lung adenocarcinomas and myeloid leukemias.[ ] [ ] [ ] In general, carcinomas (particularly from colon and pancreas) have mutations of KRAS, bladder tumors have HRAS mutations, and hematopoietic tumors bear NRAS mutations. RAS mutations are infrequent in certain other cancers, particularly those arising in the uterine cervix or breast. Several studies indicate that RAS plays an important role in mitogenesis induced by growth factors. For example, blockade of RAS function by microinjection of specific antibodies blocks the proliferative response to EGF, PDGF, and CSF-1. Normal RAS proteins are tethered to the cytoplasmic aspect of the plasma membrane, and they flip back and forth between an activated, signal-transmitting form and an inactive, quiescent state. Recently it was found that these proteins may also be found in the endoplasmic reticulum and Golgi membranes, where 58 they can be activated by growth factor binding to the plasma membrane, through a still-uncertain mechanism. [ ] In the inactive state, RAS proteins bind guanosine diphosphate (GDP); when cells are stimulated by growth factors or other receptor-ligand interactions, RAS becomes activated by exchanging GDP for GTP ( Fig. 7-32 ). Activated RAS, in turn, acts on the MAP kinase pathway by recruiting the cytosolic protein RAF-1. The MAP kinases so activated target nuclear transcription factors and thus promote mitogenesis. In normal cells, the activated signal-transmitting stage of the RAS protein is transient because its intrinsic GTPase activity hydrolyzes GTP to GDP, thereby returning RAS to its quiescent ground state (described below). The orderly cycling of the RAS protein depends on two reactions: (1) nucleotide exchange (GDP by GTP), which activates RAS protein, and (2) GTP hydrolysis, which converts the GTPbound, active RAS to the GDP-bound, inactive form. Both these processes are enzymatically regulated. The removal of GDP and its replacement by GTP during RAS activation are catalyzed by a family of guanine nucleotide-releasing proteins that are recruited to the cytosolic domain of activated growth factor receptors by adapter proteins. More importantly, the GTPase activity intrinsic to normal RAS proteins is dramatically accelerated by GTPase-activating proteins (GAPs). These widely distributed proteins bind to the active RAS and augment its GTPase activity by more than 1000-fold, leading to rapid hydrolysis of GTP to GDP and termination of signal transduction. Thus, GAPs function as "brakes" that prevent uncontrolled RAS activity. The response to this braking action of GAPs seems to falter when mutations affect the RAS gene. Mutant RAS proteins bind GAP, but their GTPase activity fails to be augmented. Hence the mutant proteins are "trapped" in their excited GTP-bound form, causing, in turn, a pathologic activation of the mitogenic signaling pathway. The importance of GTPase activation in normal growth control is underscored by the fact that a disabling mutation of neurofibromin (NF-1), a GTPase-activating protein, is also associated with neoplasia (see discussion of tumor suppressor genes below). In addition to RAS, other members of the RAS signaling cascade (RAS/RAF/MAP kinase) may also be altered in cancer cells. Thus, mutations in BRAF, one of the members of the RAF 59 60 family, have been detected in more than 60% of melanomas and in more than 80% of benign nevi.[ ] [ ] This suggests that dysregulation of the RAS/RAF/MAP kinase pathway may be one of the initiating events in the development of melanomas, although it is not sufficient by itself to cause tumorigenesis. Recent studies have revealed that, in addition to its role in transducing growth factor signals, RAS is also involved in regulation of the cell cycle. As described above, the passage of cells from G1 to the S phase is modulated by cyclins and CDKs. RAS proteins can indirectly regulate the levels of cyclins by activating the MAP kinase pathway and the AP-1 transcription factor. Because RAS is so frequently mutated in human cancers, much effort has been spent to develop anti-RAS modalities of targeted therapy. Several such strategies for cancer treatment are being evaluated. The specific targets include blockade of 297 Figure 7-32 Model for action of RAS genes. When a normal cell is stimulated through a growth factor receptor, inactive (GDP-bound) RAS is activated to a GTP-bound state. Activated RAS recruits RAF and stimulates the MAP-kinase pathway to transmit growth-promoting signals to the nucleus. The mutant RAS protein is permanently activated because of inability to hydrolyze GTP, leading to continuous stimulation of cells without any external trigger. The anchoring of RAS to the cell membrane by the farnesyl moiety is essential for its action. Figure 7-33 The chromosomal translocation and associated oncogenes in Burkitt lymphoma and chronic myelogenous leukemia. Figure 7-34 Amplification of the N-MYC gene in human neuroblastomas. The N-MYC gene, normally present on chromosome 2p, becomes amplified and is seen either as extra chromosomal double minutes or as a chromosomally integrated, homogeneous staining region. The integration involves other autosomes, such as 4, 9, or 13. (Modified from Brodeur GM: Molecular correlates of cytogenetic abnormalities in human cancer cells: implications for oncogene activation. In Brown EB (ed): Progress in Hematology, Vol 14. Orlando, FL, Grune & Stratton, 1986, pp. 229–256.) TABLE 7-9 -- Selected Tumor Suppressor Genes Involved in Human Neoplasms Subcellular Location Cell surface Gene TGF-β receptor Function Growth inhibition Tumors Associated with Somatic Mutations Carcinomas of colon Tumors Associated with Inherited Mutations Unknown E-cadherin Cell adhesion Carcinoma of stomach Familial gastric cancer Inner aspect of plasma membrane NF-1 Inhibition of RAS signal transduction and of p21 cell-cycle inhibitor Neuroblastomas Neurofibromatosis type 1 and sarcomas Cytoskeleton NF-2 Cytoskeletal stability Schwannomas and meningiomas Neurofibromatosis type 2, acoustic schwannomas and meningiomas Cytosol APC/β-catenin Inhibition of signal transduction Carcinomas of stomach, colon, pancreas; melanoma Familial adenomatous polyposis coli/ colon cancer PTEN PI-3 kinase signal transduction Endometrial and prostate cancers Unknown SMAD 2 and SMAD 4 TGF-β signal transduction Colon, pancreas tumors Unknown RB Regulation of cell cycle Retinoblastoma; osteosarcoma carcinomas of breast, colon, lung Retinoblastomas, osteosarcoma p53 Cell-cycle arrest and apoptosis in response to DNA damage Most human cancers Li-Fraumeni syndrome; multiple carcinomas and sarcomas WT-1 Nuclear transcription Wilms tumor Wilms tumor p16 (INK4a) Regulation of cell cycle by inhibition of cyclin-dependent kinases Pancreatic, breast, and esophageal cancers Malignant melanoma BRCA-1 and BRCA-2 DNA repair Unknown Carcinomas of female breast and ovary; carcinomas of male breast KLF6 Transcription factor Prostate Unknown Nucleus ( Fig. 7-36 ). When cells enter the S phase, they can continue to cell division independent of growth factors. It should be obvious from this discussion that if RB is absent (owing to gene deletions) or its ability to regulate E2F transcription factors is derailed, the molecular brakes on the cell cycle are released, and the cells move into the S phase followed by cell replication. The mutations of RB genes found in tumors are localized to a region of the RB protein, called the "RB pocket," that is involved in binding to E2F. It was mentioned previously that germ-line loss or mutations of the RB gene predispose to occurrence of retinoblastomas and to a lesser extent osteosarcomas. Furthermore, somatically acquired mutations have been described in glioblastomas, small cell carcinomas of lung, breast cancers, and bladder carcinomas. Given the presence of RB in every cell and its importance in cell-cycle control, two questions arise: (1) Why do patients with germ line mutation of the RB locus develop mainly retinoblastomas? (2) Why are inactivating mutations of RB not much more common in human cancer? The basis for the occurrence of tumors restricted to the retina in patients who inherit one defective allele of RB is not fully understood, but some possible explanations have emerged from the study of mice with targeted disruption of the RB locus. For instance, RB mutation may be a critical initiating event for retinoblastomas but may be only an accessory factor for malignancies at other sites. With respect to the second question (i.e., why the loss of RB is not more common in human tumors), the answer is much simpler: Mutations in other genes that control RB phosphorylation can mimic the effect of RB loss, and such genes are mutated in many cancers that may have normal RB genes. Thus, for example, mutational activation of cyclin D or CDK4 would favor cell proliferation by facilitating RB phosphorylation. As previously discussed, cyclin D is overexpressed in many tumors because of gene amplification or translocation. Mutational inactivation of CDK inhibitors would also drive the cell cycle by unregulated activation of cyclins and CDKs. Thus, the emerging paradigm is that loss of normal cell-cycle control is central to malignant transformation and that at least one of four key regulators of the cell cycle (p16INK4a, CYCLIN D, CDK4, RB) is dysregulated in the vast majority of human cancers. [38] In cells that harbor mutations in any one of these other genes, the function of RB is disrupted even if the RB gene itself is not mutated.[ 45] Several other pathways of cell growth regulation, some to be discussed in more detail later, also converge on RB ( Fig. 7-36 ): • TGF-β induces inhibition of cellular proliferation. This effect of TGF-β is mediated, at least in part, by up-regulation of the CDK inhibitor p27. 301 • The transforming proteins of several oncogenic animal and human DNA viruses seem to act, in part, by neutralizing the growth inhibitory activities of RB. In these cases, RB protein is functionally deleted by the binding of a viral protein and no longer acts as a cell-cycle inhibitor. Simian virus 40 and polyomavirus large T antigens, adenoviruses EIA protein, and human papillomavirus (HPV) E7 protein, all bind to the hypophosphorylated form of RB. The binding occurs in the same RB pocket that normally sequesters E2F transcription factors; in the case of HPV, the binding is particularly strong for viral types, such as HPV 16, which confer high risk for the development of 302 cervical carcinomas. Thus, the RB protein, unable to bind the E2F transcription factors, is functionally deleted, and the transcription factors are free to cause cell-cycle progression. • The p53 tumor suppressor gene exerts its growth-inhibiting effects at least in part by up-regulating the synthesis of the CDK inhibitor p21 (see Fig. 7-29 and Fig. 7-36 ). Figure 7-35 Pathogenesis of retinoblastoma. Two mutations of the RB locus on chromosome 13q14 lead to neoplastic proliferation of the retinal cells. In the familial form, all somatic cells inherit one mutant RB gene from a carrier parent. The second mutation affects the Rb locus in one of the retinal cells after birth. In the sporadic form, on the other hand, both mutations at the RB locus are acquired by the retinal cells after birth. Figure 7-36 Role of RB as a cell-cycle regulator. Various growth factors promote the formation of the cyclin D-CDK4 complex. This complex (and to some extent cyclin E-CDK2) phosphorylates RB, changing it from an active (hypophosphorylated) to an inactive state (hyperphosphorylation). RB inactivation allows the cell to pass the G1 /S restriction point. Growth inhibitors such as TGF-β and p53 and the Cip/Kip (e.g., p21, p57) and INK4a (p161NK4a and p19ARF) cell-cycle inhibitors prevent RB activation. Transforming proteins of oncogenic viruses bind hypophosphorylated RB and cause its functional inactivation. Virtually all cancers show dysregulation of the cell cycle by affecting the four genes marked by an asterisk. Figure 7-37 The role of p53 in maintaining the integrity of the genome. Activation of normal p53 by DNA-damaging agents or by hypoxia leads to cell-cycle arrest in G1 and induction of DNA repair, by transcriptional up-regulation of the cyclin-dependent kinase inhibitor p21, and the GADD45 genes, respectively. Successful repair of DNA allows cells to proceed with the cell cycle; if DNA repair fails, p53-induced activation of the BAX gene promotes apoptosis. In cells with loss or mutations of p53, DNA damage does not induce cell-cycle arrest or DNA repair, and hence genetically damaged cells proliferate, giving rise eventually to malignant neoplasms. Figure 7-38 A, The role of APC in regulating the stability and function of β-catenin. APC and β-catenin are components of the WNT signaling pathway. In resting cells (not exposed to WNT), β-catenin forms a macromolecular complex containing the APC protein. This complex leads to the destruction of β-catenin, and intracellular levels of β-catenin are low. B, When cells are stimulated by secreted WNT molecules, the destruction complex is deactivated, β-catenin degradation does not occur, and cytoplasmic levels increase. β-catenin translocates to the nucleus, where it binds to TCF, a transcription factor that activates several genes involved in the cell cycle. C, When APC is mutated or absent, the destruction of β-catenin cannot occur. βCatenin translocates to the nucleus and coactivates genes that promote the cell cycle, and cells behave as if they are under constant stimulation by the WNT pathway. Figure 7-39 Interaction between cancer susceptibility genes and DNA repair. ATM (ataxia-telangiectasia mutated) senses a double-strand break in DNA, induced by agents such as ionizing radiation. ATM and CHEK2 phosphorylate BRCA1, promoting its migration to the break site. The Fanconi's anemia protein complex (proteins A, C, E, F, G) triggers the ubiquitination and colocalization of the Fanconi protein D2 with BRCA1 at the break site. BRCA2 carries RAD51, an enzyme involved in DNA recombination repair, to the same site. BRCA1, BRCA2, and RAD51 repair the DNA break by an error-free recombination mechanism. RAD51 is a component of cell cycle check points. (Redrawn from Venkitaraman AR: A growing network of cancer-susceptibility genes. N Engl J Med 348:1917, 2003.) Figure 7-40 Cellular responses to telomere shortening. The figures show the responses of normal cells, which have intact cell-cycle checkpoints and of cells with checkpoint defects. (From Wong JMY, Collins K: Telomere maintenance and disease. Lancet 362:983, 2003.) Figure 7-42 The metastatic cascade. Schematic illustration of the sequential steps involved in the hematogenous spread of a tumor. Figure 7-43 Mechanisms of metastasis development within a primary tumor. A nonmetastatic primary tumor is shown (light blue) on the left side of all diagrams. Four models are presented: A, Metastasis is caused by rare variant clones that develop in the primary tumor; B, Metastasis is caused by the gene expression pattern of most cells of the primary tumor, referred to as a metastatic signature; C, A combination of A and B, in which metastatic variants appear in a tumor with a metastatic gene signature; D, Metastasis development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local invasiveness and resistance to immune elimination, allowing cells of the primary tumor, as in C, to become metastatic. Figure 7-44 A–D, Schematic illustration of the sequence of events in the invasion of epithelial basement membranes by tumor cells. Tumor cells detach from each other because of reduced adhesiveness, and cells then attach to the basement membrane via the laminin receptors and secrete proteolytic enzymes, including type IV collagenase and plasminogen activator. Degradation of the basement membrane and tumor cell migration follow. TABLE 7-10 -- Selected Examples of Oncogenes Activated by Translocation Malignancy Chronic myeloid leukemia Translocation (9;22)(q34;q11) Affected Genes Ab1 9q34 bcr 22q11 Acute leukemias (AML and ALL) (4;11)(q21;q23) AF4 4q21 MLL 11q23 (6;11)(q27;q23) AF6 6q27 MLL 11q23 Burkitt lymphoma (8;14)(q24;q32) c-myc 8q24 IgH 14q32 Mantle cell lymphoma (11;14)(q13;q32) Cyclin D 11q13 IgH 14q32 Follicular lymphoma (14;18)(q32;q21) IgH 14q32 bcl-2 18q21 T-cell acute lymphoblastic leukemia (8;14)(q24;q11) c-myc 8q24 TCR-α 14q11 (10;14)(q24;q11) Hox 11 10q24 TCR-α 14q11 Ewing sarcoma (11;22)(q24;q12) Fl-1 11q24 EWS 22q12 Underlined genes are involved in multiple translocations. AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia. course. The translocations associated with the ABL oncogene in chronic myeloid leukemia and with c-MYC in Burkitt lymphoma have been mentioned earlier, in conjunction with the discussion of molecular defects in cancer cells (see Fig. 7-32 ). Several other karyotype alterations in cancer cells are presented in the discussion of specific forms of neoplasia. Two types of chromosomal rearrangements can activate protooncogenes—translocations and inversions. Chromosomal translocations are much more common ( Table 7-10 ) and are discussed here. Translocations can activate protooncogenes in two ways: • In lymphoid tumors, specific translocations result in overexpression of protooncogenes by removing them from their regulatory elements. • In many hematopoietic tumors, the translocations allow normally unrelated sequences from two different chromosomes to recombine and form hybrid genes that encode growthpromoting chimeric proteins. Overexpression of a protooncogene caused by translocation is best exemplified by Burkitt lymphoma. All such tumors carry one of three translocations, each involving chromosome 8q24, where the MYC gene has been mapped, as well as one of the three immunoglobulin gene-carrying chromosomes. At its normal locus, the expression of the MYC gene is tightly controlled; it is expressed only during certain stages of the cell cycle. In Burkitt lymphoma, the most common form of translocation results in the movement of the MYC-containing segment of chromosome 8 to chromosome 14q band 32 ( Fig. 7-33 ), placing it close to the immunoglobulin heavy-chain (IgH) gene. The genetic notation for the translocation is t(8:14)(q24;q32). The molecular mechanisms of the translocation-associated activation of MYC are variable, as are the precise breakpoints within the gene. In most cases, the translocation causes mutations or loss of the regulatory sequences of the MYC gene. As the coding sequences remain intact, the gene is constitutively expressed at high levels. The gene may be translocated to the antigen receptor loci simply because these loci are accessible (i.e. in "open" chromatin) and active in developing lymphocytes. The invariable presence of the translocated MYC gene in Burkitt lymphomas attests to the importance of MYC overexpression in the pathogenesis of this tumor. There are other examples of oncogenes translocated to antigen receptor loci in lymphoid tumors. As mentioned earlier, in mantle cell lymphoma, the CYCLIN D1 gene on chromosome 11q13 is overexpressed by juxtaposition to the IgH locus on 14q32. In follicular lymphomas, a t(14;18)(q32;q21) translocation, the most common translocation in lymphoid malignancies, causes activation of the BCL-2 gene. Not unexpectedly, all these tumors in which the immunoglobulin gene is involved are of B-cell origin. In an analogous situation, overexpression of several protooncogenes in T-cell tumors results from translocations of oncogenes into the T-cell antigen receptor locus. The affected oncogenes are diverse, but in most cases, as with MYC, they encode nuclear transcription factors. The Philadelphia chromosome, characteristic of chronic myeloid leukemia and a subset of acute lymphoblastic leukemias, provides the prototypic example of an oncogene formed by fusion of two separate genes. In these cases, a reciprocal translocation between chromosomes 9 and 22 relocates 315 a truncated portion of the protooncogene c-ABL (from chromosome 9) to the BCR (break point cluster region) on chromosome 22 ( Fig. 7-33 ). The hybrid fusion gene BCR-ABL encodes a chimeric protein that has constitutive tyrosine kinase activity. As mentioned, BCR-ABL tyrosine kinase has served as a target for leukemia therapy, with remarkable success so far. Although the translocations are cytogenetically identical in chronic myeloid leukemia and acute lymphoblastic leukemias, they differ at the molecular level. In chronic myeloid leukemia, 62 63 the chimeric protein has a molecular weight of 210 kD, whereas in the more aggressive acute leukemias, a 190-kD BCR-ABL fusion protein is formed.[ ] [ ] The molecular pathways activated by the BCR-ABL protein are complex and not completely understood. It inhibits apoptosis, decreases the requirement for growth factors, binds to cytoskeleton components, decreases cell adhesion, and activates multiple pathways, including those of RAS, PI-3 kinase, and STATs ( Chapter 3 ). BCR-ABL also acts on DNA repair and may cause genomic instability that contributes to the progression of the disease. Transcription factors are often the partners in gene fusions occurring in cancer cells. For instance, the MLL (myeloid, lymphoid leukemia) gene on 11q23 is known to be involved in 25 different translocations with several different partner genes, some of which encode transcription factors (see Table 7-10 ). The Ewing Sarcoma (EWS) gene at 22q12 was first described in the t(11;22)(q24;12) reciprocal translocation present in Ewing sarcoma (a highly malignant tumor of children; Chapter 26 ) but may be translocated in other types of sarcomas. EWS is itself a transcription factor, and all of its partner genes analyzed so far also encode a transcription factor. In Ewing tumor, for example, the EWS gene fuses with the FLI 1 gene; the resultant chimeric EWS-FLI 1 protein is a member of the ETS transcription factor family, which has transforming ability. Gene Amplification Activation of protooncogenes associated with overexpression of their products may result from reduplication and amplification of their DNA sequences. Such amplification may produce 137 several hundred copies of the protooncogene in the tumor cell.[ ] The amplified genes can be readily detected by molecular hybridization with appropriate DNA probes. In some cases, the amplified genes produce chromosomal changes that can be identified microscopically. Two mutually exclusive patterns are seen: multiple small, chromosome-like structures called double minutes (dms), and homogeneous staining regions (HSRs). The latter derive from the assembly of amplified genes into new chromosomes; because the regions containing amplified genes lack a normal banding pattern, they appear homogeneous in a G-banded karyotype (see Fig. 7-34 ). The most interesting cases of amplification involve N-MYC in neuroblastoma and ERB B2 in breast cancers. N-MYC is amplified in 25% to 30% of neuroblastomas, and the amplification is associated with poor prognosis. In neuroblastomas with N-MYC amplification, the gene is present both in dms and HSRs. ERB B2 amplification occurs in about 20% of breast cancers and may represent a distinct tumor phenotype. Amplification of C-MYC, L-MYC, and N-MYC correlates with disease progression in small cell cancer of the lung. Another gene frequently amplified is CYCLIN D1 (breast carcinomas, head and neck carcinomas, and other squamous cell carcinomas). Epigenetic Changes It has become evident during the past few years that certain tumor suppressor genes may be inactivated not because of structural changes but because the gene is silenced by hypermethylation of promoter sequences without a change in DNA base sequence.[ 138] Such changes appear to be stably maintained through multiple rounds of cell division. Methylation takes place in CpG islands in DNA, but de novo methylation rarely occurs in normal tissues. However, methylation has been detected in various tumor suppressor genes in human cancers. They include p14ARF in colon and stomach cancers, p16INK4a in various types of cancers, BRCA1 in breast cancer, VHL in renal cell carcinomas, and the MLH1 mismatch repair gene in 139 colorectal cancer.[ ] Methylation also participates in the phenomenon called genomic imprinting, in which the maternal or paternal allele of a gene or chromosome is modified by methylation and is inactivated. The reverse phenomenon, that is, demethylation of an imprinted gene leading to its biallelic expression (loss of imprinting) can also occur in tumor cells. [140] Although the discussion of whether methylation of tumor suppressor genes has a causal role in cancer development continues, there has been great interest in developing potential therapeutic agents that act to demethylate DNA sequences in tumor suppressor genes. Recent data demonstrating that genomic hypomethylation causes chromosomal instability and 141] induces tumors in mice greatly strengthens the notion that epigenetic changes may directly contribute to tumor development.[ Molecular Profiles of Cancer Cells A new era in cancer research was initiated with the development of methods to measure the expression of thousands of genes in tumors and normal tissues. Among these new methods, the determination of RNA levels by microarray analysis has found wide application ( Box 7-1 ). Currently, this method can measure RNA expression from virtually all known genes ( Fig. 745 ). The expression profiles obtained from DNA microarray analysis are known as gene expression signatures or molecular profiles. The application of this technique to the study of breast cancers and leukemias has been particularly rewarding (see Box 7-1 ). It was recently found that there are breast cancer subtypes that can be identified by their molecular profiles and that the molecular signatures of some of these subtypes can help predict the course of the disease (see Box 23-1 , Chapter 23).[ 142] Analysis of acute lymphoblastic leukemia by DNA microarrays has established the molecular signatures of prognostic subtypes and uncovered novel markers associated with these subtypes.[ 143] Molecular Basis of Multistep Carcinogenesis The notion that malignant tumors arise from a protracted sequence of events is supported by epidemiologic, experimental, and molecular studies. Many eons ago, before 316 Box 7-1. Gene Expression Profiles of Human Cancers. Microarrays and Proteomics Until recently, studies of gene expression in tumors involved the analysis of individual genes. These studies have been revolutionized by the introduction of methods that can measure 208 209 the expression of thousands of genes simultaneously. [ ] [ ] The most common method for large-scale analysis of gene expression in use today is based on DNA microarray technology. In this method, DNA fragments, either cDNAs or oligonucleotides, are spotted on a glass slide or on some other solid support. As the techniques used for the spotting are similar to those employed to produce semiconductor chips for electronic products, the arrays are known as "gene chips." Chips can be purchased from commercial suppliers or produced in-house, and can contain more than 20,000 gene fragments. The fragments are typically obtained from complementary DNA (cDNA) libraries or sets of nucleotides from known and uncharacterized genes. The gene chip is then hybridized to "probes" prepared from tumor and control samples (the probes are usually cDNA copies of RNAs extracted from tumor and uninvolved tissues). Before hybridization to the chip, the probes are labeled with fluorochromes that emit different colors (e.g. red color for tumor RNA and green color for control RNA). After hybridization the chip is read using a laser scanner ( Fig. 7-45 ); each spot on the array will be red (increased expression of a gene in the tumor), green (decreased expression in the tumor) or, if there is no difference in gene expression between the tumor and control sample, the spots will be either black or yellow (depending on the type of fluorescent scanning). Sophisticated software has been developed to measure the intensity of the fluorescence for each spot and produce data sets in which genes with similar expression 210 patterns are clustered. [ ] This method of analysis, called hierarchical clustering, groups together genes according to the similarity of their gene expression patterns. The software can be linked to large sequencing and array databases available through the Web. This allows appropriate gene identification and comparison between expression profiles from various sources. A major problem in the analysis of gene expression in tumors is the heterogeneity of the tissue. In addition to the heterogeneity between tumor cells, samples may contain variable amounts of stromal connective tissue, inflammatory infiltrates, and normal tissue cells. One way to overcome this problem is to obtain nearly pure tumor cells or small tumors free from associated tissues using laser capture microdissection. In this technique, the dissection of the tumor or cells is made under a microscope through a focused laser. The dissected material is then captured or "catapulted" into a small cap and processed for RNA and DNA isolation. Gene expression profiling of tumors has multiple uses, and the number of publications using this technique has grown enormously during the past few years. Much of the work performed is not directed toward proving or disproving a proposed hypothesis. Gene expression analysis can be used to classify tumors; to predict metastatic potential, prognosis, and response to therapy; to reveal gene expression patterns that are dependent on the mutation of a single oncogene; and to analyze the effects of hormones and environmental agents on 209 141 209 210 211 cancer development. [ ] The applications of this technology keep expanding and being refined, but much has already been accomplished.[ ] [ ] [ ] [ ] We mention only a few interesting examples. Profiling of cells from adult and pediatric T-cell acute lymphoblastic leukemia has identified the patterns of gene expression in leukemic blast cells and has 211 accurately classified each prognostic subtype.[ ] The work that has received the highest publicity involves gene expression profiling of breast cancers. In addition to identifying new subtypes of breast cancers, a 70-gene prognosis profile was established. Using this type of profile, it has been reported that: (1) the profile was a powerful predictor of disease prognosis for young patients; (2) it was particularly accurate for predicting metastasis during the first 5 years after diagnosis; and (3) prognosis determined by gene expression profiles correlated highly with histologic grade and estrogen receptor status but not with lymphatic spread of the tumor.[ 211] A more recent analysis has pooled together data gathered by different 142 cancer.[ ] laboratories and has confirmed the identification of distinct subtypes of breast Given all of these remarkable results, it is time to ask whether this technology is "ready for prime time"; that is, ready for day to day clinical applications. Things are moving very fast in this area, but before clinical applications are considered, many issues need to settled. Not only do larger trials need to be conducted to prove the reliability and accuracy of the analysis but also, just as important, the procedures for handling samples, performing the analyses, and reporting the data need to be standardized, so that data obtained in various laboratories can be compared. Next on the horizon of molecular techniques for the global analysis of gene expression in cancers is proteomics, a technique used to obtain expression profiles of proteins contained in tissues, serum, or other body fluids. The original method consisted of the separation of proteins by 2-dimensional gel electrophoresis, followed by identification of individual proteins by mass spectrometry. A more recent technique, called ICAT (isotope-coding affinity tags) does not rely on electrophoresis for protein separation. In ICAT, proteins in the test and control samples are labeled with light or heavy isotopes. The differentially labeled proteins are then identified and quantified by mass spectrometry. A variation of proteomic analysis has been used to obtain protein profiles in the blood of cancer patients without identification of individual proteins.[ 215] The excitement created by the development of new techniques for the global molecular analysis of tumors has led some scientists to predict that the end of histopathology is in sight, and to consider existing approaches to tumor diagnosis as the equivalent of magical methods of divination. Indeed, it is hard to escape the excitement generated by the development of entirely new and powerful methods of molecular analysis. However, what lies ahead is not the replacement of one set of techniques by another. On the contrary, the most accurate diagnosis and prognosis of cancer will be arrived at by a combination of morphologic and molecular techniques. [ 208] 317 Figure 7-45 Schematic representation of the steps required for the analysis of global gene expression by DNA microarray. RNA is extracted from tumor and normal tissue. cDNA synthesized from each preparation is labeled with fluorescent dyes (in the example shown, normal tissue cDNA is labeled with a green dye; tumor cDNA is labeled with a red dye). The array consists of a solid support in which DNA fragments from many thousands of genes are spotted. The labeled cDNAs from tumor and normal tissue are combined and hybridized to the genes contained in the array. Hybridization signals are detected using a confocal laser scanner and downloaded to a computer for analysis (red squares, expression of the gene is higher in tumor; green square, expression of the gene is higher in normal tissue; black squares, no difference in the expression of the gene between tumor and normal tissue). In the display, the horizontal rows correspond to each gene contained in the array; each ventrical row corresponds to single samples. Figure 7-47 Schematic illustration of the pathways of malignancy initated by mutation of the gatekeeper genes (e.g., APC, NF-1, RB) or caretaker genes (e.g., hMSH2, BRCA-1, BRCA-2). Figure 7-48 Experiments demonstrating the initiation and promotion phases of carcinogenesis in mice. Group 2: application of promoter repeated at twice-weekly intervals for several months. Group 3: application of promoter delayed for several months and then applied twice weekly. Group 6: promoter applied at monthly intervals. Figure 7-49 General schema of events in chemical carcinogenesis. Note that promoters cause clonal expansion of the initiated cell, thus producing a preneoplastic clone. Further proliferation induced by the promoter or other factors causes accumulation of additional mutations and emergence of a malignant tumor. TABLE 7-11 -- Major Chemical Carcinogens Direct-Acting Carcinogens Alkylating Agents β-Propiolactone Dimethyl sulfate Diepoxybutane Anticancer drugs (cyclophosphamide, chlorambucil, nitrosoureas, and others) Acylating Agents 1-Acetyl-imidazole Dimethylcarbamyl chloride Procarcinogens That Require Metabolic Activation Polycyclic and Heterocyclic Aromatic Hydrocarbons Benz(a)anthracene Benzo(a)pyrene Dibenz(a,h)anthracene 3-Methylcholanthrene 7,12-Dimethylbenz(a)anthracene Aromatic Amines, Amides, Azo Dyes 2-Naphthylamine (β-naphthylamine) Benzidine 2-Acetylaminofluorene Dimethylaminoazobenzene (butter yellow) Natural Plant and Microbial Products Aflatoxin B1 Griseofulvin Cycasin Safrole Betel nuts Others Nitrosamine and amides Vinyl chloride, nickel, chromium Insecticides, fungicides Polychlorinated biphenyls 322 of promoters leads to proliferation and clonal expansion of initiated (mutated) cells. Initiated cells respond differently to promoters than do normal cells and hence expand selectively. Such cells (especially after RAS activation) have reduced growth factor requirements and may also be less responsive to growth inhibitory signals in their extracellular milieu. Forced to proliferate, the initiated clone of cells suffers additional mutations, developing eventually into a malignant tumor. Thus, the process of tumor promotion includes multiple steps: Proliferation of preneoplastic cells, malignant conversion, and eventually tumor progression, which depends on changes in tumor cells and the tumor stroma. The initiation-promotion sequence of chemical carcinogenesis raises an important question: Since promoters are not mutagenic, how do they contribute to tumorigenesis? Although the effects of tumor promoters are pleiotropic, induction of cell proliferation is a sine qua non of tumor promotion. TPA (tetradecanoyl phorbol-13 acetate), a phorbol ester tumor promoter, is a powerful activator of protein kinase C (see Chapter 3 ), an enzyme that phosphorylates several substrates involved in signal transduction pathways, including those activated by growth factors. The promoting effect of phenobarbital in liver carcinogenesis has been linked to stimulation of cell proliferation associated with blockage of the TGF-β pathway.[ 154] The concept that sustained cell proliferation increases the risk of mutagenesis and hence neoplastic transformation is also applicable to human carcinogenesis. For example, pathologic hyperplasia of the endometrium ( Chapter 22 ) and increased regenerative activity that accompanies chronic liver cell injury ( Chapter 18 ) are associated with the development of cancer in these organs. Carcinogenic Chemicals Before closing this discussion of chemical carcinogenesis, we briefly describe some initiators (see Table 7-11 ) and promoters of chemical carcinogenesis, with special emphasis on those that have been linked to cancer development in humans.[ 155] Direct-Acting Alkylating Agents. These agents are activation independent, and in general they are weak carcinogens. Nonetheless, they are important because many therapeutic agents (e.g., cyclophosphamide, chlorambucil, busulfan, and melphalan) fall into this category. These are used as anticancer drugs but have been documented to induce lymphoid neoplasms, leukemia, and other forms of cancer. Some alkylating agents, such as cyclophosphamide, are also powerful immunosuppressive agents and are therefore used in treatment of immunologic disorders, including rheumatoid arthritis and Wegener granulomatosis. Although the risk of induced cancer with these agents is low, judicious use of them is indicated. Alkylating agents appear to exert their therapeutic effects by interacting with and damaging DNA, but it is precisely these actions that render them carcinogenic. Polycyclic Aromatic Hydrocarbons. These agents represent some of the most potent carcinogens known. They require metabolic activation and can induce tumors in a wide variety of tissues and species. Painted on the skin, they cause skin cancers; injected subcutaneously, they induce sarcomas; introduced into a specific organ, they cause cancers locally. The polycyclic hydrocarbons are of particular interest 156 as carcinogens because they are produced in the combustion of tobacco, particularly with cigarette smoking, and are thought to contribute to the causation of lung and bladder cancers.[ ] The various components of cigarette smoke that may be associated with carcinogenicity are listed in Chapter 9 . Polycyclic aromatic hydrocarbons are also produced from animal fats in the process of broiling meats and are present in smoked meats and fish. Aromatic Amines and Azo Dyes. The carcinogenicity of most aromatic amines and azo dyes is exerted mainly in the liver, where the "ultimate carcinogen" is formed by the action of the cytochrome P-450 oxygenase systems. Thus, fed to rats, acetylaminofluorene and the azo dyes induce hepatocellular carcinomas (but not cancers of the gastrointestinal tract). An agent implicated in human cancers, β157 naphthylamine, is an exception. In the past, it was responsible for a 50-fold increased incidence of bladder cancer in heavily exposed workers in aniline dye and rubber industries.[ ] After absorption, it is hydroxylated into an active form, then detoxified by conjugation with glucuronic acid. When excreted in the urine, the nontoxic conjugate is split by the urinary enzyme glucuronidase to release the electrophilic reactant again, thus inducing bladder cancer. Regrettably, humans are one of the few species to possess urinary glucuronidase. Some of the azo dyes were developed as food coloring (e.g., butter yellow to give margarine the appearance of butter and scarlet red to impart the seductive coloration of certain foods such as maraschino cherries). These dyes are now federally regulated in the United States because of the fear that they may be dangerous to humans. Naturally Occurring Carcinogens. Among the several known chemical carcinogens produced by plants and microorganisms, the potent hepatic carcinogen aflatoxin B1 is particularly important. This mycotoxin is produced by some strains of the fungus Aspergillus flavus that thrive on improperly stored corn, rice, and peanuts. A strong correlation has been found between the dietary level of this hepatocarcinogen and the incidence of hepatocellular carcinoma in some parts of Africa and China. As discussed earlier, the aflatoxin and HBV collaborate in the production of this form of neoplasia. Nitrosamines and Amides. These carcinogens are of interest because of the possibility that they are formed in the gastrointestinal tract of humans and so may contribute to the induction of some forms of cancer, particularly gastric carcinoma. They are derived in the stomach from the reaction of nitrostable amines and nitrate used as a preservative, which is converted to nitrites by bacteria. Concerns about these agents have led many to shun processed food containing nitrate preservatives. Miscellaneous Agents. Scores of other chemicals have been indicted as carcinogens. Only a few that represent important industrial hazards are listed in Table 7-3 and are briefly mentioned here. Occupational exposure to asbestos has been associated with an increased incidence of bronchogenic carcinomas, mesotheliomas, and gastrointestinal cancers, as discussed in Chapter 15 . Concomitant cigarette smoking heightens the risk of bronchogenic carcinoma manyfold. Vinyl chloride, the monomer from which the polymer polyvinyl chloride is fabricated, was first identified as a carcinogen in animals, but investigations soon disclosed a scattered incidence 323 of the extremely rare hemangiosarcoma of the liver among workers exposed to this chemical. Chromium, nickel, and other metals, when volatilized and inhaled in industrial environments, have caused cancer of the lung. Skin cancer associated with arsenic is also well established. Similarly, there is reasonable evidence that many insecticides, such as aldrin, dieldrin, and chlordane and the polychlorinated biphenyls, are carcinogenic in animals ( Chapter 9 ). Promoters of Chemical Carcinogenesis. Certain promoters may contribute to cancers in humans. It has been argued that promoters are at least as important as initiating chemicals in carcinogenesis because cells initiated by exposure to environmental carcinogens are innocuous unless subjected to repeated assault by promoters. Tumor promotion may occur after exposure to exogenous agents, such as cigarette smoke or viral infections, that cause tissue damage and reactive hyperplasia. Perhaps more serious, because they are difficult to control, are endogenous promoters such as hormones and bile salts. Hormones such as estrogens serve in animals as promoters of liver tumors. The prolonged use of diethylstilbestrol is implicated in the production of postmenopausal endometrial carcinoma and in the causation of vaginal cancer in offspring exposed in utero ( Chapter 22 ). Intake of high levels of dietary fat has been associated with increased risk of colon cancer. This may be related to an increase in synthesis of bile acids, which have been shown to act as promoters in experimental models of colon cancer. Alcohol consumption increases the risk of development of cancers of the mouth, pharynx, and larynx by more than tenfold, probably by acting as a promoting agent ( Chapter 9 ). RADIATION CARCINOGENESIS Radiant energy, whether in the form of the UV rays of sunlight or as ionizing electromagnetic and particulate radiation, can transform virtually all cell types in vitro and induce neoplasms in vivo in both humans and experimental animals. UV light is clearly implicated in the causation of skin cancers, and ionizing radiation exposure from medical or occupational exposure, nuclear plant accidents, and atomic bomb detonations have produced a variety of forms of malignant neoplasia. Although the contribution of radiation to the total human burden of cancer is probably small, the well-known latency of radiant energy and its cumulative effect require extremely long periods of observation and make it difficult to ascertain its full significance. An increased incidence of breast cancer has become apparent decades later among women exposed during childhood to the atomic bomb. The incidence peaked during 1988–1992 and then 158 declined during the period 1993–1997.[ ] Moreover, radiation's possible additive or synergistic effects with other potential carcinogenic influences add another dimension to the picture. The effects of UV light on DNA differ from those of ionizing radiation. The cellular and molecular effects of ionizing radiation are discussed in Chapter 9 . Ultraviolet Rays There is ample evidence from epidemiologic studies that UV rays derived from the sun induce an increased incidence of squamous cell carcinoma, basal cell carcinoma, and possibly 159 malignant melanoma of the skin.[ ] The degree of risk depends on the type of UV rays, the intensity of exposure, and the quantity of light-absorbing "protective mantle" of melanin in the skin. Persons of European origin who have fair skin that repeatedly gets sunburned but stalwartly refuses to tan and who live in locales receiving a great deal of sunlight (e.g., Queensland, Australia, close to the equator) have among the highest incidence of skin cancers in the world. The UV portion of the solar spectrum can be divided into three wavelength ranges: UVA (320 to 400 nm), UVB (280 to 320 nm), and UVC (200 to 280 nm). Of these, UVB is believed to be responsible for the induction of cutaneous cancers. UVC, although a potent mutagen, is not considered significant because it is filtered out by the ozone shield around the earth (hence the concern about ozone depletion). UV rays have a number of effects on cells, including inhibition of cell division, inactivation of enzymes, induction of mutations and, in sufficient dosage, death of cells. The carcinogenicity of UVB light is attributed to its formation of pyrimidine dimers in DNA. This type of DNA damage is repaired by the nucleotide excision repair (NER) pathway. There are five steps in NER: (1) recognition of the DNA lesion, (2) incision of the damaged strand on both sites of the lesion, (3) removal of the damaged nucleotide, (4) synthesis of a nucleotide patch, and (5) its ligation. In mammalian cells, the process may involve 30 or more proteins. It is postulated that with excessive sun exposure, the capacity of the NER pathway is overwhelmed; hence, some DNA damage remains unrepaired. This leads to large transcriptional errors and, in some instances, cancer. The importance of the NER pathway of DNA repair is most graphically illustrated by a study of patients with the hereditary disorder xeroderma pigmentosum. This autosomal recessive disorder is characterized by extreme photosensitivity, a 2000-fold increased risk of skin cancer in sun-exposed skin and, in some cases, neurologic abnormalities. The molecular basis of the degenerative changes in sun-exposed skin and occurrence of cutaneous tumors rests on an inherited inability to repair UV-induced DNA damage. Xeroderma pigmentosum is a genetically heterogeneous condition, with at least seven different variants. Each of these is caused by a mutation in one of several genes involved in NER.[ 160] As with other carcinogens, UVB also causes mutations in oncogenes and tumor suppressor genes. In particular, mutant forms of the RAS and p53 genes have been detected both in human skin cancers and in UVB-induced cancers in mice. These mutations occur mainly at dipyrimidine sequences within the DNA, thus implicating UVB-induced genetic damage in the causation of skin cancers. In animal models, p53 mutations occur early after exposure to UVB, before the appearance of tumors. Ionizing Radiation Electromagnetic (x-rays, γ rays) and particulate (α particles, β particles, protons, neutrons) radiations are all carcinogenic. The evidence is so voluminous that a few examples suffice. Many of the pioneers in the development of X-rays developed skin cancers. Miners of radioactive elements in central Europe and the Rocky Mountain region of the United States have a tenfold increased incidence of lung cancers. Most telling is the follow-up of survivors of the atomic bombs dropped on 324 Hiroshima and Nagasaki. Initially, there was a marked increase in the incidence of leukemias—principally acute and chronic myelocytic leukemia—after an average latent period of about 7 years. Subsequently the incidence of many solid tumors with longer latent periods (e.g., breast, colon, thyroid, and lung) increased. Residents of the Marshall Islands were exposed on one occasion to accidental fallout from a hydrogen bomb test that contained thyroid-seeking radioactive iodines. As many as 90% of the children under age 10 years on Rongelap Island developed thyroid nodules within 15 years, and about 5% of these nodules proved to be thyroid carcinomas. A marked increase in the incidence of thyroid cancer has also been noted in areas exposed to the fallout from the nuclear power plant accident in Chernobyl in 1986. In addition to approximately 30 deaths that 161] occurred at the time of the accident, more than 2000 cases of thyroid cancers have been recorded in children living in the area.[ Cytogenetic studies have detected an elevated frequency 162 accident.[ ] of chromosomal alterations in persons who did cleanup work at the power plant after the It is evident that radiant energy—whether absorbed in the pleasant form of sunlight, through the best intentions of a physician, or by tragic exposure to an atomic bomb blast or radiation released by nuclear plant accidents—has awesome carcinogenic potential. Even therapeutic irradiation has been documented to be carcinogenic. Thyroid cancers have developed in approximately 9% of those exposed during infancy and childhood to head and neck radiation. The previous practice of treating ankylosing spondylitis with therapeutic irradiation yielded a 10- to 12-fold increase in the incidence of leukemia years later. In humans, there is a hierarchy of vulnerability of different tissues to radiation-induced cancers. Most frequent are the leukemias, except for chronic lymphocytic leukemia, which, for unknown reasons, almost never develops after radiation. Cancer of the thyroid follows closely but only in the young. In the intermediate category are cancers of the breast, lungs, and salivary glands. In contrast, skin, bone, and the gastrointestinal tract are relatively resistant to radiation-induced neoplasia, even though the gastrointestinal epithelial cells are vulnerable to the acute cell-killing effects of radiation, and the skin is in the pathway of all external radiation. Nonetheless, the physician dare not forget: practically any cell can be transformed into a cancer cell by sufficient exposure to radiant energy. MICROBIAL CARCINOGENESIS A large number of DNA and RNA viruses have proved to be oncogenic in a wide variety of animals, ranging from amphibia to primates, and the evidence grows stronger that certain forms of human cancer are of viral origin. In the following discussion, the better-characterized and most intensively studied human oncogenic viruses are presented first. This is followed by a brief account of the association between infection by the bacterium Helicobacter pylori and gastric tumors. Oncogenic DNA Viruses 163 Several DNA viruses have been associated with the causation of cancer in animals.[ ] Some, such as adenoviruses, cause tumors only in laboratory animals, whereas others, such as the bovine papillomaviruses, cause benign as well as malignant neoplasms in their natural hosts. Of the various human DNA viruses, four (papillomaviruses [HPV], Epstein-Barr virus [EBV], hepatitis B virus [HBV], and Kaposi sarcoma herpesvirus [KSHV]) are of particular interest because they have been implicated in the causation of human cancer. KSHV is discussed in Chapter 6 and Chapter 11 . Although not a DNA virus, hepatitis C virus (HCV) is also associated with cancer. Before we discuss the role of these viruses in carcinogenesis, a few general comments relating to transformation by DNA viruses are offered: • The genomes of oncogenic DNA viruses integrate into and form stable associations with the host cell genome. The virus is unable to complete its replicative cycle because the viral genes essential for completion of replication are interrupted during integration of viral DNA. Thus, the virus can remain in a latent state for years. • Those viral genes that are transcribed early in the viral life cycle (early genes) are important for transformation, and are expressed in transformed cells. Human Papillomavirus. Approximately 70 genetically distinct types of HPV have been identified. Some types (e.g., 1, 2, 4, and 7) cause benign squamous papillomas (warts) in humans. Human papillomaviruses have been implicated in the genesis of several cancers, particularly squamous cell carcinoma of the cervix and anogenital region, and in some cases, to the causation of oral and laryngeal cancers ( Chapter 16 ).[ 164] Epidemiologic studies suggest that carcinoma of the cervix is caused by a sexually transmitted agent, and HPV is the culprit. DNA sequences of HPV 16 and 18 and, less commonly, HPV 31, 33, 35, and 51 are found in approximately 85% of invasive squamous cell cancers and their presumed precursors (severe dysplasias and carcinoma in situ). In contrast to cervical cancers, genital warts with low malignant potential are associated with distinct HPV types, predominantly HPV 6 and HPV 11 ("low-risk" types). Molecular analyses of HPV-associated carcinomas and benign genital warts reveal differences that may be pertinent to the transforming activity of these viruses. In benign warts and in preneoplastic lesions, the HPV genome is maintained in an episomal (nonintegrated) form, whereas in cancers, the viral DNA is usually integrated into the host cell genome. This suggests that integration of viral DNA is important in malignant transformation. Although the site of viral integration in host chromosomes is random (the viral DNA is found at different locations in different cancers), the pattern of integration is clonal; that is, the site of integration is identical within all cells of a given cancer. This would not occur if HPV were merely a passenger that infects cells after transformation. Furthermore, the viral DNA is interrupted at a fairly constant site in the process of integration: It is almost always within the E1/E2 open reading frame of the viral genome. Because the E2 region of the viral DNA normally represses the transcription of the E6 and E7 early viral genes, its interruption causes over-expression of the E6 and E7 proteins of HPV 16 and HPV 18. The oncogenic potential of HPV 16 and HPV 18 can be related to these two early viral gene products, which act in conjunction to immortalize and transform cells.[ 162] [165] The replication of DNA viruses is dependent on the replication machinery of 325 166 the host cells, and E6 and E7 act to overcome the activity of cell-cycle inhibitors ( Fig. 7-50 ).[ ] E6 binds to p53 and E7 binds to RB, inducing the degradation of these proteins. In addition, E7 can interfere with p53 transcriptional activity and also inactivate p21. Thus, E6 and E7 block p53 and RB cell cycle suppression pathways. The affinity of these viral proteins for the products of tumor suppressor genes differs depending on the oncogenic potential of HPV. E6 proteins derived from high-risk HPV (HPV 16, 18, and 31) inactivate p53 by 167 enhancing its degradation through ubiquitin-dependent proteolysis.[ ] E6 proteins of low-risk HPV (HPV 6 and 11) bind p53 with low affinity and have no effect on p53 stability. E7 proteins from high-risk HPV strongly bind to RB, disrupting the E2F/RB complex and promoting the degradation of RB. By contrast, E7 proteins from low-risk HPV have lower affinity for RB and have a weak capacity to transform cells. Thus, E6 and E7 proteins of high-risk HPV disable two important tumor suppressor proteins that regulate the cell cycle. In HPVinduced tumors, p53 mutations are extremely uncommon, presumably because loss of p53 function is accomplished by binding to the E6 oncoprotein. This binding not only blocks the inhibitory effect of p53 on the cell cycle but also interferes with p53 activation after DNA damage, a mechanism that allows DNA repair or elimination of cells with genomic damage. Moreover, E6 may have other effects independent of its binding of p53, such as the activation of telomerase and tyrosine kinases.[ 164] The E6-p53 interaction may also offer some clues regarding risk factors for cervical cancer development in infected persons. Human p53 is polymorphic at amino acid 72, encoding either a proline or arginine residue at that position. It turns out that the arginine-containing p53 at position 72 is much more susceptible to degradation by E6. The arginine form is found more frequently in infected individuals with cervical Figure 7-50 Effect of HPV proteins E6 and E7 on the cell cycle. E6 and E7 enhance p53 degradation, causing a block in apoptosis and decreased activity of the p21 cell cycle inhibitor. E7 associates with p21 and prevents its inhibition of the Cyclin/CDK4 complex; E7 can bind to RB, removing cell cycle restriction. The net effect of HPV E6 and E7 proteins is to block apoptosis and remove the restrains to cell proliferation (see Fig. 7-29 ). (Modified from Münger K, Howley PM: Human papillomavirus immortalization and transformation functions. Virus Research 89:213–228, 2002.) Figure 7-51 Schema depicting the possible evolution of Epstein-Barr virus (EBV)-induced Burkitt lymphoma. Figure 7-52 Tumor antigens recognized by CD8+ T cells. (Modified from Abbas AK, Lichtman AH: Cellular and Molecular Immunology, 5th ed. Philadelphia, WB Saunders, 2003.) Figure 7-53 Mechanisms by which tumors evade the immune system. (Reprinted from Abbas AK, Lichtman AH: Cellular and Molecular Immunology, 5th ed. Philadelphia, WB Saunders, 2003.) TABLE 7-12 -- Paraneoplastic Syndromes Clinical Syndromes Major Forms of Underlying Cancer Causal Mechanism Endocrinopathies Cushing syndrome Small cell carcinoma of lung ACTH or ACTH-like substance Pancreatic carcinoma Neural tumors Syndrome of inappropriate antidiuretic hormone secretion Small cell carcinoma of lung; intracranial neoplasms Antidiuretic hormone or atrial natriuretic hormones Hypercalcemia Squamous cell carcinoma of lung Parathyroid hormone-related protein (PTHRP), TGF-α, TNF, IL-1 Breast carcinoma Renal carcinoma Adult T-cell leukemia/lymphoma Ovarian carcinoma Hypoglycemia Fibrosarcoma Insulin or insulin-like substance Other mesenchymal sarcomas Hepatocellular carcinoma Carcinoid syndrome Bronchial adenoma (carcinoid) Serotonin, bradykinin Pancreatic carcinoma Gastric carcinoma Polycythemia Renal carcinoma Erythropoietin Cerebellar hemangioma Hepatocellular carcinoma Nerve and Muscle Syndromes Myasthenia Bronchogenic carcinoma Immunologic Disorders of the central and peripheral nervous systems Breast carcinoma Dermatologic Disorders Acanthosis nigricans Gastric carcinoma Immunologic; secretion of epidermal growth factor Lung carcinoma Uterine carcinoma Dermatomyositis Osseous, Articular, and Soft Tissue Changes Bronchogenic, breast carcinoma Immunologic Hypertrophic osteoarthropathy and clubbing of the fingers Bronchogenic carcinoma Unknown Pancreatic carcinoma Tumor products (mucins that activate clotting) Vascular and Hematologic Changes Venous thrombosis (Trousseau phenomenon) Bronchogenic carcinoma Other cancers Nonbacterial thrombotic endocarditis Advanced cancers Hypercoagulability Anemia Thymic neoplasms Unknown Various cancers Tumor antigens, immune complexes Others Nephrotic syndrome ACTH, adrenocorticotropic hormone; TGF, transforming growth factor; TNF, tumor necrosis factor; IL, interleukin. are small. It is thought that PTHRP regulates calcium transport in the lactating breast and across the placenta. Tumors most often associated with paraneoplastic hypercalcemia are carcinomas of the breast, lung, kidney, and ovary. In breast cancers, PTHRP production is associated with osteolytic bone disease, bone metastasis, and humoral hypercalcemia. The most common lung neoplasm associated with hypercalcemia is the squamous cell bronchogenic carcinoma, rather than small cell cancer of the lung (more often associated with endocrinopathies). In addition to PTHRP, several other factors, such as IL-1, TGF-α, TNF, and dihydroxyvitamin D, have also been implicated in causing the hypercalcemia of malignancy. The neuromyopathic paraneoplastic syndromes take diverse forms, such as peripheral neuropathies, cortical cerebellar degeneration, a polymyopathy resembling polymyositis, and a myasthenic syndrome similar to myasthenia gravis. The cause of these syndromes is poorly understood. In some cases, antibodies, 335 presumably induced against tumor cells that cross-react with neuronal cells, have been detected. It is postulated that some neural antigens are ectopically expressed by visceral cancers. For some unknown reason, the immune system recognizes these antigens as foreign and mounts an immune response. Acanthosis nigricans is characterized by gray-black patches of verrucous hyperkeratosis on the skin. This disorder occurs rarely as a genetically determined disease in juveniles or adults ( Chapter 25 ). In addition, in about 50% of the cases, particularly in those over age 40, the appearance of such lesions is associated with some form of cancer. Sometimes the skin changes appear before discovery of the cancer. Hypertrophic osteoarthropathy is encountered in 1% to 10% of patients with bronchogenic carcinomas. Rarely, other forms of cancer are involved. This disorder is characterized by (1) periosteal new bone formation, primarily at the distal ends of long bones, metatarsals, metacarpals, and proximal phalanges; (2) arthritis of the adjacent joints; and (3) clubbing of the digits. Although the osteoarthropathy is seldom seen in non-cancer patients, clubbing of the fingertips may be encountered in liver diseases, diffuse lung disease, congenital cyanotic heart disease, ulcerative colitis, and other disorders. The cause of hypertrophic osteoarthropathy is unknown. Several vascular and hematologic manifestations may appear in association with a variety of forms of cancer. As mentioned in the discussion of thrombosis ( Chapter 4 ), migratory thrombophlebitis (Trousseau syndrome) may be encountered in association with deep-seated cancers, most often carcinomas of the pancreas or lung. Disseminated intravascular coagulation may complicate a diversity of clinical disorders ( Chapter 13 ). Acute disseminated intravascular coagulation is most commonly associated with acute promyelocytic leukemia and prostatic adenocarcinoma. Bland, small, nonbacterial fibrinous vegetations sometimes form on the cardiac valve leaflets (more often on left-sided valves), particularly in patients with advanced mucin-secreting adenocarcinomas. These lesions, called nonbacterial thrombotic endocarditis, are described further in Chapter 12 . The vegetations are potential sources of emboli that can further complicate the course of cancer. GRADING AND STAGING OF TUMORS Prognosis of the course of the disease and the determination of efficacy of various forms of cancer treatment require a high degree of similarity among the tumors being considered. Systems have been developed to express, at least in semiquantitative terms, the level of differentiation, or grade, and extent of spread of a cancer within the patient, or stage, as parameters of the clinical gravity of the disease. Grading of a cancer is based on the degree of differentiation of the tumor cells and the number of mitoses within the tumor as presumed correlates of the neoplasm's aggressiveness. Thus, cancers are classified as grades I to IV with increasing anaplasia. Criteria for the individual grades vary with each form of neoplasia and so are not detailed here, but all attempt, in essence, to judge the extent to which the tumor cells resemble or fail to resemble their normal counterparts. Although histologic grading is useful, the correlation between histologic appearance and biologic behavior is less than perfect. In recognition of this problem and to avoid spurious quantification, it is common practice to characterize a particular neoplasm in descriptive terms, for example, well-differentiated, mucin-secreting adenocarcinoma of the stomach, or highly undifferentiated, retroperitoneal malignant tumor—probably sarcoma. In general, with a few exceptions, such as soft tissue sarcomas, grading of cancers has proved of less clinical value than has staging. The staging of cancers is based on the size of the primary lesion, its extent of spread to regional lymph nodes, and the presence or absence of blood-borne metastases. Two major staging systems are currently in use, one developed by the Union Internationale Contre Cancer (UICC) and the other by the American Joint Committee (AJC) on Cancer Staging. The UICC employs a classification called the TNM system—T for primary tumor, N for regional lymph node involvement, and M for metastases. The TNM staging varies for each specific form of cancer, but there are general principles. With increasing size, the primary lesion is characterized as T1 to T4. T0 is added to indicate an in situ lesion. N0 would mean no nodal involvement, whereas N1 to N3 would denote involvement of an increasing number and range of nodes. M0 signifies no distant metastases, whereas M1 or sometimes M2 indicates the presence of blood-borne metastases and some judgment as to their number. The AJC employs a somewhat different nomenclature and divides all cancers into stages 0 to IV, incorporating within each of these stages the size of the primary lesions as well as the presence of nodal spread and distant metastases. The staging systems and additional details are mentioned in appropriate chapters, in conjunction with the discussion of specific tumors. It merits emphasis here, however, that staging of neoplastic disease has assumed great importance in the selection of the best form of therapy for the patient. It bears repeating that staging has proved to be of greater clinical value than grading. In some cases, such as for lung cancers, staging has been greatly aided by imaging techniques such as positron emission 55] tomography.[ LABORATORY DIAGNOSIS OF CANCER Every year the approach to laboratory diagnosis of cancer becomes more complex, more sophisticated, and more specialized. For virtually every neoplasm mentioned in this text, the experts have characterized a number of subcategories; we must walk, however, before we can run. Each of the following sections attempts to present the state of the art, avoiding details of method. Histologic and Cytologic Methods. The laboratory diagnosis of cancer is, in most instances, not difficult. The two ends of the benign-malignant spectrum pose no problems; however, in the middle lies a gray zone where one should tread cautiously. The focus here is on the roles of the clinician (often a surgeon) and the pathologist in facilitating the correct diagnosis. Clinical data are invaluable for optimal pathologic diagnosis, but often clinicians tend to underestimate the value of the clinical data. Radiation changes in the skin or mucosa can be similar to those associated with cancer. Sections taken from a healing fracture can mimic an osteosarcoma. Moreover the 336 laboratory evaluation of a lesion can be only as good as the specimen made available for examination. It must be adequate, representative, and properly preserved. Several sampling approaches are available: (1) excision or biopsy, (2) needle aspiration, and (3) cytologic smears. When excision of a small lesion is not possible, selection of an appropriate site for biopsy of a large mass requires awareness that the margins may not be representative and the center largely necrotic. Appropriate preservation of the specimen is obvious, yet it involves such actions as prompt immersion in a usual fixative (commonly formalin solution, but other fluids can be used), preservation of a portion in a special fixative (e.g., glutaraldehyde) for electron microscopy, or prompt refrigeration to permit optimal hormone, receptor, or other types of molecular analysis. Requesting "quick-frozen section" diagnosis is sometimes desirable, for example, in determining the nature of a mass lesion or in evaluating the margins of an excised cancer to ascertain that the entire neoplasm has been removed. This method permits histologic evaluation within minutes. In experienced, competent hands, frozen-section diagnosis is highly accurate, but there are particular instances in which the better histologic detail provided by the more time-consuming routine methods is needed—for example, when extremely radical surgery, such as the amputation of an extremity, may be indicated. Better to wait a day or two despite the drawbacks, than to perform inadequate or unnecessary surgery. Fine-needle aspiration of tumors is another approach that is widely used. The procedure involves aspirating cells and attendant fluid with a small-bore needle, followed by cytologic examination of the stained smear. This method is used most commonly for the assessment of readily palpable lesions in sites such as the breast, thyroid, and lymph nodes. Modern imaging techniques enable the method to be extended to lesions in deep-seated structures, such as pelvic lymph nodes and pancreas. Fine-needle aspiration is less invasive and more rapidly performed than are needle biopsies. In experienced hands, it is an extremely reliable, rapid, and useful technique. Cytologic (Pap) smears provide yet another method for the detection of cancer ( Chapter 22 ). This approach is widely used Figure 7-54 A normal cervicovaginal smear shows large, flattened squamous cells and groups of metaplastic cells; interspersed are some neutrophils. There are no malignant cells. (Courtesy of Dr. P.K. Gupta, University of Pennsylvania, Philadelphia, PA.) Figure 7-55 An abnormal cervicovaginal smear shows numerous malignant cells that have pleomorphic, hyperchromatic nuclei; interspersed are some normal polymorphonuclear leukocytes. (Courtesy of Dr. P.K. Gupta, University of Pennsylvania, Philadelphia, PA.) Figure 7-56 Anticytokeratin immunoperoxidase stain of a tumor of epithelial origin (carcinoma). (Courtesy of Dr. Melissa Upton, University of Washington, Seattle, WA.) TABLE 7-13 -- Selected Tumor Markers Markers Associated Cancers Hormones Human chorionic gonadotropin Trophoblastic tumors, nonseminomatous testicular tumors Calcitonin Medullary carcinoma of thyroid Catecholamine and metabolites Pheochromocytoma and related tumors Ectopic hormones See Paraneoplastic Syndromes in Table 7-12 Oncofetal Antigens α-Fetoprotein Liver cell cancer, nonseminomatous germ cell tumors of testis Carcinoembryonic antigen Carcinomas of the colon, pancreas, lung, stomach, and heart Isoenzymes Prostatic acid phosphatase Prostate cancer Neuron-specific enolase Small cell cancer of lung, neuroblastoma Specific Proteins Immunoglobulins Multiple myeloma and other gammopathies Prostate-specific antigen and prostate-specific membrane antigen Prostate cancer Mucins and Other Glycoproteins CA-125 Ovarian cancer CA-19-9 Colon cancer, pancreatic cancer CA-15-3 Breast cancer New Molecular Markers p53, APC, RAS mutations in stool and serum Colon cancer p53 and RAS mutations in stool and serum Pancreatic cancer p53 and RAS mutations in sputum and serum Lung cancer p53 mutations in urine Bladder cancer development of tests to detect cancer markers in blood and body fluids is an active area of research. 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Wulfkuhle JD, et al: Proteomic approaches to the diagnosis, treatment, and monitoring of cancer. Adv Exp Med Biol 532:59, 2003 343 Chapter 8 - Infectious Diseases Alexander J. McAdam MD, PhD Arlene H. Sharpe MD, PhD * *The contributions of Dr. John Samuelson and Dr. Franz von Lichtenberg to the previous editions are gratefully acknowledged. 344 General Principles of Microbial Pathogenesis Despite the availability and use of effective vaccines and antibiotics, infectious diseases remain an important cause of death in the United States and worldwide. In the United States, two of 1 the top 10 leading causes of death are infectious diseases (pneumonia and influenza, and septicemia).[ ] Infectious diseases are particularly important causes of death among the elderly and people with acquired immunodeficiency syndrome (AIDS), those with chronic diseases, and those receiving immunosuppressive drugs. In developing countries, unsanitary living conditions and malnutrition contribute to a massive burden of infectious diseases that kills more than 10 million people each year. Most of these deaths are among children, especially from respiratory and diarrheal infections.[ 2] HISTORY The history of infectious disease pathology is intertwined with that of microbiology. Some of the major historical events in these fields are briefly described here to provide a perspective for the concepts of pathogenesis to be discussed later. Some important experiments that were performed in the past would not be ethically acceptable today. Louis Pasteur and Robert Koch were pioneers in establishing the microbiologic etiology of infectious diseases. Pasteur is credited with proving that microorganisms can cause disease (the germ theory of disease). Pasteur also created the first attenuated vaccines, including a rabies vaccine for humans in 1885. In 1882, Koch championed criteria for linking a specific microorganism to a disease. Koch's postulates require that (1) the organism is found in the lesions of the disease, (2) the organism can be isolated as single colonies on solid media, (3) inoculation of the organism causes lesions in experimental animals, and (4) the organism can be recovered from the experimental animal. Koch also isolated the bacteria that cause tuberculosis (Mycobacterium tuberculosis) and anthrax (Bacillus anthracis). Ronald Ross, an English military physician posted in India, demonstrated in 1897 that mosquitoes carry malaria. At the time, it was believed that malaria was caused by breathing the air near swamps ("malaria" comes from the Italian for "bad air"). Ross's demonstration that Anopheles mosquitoes transmit malaria led to public health efforts to reduce malaria through control of mosquitoes. This was successful in the United States, but malaria continues to be a major health problem in many parts of the world. Walter Reed, an American military physician, led a team of investigators in Cuba in 1900 who demonstrated that yellow fever, like malaria, is transmitted by the bite of mosquitoes. Military volunteers allowed themselves to be bitten by mosquitoes that had previously bitten people sick with yellow fever. Following Reed's result, Dr. James Carroll showed in 1901 that yellow fever was caused by a virus. This was the first demonstration that a virus causes disease in humans. F. Peyton Rous found the first evidence for an infectious cause of cancer in 1909. In 1911, Rous demonstrated that a virus causes sarcoma in chickens. Although a viral cause has not been found for most human cancers, we now know that viruses can contribute to the development of some; such associations include human papillomaviruses and cervical cancer. The dawn of modern microbiology, which is based on molecular genetics, came in 1944, when Oswald Avery demonstrated that transfer of DNA from virulent to avirulent Streptococcus pneumoniae transformed the latter into a virulent phenotype. This showed that DNA is the genetic material, leading to an explosion of research in molecular genetics. Today, the entire genomic sequences of many species, including microbes and humans, are known, and this holds great promise for future research into the pathogenesis, diagnosis, and treatment of infectious diseases. Knowledge of the genomes of the host and pathogens promise to produce a much richer description of the host response to infectious 345 agents than the morphologic descriptions of antimicrobial responses in this chapter. NEW AND EMERGING INFECTIOUS DISEASES Although infectious diseases such as leprosy have been known since biblical times and parasitic schistosomes and mycobacteria have been demonstrated in Egyptian mummies, a surprising number of new infectious agents continue to be discovered ( Table 8-1 ). The infectious causes of some diseases with significant morbidity and mortality (e.g., Helicobacter pylori gastritis, hepatitis B and hepatitis C, human metapneumovirus respiratory disease, and Legionnaire's pneumonia) were previously unrecognized because the infectious agents are difficult to culture. Some infectious agents are genuinely new to humans, e.g., human immunodeficiency virus (HIV), which causes the acquired immunodeficiency syndrome (AIDS); Borrelia burgdorferi, which causes Lyme disease; and the coronavirus that may cause severe acute respiratory syndrome (SARS) ( Chapter 15 ). Other infections are much more TABLE 8-1 -- Some Recently Recognized Infectious Agents and Manifestations 1977 Ebola virus Epidemic hemorrhagic fever Hantaan virus Hemorrhagic fever with renal disease Legionella pneumophila Legionnaire's disease Campylobacter jejuni Enteritis 1980 HTLV-I T-cell lymphoma or leukemia 1981 Staphylococcus aureus Toxic shock syndrome 1982 HTLV-II Hairy cell leukemia Escherichia coli O 157:H7 Hemolytic-uremic syndrome Borrelia burgdorferi Lyme disease HIV AIDS Helicobacter pylori Gastric ulcers 1985 Enterocytozoon bieneusi Chronic diarrhea 1988 HHV-6 Roseola subitum 1983 Hepatitis E Enterically transmitted hepatitis Hepatitis C Hepatitis C Ehrlichia chaffeensis Human monocytic ehrlichiosis Vibrio cholerae O 139 New epidemic cholera strain Bartonella henselae Cat-scratch disease 1993 Encephalitozoon cuniculi Opportunistic infections 1994 Anaplasma phagocytophilium Human granulocytic ehrlichiosis (anaplasmosis) 1995 KSHV (HHV-8) Kaposi sarcoma in AIDS 2001 Human metapneumovirus Respiratory infections 2002 West Nile virus Acute flaccid paralysis 2003 SARS coronavirus Severe acute respiratory syndrome 1989 1992 Adapted from Lederberg J: Infectious disease as an evolutionary paradigm. Emerg Infect Dis 3:417, 1997. commonly seen because of immunosuppression caused by AIDS (e.g., cytomegalovirus [CMV], Kaposi sarcoma herpesvirus, Mycobacterium avium-intracellulare, Pneumocystis jiroveci 3 4 (carinii), and Cryptosporidium parvum).[ ] [ ] Finally, infectious diseases that are common in one area may be introduced into a new area. West Nile virus was common in Europe, Asia, and Africa when it was first described in the United States in 1999. Human demographics and behavior are among the many factors that contribute to the emergence of infectious diseases. AIDS has been predominantly (but not exclusively) a disease of homosexuals and drug abusers in the United States and Western countries, while in Africa, AIDS is predominantly a heterosexual disease that is much more frequent in areas where men 5 remain uncircumcised.[ ] Changes in the environment occasionally drive rates of infectious diseases. Reforestation of the eastern United States has led to massive increases in the 6 populations of deer and mice, which carry the ticks that transmit Lyme disease, babesiosis, and ehrlichiosis. [ ] Failure of DDT to control the mosquitoes that transmit malaria and the development of drug-resistant parasites have dramatically increased the morbidity and mortality of Plasmodium falciparum in Asia, Africa, and Latin America. Microbial adaptation to widespread antibiotic use contributed to the development of new drug-resistant strains of Mycobacterium tuberculosis, Neisseria gonorrhoeae, Staphylococcus aureus, and Enterococcus faecium. AGENTS OF BIOTERRORISM Sadly, the anthrax attacks in the United States in 2001 transformed the theoretical threat of bioterrorism into reality. The Centers for Disease Control and Prevention (CDC) have evaluated the microorganisms that pose the greatest danger as weapons on the basis of how efficiently disease can be transmitted, how hard the microorganisms are to produce and distribute, how well they can be defended against, and how likely they are to alarm the public and produce widespread fear. The CDC has ranked bioweapons into three categories, A, B, and C, based on these criteria. These agents are listed in Table 8-2 . [ 7] Category A agents are the highest-risk agents and can be readily disseminated or transmitted from person to person, can cause high mortality with potential for major public health impact, might cause public panic and social disruption, and require special action for public health preparedness. For example, smallpox is a category A agent owing to its high transmissibility in any climate or season, case mortality rate of 30% or greater, and lack of effective antiviral therapy. This agent can be easily disseminated because of the stability of the virus in aerosol form and the very small dose needed for infection. Smallpox naturally spreads from person to person mainly by respiratory aerosol or by direct contact with virus in skin lesions or contaminated clothing or bedding. Symptoms appear after 7 to 17 days. Initially, there is high fever, headache, and backache, followed by the appearance of the rash, which first appears on the mucosa of the mouth and pharynx, face, and forearms and later spreads to the trunk and legs and becomes vesicular and later pustular. Because people are infectious during the incubation period, this virus has the potential to continue to spread throughout an unprotected population. Since vaccination ended in the United States in 1972 and vaccination immunity has waned, the population is 346 TABLE 8-2 -- Potential Agents of Bioterrorism Category A Diseases/Agents * Category B Diseases/Agents • Anthrax (Bacillus anthracis) • Brucellosis (Brucella species) • Botulism (Clostridium botulinum toxin) • Epsilon toxin of Clostridium perfringens • Plague (Yersinia pestis) • Food safety threats (e.g., Salmonella species, Escherichia coli 0157: H7, Shigella) Category C Diseases/Agents • Emerging infectious disease threats such as Nipah virus and Hantavirus • Smallpox (Variola major virus) • Tularemia (Francisella tularensis) • Glanders (Burkholderia mallei) • Viral hemorrhagic fevers (filoviruses [e.g., Ebola, Marburg], arenaviruses [Lassa fever virus and New World arenaviruses], bunyaviruses [e.g. Crimean-Congo hemorrhagic fever and Rift Valley Fever viruses] • Melioidosis (Burkholderia pseudomallei) • Psittacosis (Chlamydia psittaci) • Q fever (Coxiella burnetti) • Ricin toxin from Ricinus communis (castor beans) • Staphylococcal enterotoxin B • Typhus fever (Rickettsia prowazekii) • Viral encephalitis (alphaviruses [e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis]) • Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum) *Adapted from Centers for Disease Control Information. highly susceptible to smallpox. Recent concern that smallpox could be used for bioterrorism has led to a return of vaccination for selected groups in the U.S. and Israel. Category B agents are moderately easy to disseminate, produce moderate morbidity but low mortality, and require specific diagnostic and disease surveillance. Many of these agents are foodborne or waterborne. Category C agents include emerging pathogens that could be engineered for mass dissemination because of availability, ease of production and dissemination, potential for high morbidity and mortality, and great impact on health. CATEGORIES OF INFECTIOUS AGENTS Infectious agents belong to a wide range of classes and vary in size from the ∼27-kD nucleic acid-free prion to 20-nm poliovirus to 10-m tapeworms ( Table 8-3 ). TABLE 8-3 -- Classes of Human Pathogens and Their Habitats Taxonomic Size Site of Propagation Sample Species Disease Viruses 20–300 nm Obligate intracellular Poliovirus Poliomyelitis Chlamydiae 200–1000 nm Obligate intracellular Chlamydia trachomatis Trachoma, urethritis Rickettsiae 300–1200 nm Obligate intracellular Rickettsia prowazekii Typhus fever Mycoplasmas 125–350 nm Extracellular Mycoplasma pneumoniae Atypical pneumonia Bacteria 0.8–15 µm Cutaneous Staphylococcus aureus Wound Mucosal Vibrio cholerae Cholera Extracellular Streptococcus pneumoniae Pneumonia Facultative intracellular Mycobacterium tuberculosis Tuberculosis Cutaneous Trichophyton sp. Tinea pedis (athlete's foot) Mucosal Candida albicans Thrush Extracellular Sporothrix schenckii Sporotrichosis Facultative intracellular Histoplasma capsulatum Histoplasmosis Mucosal Giardia lamblia Giardiasis Extracellular Trypanosoma gambiense Sleeping sickness Facultative intracellular Trypanosoma cruzi Chagas disease Obligate intracellular Leishmania donovani Kala-azar Mucosal Enterobius vermicularis Enterobiasis Extracellular Wuchereria bancrofti Filariasis Intracellular Trichinella spiralis Trichinosis Fungi Protozoa Helminths Prions 2–200 µm 1–50 µm 3 mm–10 m 8 Prions are apparently composed of abnormal forms of a host protein, termed prion protein (PrP).[ ] These agents cause transmissible spongiform encephalopathies, including kuru (associated with human cannibalism), Creutzfeldt-Jakob disease (CJD; associated with corneal transplants), bovine spongiform encephalopathy (BSE; better known as mad cow disease), 9 and variant Creutzfeldt-Jakob disease (vCJD; likely transmitted to humans from BSE-infected cattle). [ ] PrP is normally found in neurons. Diseases occur when the prion protein undergoes a conformational change that confers resistance to proteases. The protease-resistant PrP promotes conversion of the normal protease-sensitive PrP to the abnormal form, explaining the infectious nature of these diseases. Accumulation of abnormal PrP leads to neuronal damage and distinctive spongiform pathologic changes in the brain. 347 Spontaneous or inherited mutations in PrP, which make PrP protease resistant, have been observed in the sporadic and familial forms of CJD, respectively. These diseases are discussed in detail in Chapter 28 . Viruses Viruses are obligate intracellular parasites that depend on the host cell's metabolic machinery for their replication. They consist of a nucleic acid genome surrounded by a protein coat (called a capsid) that is sometimes encased in a lipid membrane. Viruses are classified by their nucleic acid genome (DNA or RNA but not both), the shape of the capsid (icosahedral or helical), the presence or absence of a lipid TABLE 8-4 -- Selected Human Viral Diseases and Their Pathogens Viral Pathogen Virus Family Genomic Type Disease Expression Respiratory Adenovirus Adenoviridae DS DNA Upper and lower respiratory tract infections, conjunctivitis, diarrhea Rhinovirus Picornaviridae SS RNA Upper respiratory tract infection Coxsackievirus Picornaviridae SS RNA Pleurodynia, herpangina, hand-foot-and-mouth disease, SARS Coronavirus Coronaviridae SS RNA Upper respiratory tract infection Influenza viruses A, B Orthomyxoviridae SS RNA Influenza Respiratory syncytial virus Paramyxoviridae SS RNA Bronchiolitis, pneumonia Mumps virus Paramyxoviridae SS RNA Mumps, pancreatitis, orchitis Rotavirus Reoviridae DS RNA Childhood diarrhea Norwalk agent Caliciviridae SS RNA Gastroenteritis Hepatitis A virus Picornaviridae SS RNA Acute viral hepatitis Hepatitis B virus Hepadnaviridae DS DNA Acute or chronic hepatitis Digestive Hepatitis D virus Viroid-like SS RNA With HBV, acute or chronic hepatitis Hepatitis C virus Flaviviridae SS RNA Acute or chronic hepatitis Hepatitis E virus Norwalk-like SS RNA Enterically transmitted hepatitis Measles virus Paramyxoviridae SS RNA Measles (rubeola) Rubella virus Togaviridae SS RNA German measles (rubella) Parvovirus Parvoviridae SS DNA Erythema infectiosum, aplastic anemia Vaccinia virus Poxviridae DS DNA Smallpox vaccine Varicella-zoster virus Herpesviridae DS DNA Chickenpox, shingles Herpes simplex virus 1 Herpesviridae DS DNA "Cold sore" Herpes simplex virus 2 Herpesviridae DS DNA Genital herpes Cytomegalovirus Herpesviridae DS DNA Cytomegalic inclusion disease Epstein-Barr virus Herpesviridae DS DNA Infectious mononucleosis HTLV-I Retroviridae SS RNA Adult T-cell leukemia; tropical spastic paraparesis HIV-1 and HIV-2 Retroviridae SS RNA AIDS Dengue virus 1–4 Togaviridae SS RNA Dengue, hemorrhagic fever Yellow fever virus Togaviridae SS RNA Yellow fever Regional hemorrhagic fever viruses Filoviridae SS RNA Ebola, Marburg disease Hantavirus SS RNA Korean, U.S. pneumonia Papovaviridae DS DNA Condyloma; cervical carcinoma Poliovirus Picornaviridae SS RNA JC virus Papovaviridae DS DNA Progressive multifocal leukoencephalopathy (opportunistic) Arboviral encephalitis viruses Togaviridae SS RNA Systemic with Skin Eruptions Systemic with Hematopoietic Disorders Arboviral and Hemorrhagic Fevers Warty Growths Papillomavirus Central Nervous System Poliomyelitis Eastern, Western, Venezuelan, St. Louis, DS, double-stranded; SS, single-stranded. envelope, their mode of replication, the preferred cell type for replication (called tropism), or the type of pathology ( Table 8-4 ). Because viruses are only 20 to 300 nm in size, they are best visualized with the electron microscope ( Fig. 8-1 ). However, some viral particles aggregate within the cells they infect and form characteristic inclusion bodies, which may be seen with the light microscope and are useful for diagnosis. For example, cytomegalovirus (CMV)-infected cells are enlarged and show a large eosinophilic nuclear inclusion and smaller basophilic cytoplasmic inclusions; herpesviruses form a large nuclear inclusion surrounded by a clear halo; and both smallpox and rabies viruses form characteristic cytoplasmic inclusions. Many viruses do not give rise to inclusions (e.g., Epstein Barr virus [EBV]). 348 Figure 8-1 The variety of viral structures, as seen by electron microscopy. A, Adenovirus, an icosahedral nonenveloped DNA virus with fibers. B, Epstein Barr virus, an icosahedral enveloped DNA virus. C, Rotavirus, a nonenveloped, wheel-like, RNA virus. D, Paramyxovirus, a spherical enveloped RNA virus. RNA is seen spilling out of the disrupted virus. (Photos courtesy of Science Source; © Photo Researchers, Inc., New York, New York.) TABLE 8-5 -- Examples of Bacterial, Spirochetal, and Mycobacterial Diseases Clinical or Microbiologic Category Infections by pyogenic cocci Gram-negative infections, common Contagious childhood bacterial diseases Enteropathic infections Species Frequent Disease Presentations Staphylococcus aureus, S. epidermidis Abscess, cellulitis, pneumonia, septicemia Streptococcus pyogenes, β-hemolytic Upper respiratory tract infection, erysipelas, scarlet fever, septicemia Streptococcus pneumoniae (pneumoccoccus) Lobar pneumonia, meningitis Neisseria meningitidis (meningococcus) Cerebrospinal meningitis Neisseria gonorrhoeae (gonococcus) Gonorrhea * Escherichia coli Urinary tract infection, wound infection, abscess, pneumonia, septicemia, endotoxemia, endocarditis * Klebsiella pneumoniae * Enterobacter (Aerobacter) aerogenes * Proteus spp. (P. mirabilis, P. morgagni) * Serratia marcescens * Pseudomonas spp. (P. aeruginosa) Bacteroides spp. (B. fragilis) Anaerobic infection Legionella spp. (L. pneumophila) Legionnaires disease Haemophilus influenzae Meningitis, upper and lower respiratory tract infections Bordetella pertussis Whooping cough Corynebacterium diphtheriae Diphtheria Enteropathogenic E. coli Invasive or noninvasive gastroenterocolitis, some with septicemia Shigella spp. Vibrio cholerae Campylobacter fetus, C. jejuni Yersinia enterocolitica Salmonella spp. (1000 strains) Clostridial infections Zoonotic bacterial infections Human treponemal infections Mycobacterial infections Actinomycetaceae Salmonella typhi Typhoid fever Clostridium tetani Tetanus (lockjaw) Clostridium botulinum Botulism (paralytic food poisoning) Clostridium perfringens, C. septicum Gas gangrene, necrotizing cellulitis * Pseudomembranous colitis Clostridium difficile Bacillus anthracis Anthrax (malignant pustule) * Listeria meningitis, listeriosis Listeria monocytogenes Yersinia pestis Bubonic plague Francisella tularensis Tularemia Brucella melitensis, B. suis, B. abortus Brucellosis (undulant fever) Burkholderia mallei, B. pseudomallei Glanders, melioidosis Leptospira spp. (many groups) Leptospirosis, Weil disease Borrelia recurrentis Relapsing fever Borrelia burgdorferi Lyme borreliosis Bartonella henselae Cat-scratch disease; bacillary angiomatosis Spirillum minus, Streptobacillus moniliformis Rat-bite fever Treponema pallidum Venereal, endemic syphilis (bejel) Treponema pertenue Yaws (frambesia) Treponema carateum (T. herrejoni) Pinta (carate, mal del pinto) * Tuberculosis Mycobacterium tuberculosis, M. bovis (Koch bacillus) M. leprae (Hansen bacillus) Leprosy * Atypical mycobacterial infections M. kansasii, M. avium, M. intracellulare M. ulcerans Buruli ulcer * Nocardiosis Nocardia asteroides Actinomyces israelii *Important opportunistic infections. Actinomycosis include Staphylococcus epidermidis and Propionibacterium acnes, the cause of acne. Aerobic and anaerobic bacteria in the mouth, particularly Streptococcus mutans, contribute to dental plaque, a major cause of tooth decay. In the colon, 99.9% of bacteria are anaerobic, including Bacteroides species. Many bacteria remain extracellular when they invade the body, while others can survive and replicate either outside or inside of host cells (facultative intracellular bacteria) and some grow only inside host cells (obligate intracellular bacteria). Chlamydiae, Rickettsiae, Mycoplasmas These microbes are grouped together because, like other bacteria, they divide by binary fission and are sensitive to antibiotics, but they lack certain structures (e.g., Mycoplasma lack a cell wall) or metabolic capabilities (e.g., Chlamydia cannot synthesize adenosine triphosphate [ATP]). Chlamydia and Rickettsiae are obligate intracellular organisms that replicate in membrane-bound vacuoles in epithelial cells and the 350 Figure 8-2 Molecules on the surface of Gram-negative and Gram-positive bacteria involved in pathogenesis. Not shown is the type 3 secretory apparatus of Gram-negative bacteria (see text). Figure 8-3 The variety of bacterial morphology. A, Gram stain of sputum from patient with pneumonia. There are Gram-positive cocci in clusters (Staphylococcus aureus) with degenerating neutrophils. B, Gram stain of sputum from a patient with pneumonia. Gram-positive, elongated cocci in pairs and short chains (Streptococcus pneumoniae) and a neutrophil is seen. C, Gram stain of Clostridium sordellii grown in culture. A mixture of Gram-positive and Gram-negative rods, many of which have subterminal spores (clear areas), are present. Clostridia species often stain as both Gram-positive and negative, although they are true Gram-positive bacteria. D, Gram stain of a bronchoalveolar lavage specimen showing Gramnegative intracellular rods typical of Enterobacteriaceae such as Klebsiella pneumoniae or Escherichia coli. E, Gram stain of urethral discharge from a patient with gonorrhea. Many Gramnegative diplococci (Neisseria gonorrhoeae) are present within a neutrophil. F, Silver stain of brain tissue from a patient with Lyme disease meningoencephalitis. Two helical spirochetes (Borrelia burgdorferi) are indicated by arrows. The panels are at different magnifications. (D, Courtesy of Dr. Karen Krisher, Clinical Microbiology Institute, Wilsonville, OR. All other panels courtesy of Dr. Kenneth Van Horn.) TABLE 8-6 -- Protozoa Pathogenic for Humans Species Order Form, Size Disease Luminal or Epithelial Entamoeba histolytica Amebae Trophozoite 15–20 µm Amebic dysentery; liver abscess Balantidium coli Ciliates Trophozoite 50–100 µm Colitis Naegleria fowleri Ameboflagellates Trophozoite 10–20 µm Meningoencephalitis Acanthamoeba sp. Ameboflagellates Trophozoite 15–30 µm Meningoencephalitis or ophthalmitis Giardia lamblia Mastigophora Trophozoite 11–18 µm Diarrheal disease, malabsorption Isospora belli Coccidia Oocyst 10–20 µm Chronic enterocolitis or malabsorption or both Cryptosporidium sp. Coccidia Oocyst 5–6 µm Trichomonas vaginalis Mastigophora Trophozoite 10–30 µm Urethritis, vaginitis Plasmodium species Hemosporidia Trophozoites, schizonts, gametes (all small and inside red cells) Malaria Babesia microti, B. bovis Hemosporidia Trophozoites inside red cells Babesiosis Trypanosoma species Hemoflagellates Trypomastigote 14–33 µm African sleeping sickness Trypanosoma cruzi Hemoflagellates Trypomastigote 20 µm Chagas disease Leishmania donovani Hemoflagellates Amastigote 2 µm Kala-azar Leishmania species Hemoflagellates Amastigote 2 µm Cutaneous and mucocutaneous leishmaniasis Toxoplasma gondii Coccidia Tachyzoite 4–6 µm (cyst larger) Toxoplasmosis Bloodstream Intracellular immunosuppressed individuals do opportunistic fungi give rise to life-threatening infections characterized by tissue necrosis, hemorrhage, and vascular occlusion, with minimal to no inflammatory response. In addition, AIDS patients are victims of the opportunistic fungus Pneumocystis jiroveci (carinii). Protozoa Parasitic protozoa are single-celled eukaryotes that are major causes of disease and death in developing countries ( Table 8-6 ). Protozoa can replicate intracellularly within a variety of cells (e.g., Plasmodium in red blood cells, Leishmania in macrophages) or extracellularly in the urogenital system, intestine, or blood. Trichomonas vaginalis are flagellated protozoal parasites that are sexually transmitted and can colonize the vagina and male urethra. The most prevalent intestinal protozoans, Entamoeba histolytica and Giardia lamblia, have two forms: (1) motile trophozoites that attach to the intestinal epithelial wall and may invade and (2) immobile cysts that are resistant to stomach acids and are infectious when ingested. Blood-borne protozoa (e.g., Plasmodium, Trypanosoma, and Leishmania) are transmitted by insect vectors, in which they replicate before being passed to new human hosts. Toxoplasma gondii is acquired either by contact with oocyst-shedding kittens or by eating cyst-ridden, undercooked meat. Helminths Parasitic worms are highly differentiated multicellular organisms. Their life cycles are complex; most alternate between sexual reproduction in the definitive host and asexual multiplication in an intermediary host or vector. Thus, depending on parasite species, humans may harbor either adult worms (e.g., Ascarus lumbricoides) or immature stages (e.g., Toxocara canis) or asexual larval forms (e.g., Echinococcus species). Once adult worms take up residence in humans, 352 they do not multiply but generate eggs or larvae destined for the next phase of the cycle. An exception is Strongyloides stercoralis, the larvae of which can become infectious in the gut and cause overwhelming autoinfection in immunosuppressed persons. There are two important consequences of the lack of replication of adult worms: (1) Disease is often caused by inflammatory responses to the eggs or larvae rather than to the adults (e.g., schistosomiasis), and (2) disease is in proportion to the number of organisms that have infected the individual (e. g., 10 hookworms cause little disease, whereas 1000 hookworms cause severe anemia by consuming 100 mL of blood per day). Ectoparasites Ectoparasites are insects (lice, bedbugs, fleas) or arachnids (mites, ticks, spiders) that attach to and live on or in the skin. Arthropods may produce disease directly by damaging the human host or indirectly by serving as the vectors for transmission of an infectious agent into a human host. Some arthropods may cause itching and excoriations (e.g., pediculosis caused by lice attached to hair shafts, or scabies caused by mites burrowing into the stratum corneum). At the site of the bite, mouthparts may be found associated with a mixed infiltrate of lymphocytes, macrophages, and eosinophils. In addition, attached arthropods can be vectors for other pathogens. For example, deer ticks transmit the Lyme disease spirochete Borrelia burgdorferi. TRANSMISSION AND DISSEMINATION OF MICROBES Host Barriers to Infection The outcome of infection is determined by the ability of the microbe to infect, colonize, and damage host tissues and the ability of host defense mechanisms to eradicate the infection. Host 11 barriers to infection prevent microbes from entering the body and consist of innate and adaptive immune defenses [ ] (see Fig. 6-1 , Chapter 6). Innate immune defense mechanisms exist before infection and respond rapidly to microbes. These mechanisms include physical barriers to infection, phagocytic cells and natural killer cells, and plasma proteins, including the complement system proteins and other mediators of inflammatory responses (cytokines, collectins, acute phase reactants). Adaptive immune responses are stimulated by exposure to microbes and increase in magnitude, speed, and effectiveness with successive exposures to microbes. Adaptive immunity is mediated by T and B lymphocytes and their products ( Chapter 6 ). Microbes can enter the host by inhalation, ingestion, sexual transmission, insect or animal bites, or injection. The first barriers to infection are intact host skin and mucosal surfaces and their secretory products. In general, respiratory, gastrointestinal, or genitourinary tract infections occur in healthy persons and are caused by relatively virulent microorganisms that are capable of damaging or penetrating intact epithelial barriers. In contrast, most skin infections in healthy persons are caused by less virulent organisms entering the skin through damaged sites (cuts and burns). Skin. The dense, keratinized outer layer of skin is a natural barrier to infection, and the low pH of the skin (about 5.5) and the presence of fatty acids inhibit growth of microorganisms other than residents of the normal flora. Human skin is normally inhabited by a variety of bacterial and fungal species, including some potential opportunists, such as Staphyloccus epidermidis and Canadida albicans. Although skin is usually an effective barrier, certain types of fungi (dermatophytes) can infect the stratum corneum, hair, and nails, and a few microorganisms are able to traverse the unbroken skin. For example, Schistosoma larvae released from freshwater snails penetrate swimmers' skin by releasing collagenase, elastase, and other enzymes that dissolve the extracellular matrix. Most microorganisms, however, penetrate through breaks in the skin, including superficial pricks (fungal infections), wounds (staphylococci), burns (Pseudomonas aeruginosa), and diabetic and pressure-related foot sores (multibacterial infections). Intravenous catheters in hospitalized patients can produce local or systemic infection (bacteremia). Needle sticks can expose the recipient to potentially infected blood and may transmit HBV, HCV, or HIV. Some pathogens penetrate the skin via an insect or animal bite. For instance, bites by fleas, ticks, mosquitoes, mites, and lice break the skin and transmit arboviruses (causes of yellow fever and encephalitis), rickettsiae (Rocky Mountain spotted fever), bacteria (plague, Lyme disease), protozoa (malaria, leishmaniasis), and helminths (filariasis). Animal bites can lead to infections with bacteria or with rabies virus. Gastrointestinal Tract. Most gastrointestinal pathogens are transmitted by food or drink contaminated with fecal material. Where hygiene fails, diarrheal disease becomes rampant. Acidic gastric secretions are important defenses within the gastrointestinal tract and are lethal for many gastrointestinal pathogens.[ 11 10[ ] 11] Healthy volunteers do not become infected by Vibrio cholerae unless they are fed organisms, whereas volunteers given Vibrio cholerae and sodium bicarbonate have a 10,000-fold increase in susceptibility to cholera. In contrast, some ingested agents, such as Shigella and Giardia cysts, are relatively resistant to gastric acid; hence, as few as 100 organisms of each are sufficient to cause illness. Other normal defenses within the gastrointestinal tract include (1) the viscous mucous layer covering the gut, (2) lytic pancreatic enzymes and bile detergents, (3) mucosal antimicrobial peptides called defensins, (4) normal flora, and (5) secreted IgA antibodies. IgA antibodies are made by B cells located in mucosa-associated lymphoid tissues (MALT). These lymphoid aggregates are covered by a single layer of specialized epithelial cells called M cells. M cells are important for transport of antigens to MALT and for binding and uptake of numerous gut pathogens, including poliovirus, enteropathic Escherichia coli, Vibrio cholerae, Salmonella typhi, and Shigella flexneri. [ 12] Infections via the gastrointestinal tract occur when local defenses are weakened or the organisms develop strategies to overcome these defenses. Host defenses are weakened by low gastric acidity, by antibiotics that unbalance the normal bacterial flora (e.g., in pseudomembranous colitis), or when there is stalled peristalsis or mechanical obstruction (e.g., in blind loop syndrome). Most enveloped viruses are killed by the bile and digestive enzymes, but nonenveloped viruses may be resistant (e.g., the hepatitis A virus, rotaviruses, reoviruses, and Norwalk agents). 353 Enteropathogenic bacteria elicit gastrointestinal disease by a variety of mechanisms: • While growing on contaminated food, certain staphylococcal strains release powerful enterotoxins that cause food poisoning symptoms without any bacterial multiplication in the gut. • V. cholerae and toxigenic E. coli multiply inside the mucous layer overlying the gut epithelium and release exotoxins that cause the gut epithelium to secrete high volumes of watery diarrhea. • Shigella, Salmonella, and Campylobacter invade and damage the intestinal mucosa and lamina propria and so cause ulceration, inflammation, and hemorrhage, clinically 13 manifested as dysentery.[ ] • S. typhi passes from the damaged mucosa through Peyer patches and mesenteric lymph nodes and into the bloodstream, resulting in a systemic infection. Fungal infection of the gastrointestinal tract occurs mainly in immunologically compromised patients. Candida, part of the normal gastrointestinal flora, shows a predilection for stratified squamous epithelium, causing oral thrush or membranous esophagitis, but may also disseminate to the stomach, lower gastrointestinal tract, and systemic organs. The cyst forms of intestinal protozoa are essential for their transmission because cysts resist stomach acid. In the gut, cysts convert to motile trophozoites and attach to sugars on the intestinal epithelia through surface lectins. Thereafter, there is wide species variation. Giardia lamblia attaches to the epithelial brush border, whereas cryptosporidia are taken up by enterocytes, in which they form gametes and spores. Entamoeba histolytica causes contact-mediated cytolysis through a channel-forming pore protein and thereby ulcerates and invades the colonic mucosa. Intestinal helminths, as a rule, cause disease only when they are present in large numbers or in ectopic sites, for example, by obstructing the gut or invading and damaging the bile ducts (Ascaris lumbricoides). Hookworms may cause iron deficiency anemia by chronic loss of blood sucked from intestinal villi; the fish tapeworm Diphyllobothrium latum can deplete its host of vitamin B12 , giving rise to an illness resembling pernicious anemia. Finally, the larvae of several helminth parasites pass through the gut briefly on their way toward another organ habitat; for example, Trichinella spiralis larvae preferentially encyst in muscle, Echinococcus species larvae in the liver or lung. Respiratory Tract. Some 10,000 microorganisms, including viruses, bacteria, and fungi, are inhaled daily by every city inhabitant. The distance these microorganisms travel into the respiratory system is 11 inversely proportional to their size.[ ] Large microbes are trapped in the mucociliary blanket that lines the nose and the upper respiratory tract. Microorganisms are trapped in the mucus secreted by goblet cells and are then transported by ciliary action to the back of the throat, where they are swallowed and cleared. Organisms smaller than 5 µm travel directly to the alveoli, where they are phagocytosed by alveolar macrophages or by neutrophils recruited to the lung by cytokines. Damage to the mucociliary defense results from repeated insults in smokers and patients with cystic fibrosis, while acute injury occurs in intubated patients and in those who aspirate gastric acid. Successful respiratory microbes evade the mucociliary defenses in part by attaching to epithelial cells in the lower respiratory tract and pharynx. For example, influenza viruses possess hemagglutinin proteins that project from the surface of the virus and bind to sialic acid on the surface of epithelial cells. This attachment induces the host cell to engulf the virus, leading to viral entry and replication within the host cell. However, sialic acid binding prevents newly synthesized viruses from leaving the host cell. Influenza viruses have another cell surface protein, neuraminidase, which cleaves sialic acid and allows virus to release from the host cell. Neuraminidase also lowers the viscosity of mucus and facilitates viral transit within the respiratory tract. Interestingly, some anti-influenza drugs are sialic acid analogs that inhibit neuraminidase and prevent viral release from host cells. Certain respiratory bacterial pathogens can impair ciliary activity. For instance, Haemophilus influenza and Bordetella pertussis elaborate toxins that paralyze mucosal cilia; Pseudomonas aeruginosa, a cause of severe respiratory infection in persons with cystic fibrosis, and Mycoplasma pneumoniae produce ciliostatic substances. Some bacteria such as Streptococcus pneumoniae or Staphylococcus species lack specific adherence factors and often gain access after viral infection causes loss of ciliated epithelium, making individuals who have had viral respiratory infection more susceptible to secondary bacterial respiratory infection. Mycobacterium tuberculosis, in contrast, gains its foothold in normal alveoli because it is able to escape phagocytic killing by macrophages. Growth requirements for microorganisms can determine their site of infection in the respiratory tract. For example, rhinoviruses, which cause the common cold, grow optimally at 33°C, the temperature of the nasal mucosa, but grow poorly at 37°C, the temperature of the lower respiratory tract. Finally, opportunistic fungi infect the lungs when cellular immunity is depressed or when leukocytes are reduced in number (e.g., P. jiroveci [carinii] in AIDS patients and Aspergillus species in chemotherapy patients). Urogenital Tract. 11 The urinary tract is almost always invaded from the exterior via the urethra.[ ] The regular flushing of the urinary tract with urine serves as a defense against invading microorganisms. Urine in the bladder is normally sterile, and successful pathogens (e.g., gonococci, E. coli) adhere to the urinary epithelium. Anatomy is an important factor for infection. Women have more than 10 times as many urinary tract infections (UTIs) as men, because the distance between the urinary bladder and skin (i.e., the length of the urethra) is 5 cm, in contrast to 20 cm in men. Obstruction of urinary flow and/or reflux can compromise normal defenses and increase susceptibility to UTIs. UTIs can spread retrogradely from the bladder to the kidney and cause acute and chronic pyelonephritis, which is the major preventable cause of renal failure. From puberty until menopause, the vagina is protected from pathogens by a low pH resulting from catabolism of glycogen in the normal epithelium by lactobacilli. Antibiotics can kill the lactobacilli and make the vagina susceptible to infection. To be successful as pathogens, microorganisms have developed specific mechanisms for attaching to vaginal or cervical mucosa or enter via local breaks in the mucosa during sex (genital warts, syphilis). 354 Spread and Dissemination of Microbes 11 Some microorganisms proliferate locally, at the site of infection, whereas others penetrate the epithelial barrier and spread to other sites via the lymphatics, the blood, or nerves[ ] ( Fig. 8-4 ). Some of the superficial pathogens stay confined to the lumen of hollow viscera (e.g., cholera); others adhere to or proliferate exclusively in or on epithelial cells (e.g., papillomaviruses, dermatophytes). A variety of pathogenic bacteria, fungi, and helminths are invasive by virtue of their motility or ability to secrete lytic enzymes (e.g., streptococci and staphylococci secrete hyaluronidase, which degrades the extracellular matrix between host cells). Microbial spread initially follows tissue planes of least resistance and regional lymphatic and vascular anatomy. For example, staphylococcal infections may progress from a localized abscess or furuncle to regional lymphadenitis that sometimes leads to bacteremia and colonization of distant organs (heart, liver, brain, kidney, bone). Within the blood, microorganisms may be transported free or within host cells. Some viruses (e.g., poliovirus and HBV), most bacteria and fungi, some protozoa (e.g., African trypanosomes), and all helminths are transported free in the plasma. Leukocytes can carry herpesviruses, HIV, mycobacteria, and Leishmania and Toxoplasma organisms. Certain viruses (e.g., Colorado tick fever virus) and parasites (Plasmodium and Babesia) are carried by red blood cells. Viruses also may propagate Figure 8-4 Routes of entry, dissemination, and release of microbes from the body. (Adapted from Mims CA: The Pathogenesis of Infectious Disease, 4th ed. San Diego, CA, Academic Press, 1996.) TABLE 8-7 -- Classification of Important Sexually Transmitted Diseases Disease or Syndrome and Population Principally Affected Pathogens Males Both Females Viruses ••Herpes simplex virus Primary and recurrent herpes, neonatal herpes ••Hepatitis B virus Hepatitis ••Human papillomavirus Cancer of penis (some cases) ••Human immunodeficiency virus Condyloma acuminatum Cervical dysplasia and cancer, vulvar cancer Acquired immunodeficiency syndrome Chlamydiae ••Chlamydia trachomatis Urethritis, epididymitis, proctitis Lymphogranuloma venereum Urethral syndrome, cervicitis, bartholinitis, salpingitis and sequelae Urethritis, proctitis, pharyngitis, disseminated gonococcal infection Cervicitis, endometritis, bartholinitis, salpingitis, and sequelae (infertility, ectopic pregnancy, recurrent salpingitis) Mycoplasmas ••Ureaplasma urealyticum Urethritis Bacteria ••Neisseria gonorrhoeae Epididymitis, prostatitis, urethral stricture Treponema pallidum Syphilis Haemophilus ducreyi Chancroid Calymmatobacterium granulomatis Granuloma inguinale (donovanosis) Shigella * Enterocolitis Campylobacter * Enterocolitis Protozoa Vaginitis ••Trichomonas vaginalis Urethritis, balanitis ••Entamoeba histolytica * Amebiasis ••Giardia lamba * Giardiasis Modified and updated from Krieger JN: Biology of sexually transmitted diseases. Urol Clin North Am 11:15, 1984. *Most important in homosexual populations. Syphilis is discussed later in this chapter, and other STIs are described in Chapter 21 and Chapter 22 . HOW MICROORGANISMS CAUSE DISEASE Infectious agents establish infection and damage tissues in three ways: • They can contact or enter host cells and directly cause cell death. • They may release toxins that kill cells at a distance, release enzymes that degrade tissue components, or damage blood vessels and cause ischemic necrosis. • They can induce host cellular responses that, although directed against the invader, cause additional tissue damage, usually by immune-mediated mechanisms. Thus, as we discussed in Chapter 2 and Chapter 6 , the defensive responses of the host are a two-edged sword: They are necessary to overcome the infection but at the same time may directly contribute to tissue damage. Here we describe some of the mechanisms whereby viruses and bacteria damage host tissues. Mechanisms of Viral Injury Viruses can directly damage host cells by entering them and replicating at the host's expense. The predilection for viruses to infect certain cells and not others is called tissue tropism and is determined by several factors, including (1) host cell receptors for the virus, (2) cellular transcription factors that recognize viral enhancer and promoter sequences, (3) anatomic barriers, and (4) local temperature, pH, and host defenses.[ 15] Each of these is described briefly. A major determinant of tissue tropism is the presence of viral receptors on host cells. Viruses possess specific cell-surface proteins that bind to particular host cell-surface proteins. Many viruses use normal cellular receptors of the host to enter cells. For example, HIV gp120 binds to CD4 on T cells and to the chemokine receptors CXCR4 (mainly on T cells) and CCR5 (mainly on macrophages). Rhinoviruses bind to the same site on ICAM-1 as LFA-1, an integrin on the surface of lymphocytes that is an important adhesion molecule for lymphocyte 16] activation and migration.[ hemagglutinin. In some cases, host proteases are needed to enable binding of virus to host cells; for instance, a host protease cleaves and activates the influenza virus 357 Another determinant of viral tropism is the ability of the virus to replicate inside some cells but not in others, and this is related to the presence of cell-type—specific transcription factors. For example, the JC virus, which causes leukoencephalopathy ( Chapter 28 ), is restricted to oligodendroglia in the central nervous system because the promoter and enhancer DNA sequences upstream from the viral genes are active in glial cells but not in neurons or endothelial cells. Physical barriers also can contribute to tissue tropism. For example, enteroviruses replicate in the intestine in part because they can resist inactivation by acids, bile, and digestive enzymes. Rhinoviruses replicate only within the upper respiratory tract because they survive optimally at the lower temperature of the upper respiratory tract. Once viruses are inside host cells, they can kill the cells and/or cause tissue damage in a number of ways ( Fig. 8-5 ): • Viruses may inhibit host cell DNA, RNA, or protein synthesis. For example, poliovirus inactivates cap-binding protein, which is essential for translation of host cell mRNAs, but leaves translation of poliovirus mRNAs unaffected. • Viral proteins may insert into the host cell's plasma membrane and directly damage its integrity or promote cell fusion (HIV, measles virus, and herpesviruses). • Viruses may lyse host cells. For example, respiratory epithelial cells are killed by influenza virus replication, liver cells by yellow fever virus, and neurons by poliovirus and rabies virus. • Viruses may manipulate programmed cell death (apoptosis). Some virus-encoded proteins (including TAT and gp120 of HIV, adenovirus E1A) can induce cell death. In contrast, some viruses encode one or more genes that inhibit apoptosis (e.g., homologues of the cellular bcl-2 gene), suggesting that apoptotic cell death may be a protective host response to eliminate virus-infected cells. It has been hypothesized that viral antiapoptotic strategies may enhance viral replication, promote persistent viral infections, or promote virus17 induced cancers.[ ] • Viral proteins on the surface of the host cells may be recognized by the immune system, and the host lymphocytes may attack the virus-infected cells. Acute liver failure during hepatitis B infection may be accelerated by cytotoxic T lymphocyte (CTL)-mediated destruction of infected hepatocytes (a normal response to clear the infection). FAS ligand on 18 CTLs, which bind to FAS receptors on the surface of hepatocytes, also can induce apoptosis in target cells.[ ] • Viruses may damage cells involved in host antimicrobial defense, leading to secondary infections. For example, viral damage to respiratory epithelium predisposes to the subsequent development of pneumonia by Streptococcus pneumoniae and Haemophilus influenzae. HIV depletes CD4+ helper lymphocytes and thereby causes opportunistic infections. • Viral killing of one cell type may cause the death of other cells that depend on them. For example, denervation by the attack of poliovirus on motor neurons causes atrophy and sometimes death of distal skeletal muscle supplied by such neurons. • Some viruses can cause cell proliferation and transformation (e.g., EBV, HBV, human papillomavirus, or HTLV-1), resulting in cancer. The mechanisms of viral transformation are numerous and are discussed in Chapter 7 . Figure 8-5 Mechanisms by which viruses cause injury to cells. TABLE 8-8 -- Pathogens with Significant Antigenic Variation Rhinoviruses Colds Influenza virus Influenza Neisseria gonorrhoeae Gonorrhea Borrelia hermsii Relapsing fever Borrelia burgdorferi Lyme disease Trypanosoma brucei African sleeping sickness Giardia lamblia Giardiasis Plasmodium falciparum Severe malaria 360 33 neutrophils and macrophages.[ ] The carbohydrate capsule on the surface of all the major bacteria that cause pneumonia or meningitis (pneumococcus, meningococcus, Haemophilus influenzae) makes them more virulent by shielding bacterial antigens and by preventing phagocytosis of the organisms by neutrophils. For example, E. coli with the sialic acid-containing K1 capsule causes meningitis in newborns. Sialic acid will not bind C3b, which is critical for activation of the alternative complement pathway, so the bacteria escape from complementmediated lysis and opsonization-directed phagocytosis. Many bacteria make toxic proteins that kill phagocytes, prevent their migration, or diminish their oxidative burst. Bacteria also can circumvent immune defenses by covering themselves with host proteins. S. aureus are covered by protein A molecules that bind the Fc portion of antibodies and so inhibit phagocytosis. Neisseria, Haemophilus, and Streptococcus all secrete proteases that degrade antibodies. Another successful strategy for circumventing phagocytic defense mechanisms is to replicate within phagocytic cells. A number of viruses, rickettsias, some intracellular bacteria (including mycobacteria, Listeria, and Legionella), fungi (e.g., Cryptococcus neoformans), and protozoa (e.g., leishmania, trypanosomes, toxoplasmas) can multiply within phagocytes. 15 35 36 Viruses can produce molecules that inhibit innate immunity.[ ] [ ] [ ] Some viruses (e.g., herpesviruses and poxviruses) produce proteins that block complement activation. Viruses have developed a large number of strategies to combat interferons (IFN), an early host defense against viruses. Some viruses produce soluble homologues of IFN-α/β or IFN-γ receptors that inhibit actions of extracellular IFNs, or produce proteins that inhibit intracellular JAK/STAT signaling downstream of IFN receptors or inactivate or inhibit dsRNA-dependent protein kinase (PKR), a key mediator of the antiviral effects of IFN. Viruses also can produce homologues of chemokines or chemokine receptors, and these can function as antagonists and inhibit recruitment of inflammatory cells to favor survival of viruses. Viruses also can produce soluble cytokine mimics (e.g., EBV produces a homologue of the immunosuppressive cytokine IL10) or soluble cytokine receptor homologues. Some microbes can decrease recognition of infected cells by CD4+ helper T cells and CD8+ cytotoxic T cells. For example, several DNA viruses (e.g., herpesviruses, including HSV, 36 37 HCMV, and EBV) can bind to or alter localization of MHC class I proteins, impairing peptide presentation to CD8+ T cells[ ] [ ] ( Fig. 8-6 ). Downregulation of MHC class I molecules might make it likely that virus-infected cells would be targets for NK cells. However, herpesviruses also express MHC class I homologues that act as effective inhibitors of NK cells by engaging killer inhibitory receptors ( Chapter 6 ). Similarly, herpesviruses can target MHC class II molecules for degradation, impairing antigen presentation to CD4+ T helper cells. Viruses also can infect lymphocytes and directly compromise their function. HIV infects CD4+ T cells, macrophages, and dendritic cells, and EBV infects B lymphocytes. INFECTIONS IN IMMUNOSUPPRESSED HOSTS Different types of immunosuppression affect different cells of the immune system. The opportunistic infections that an immunosuppressed person contracts depend on the types of Figure 8-6 Inhibition of MHC expression by viruses. The steps at which different viruses inhibit the class I MHC antigen presentation pathway are shown. (Modified with permission from Abbas AK, Lichtman AH: Cellular and Molecular Immunology, 5th ed., Philadelphia, Saunders, 2003.) TABLE 8-9 -- Special Techniques for Diagnosing Infectious Agents Gram stain Most bacteria Acid-fast stain Mycobacteria, nocardiae (modified) Silver stains Fungi, legionellae, pneumocystis Periodic acid-Schiff Fungi, amebae Mucicarmine Cryptococci Giemsa Campylobacteria, leishmaniae, malaria parasites Antibody probes Viruses, rickettsiae Culture All classes DNA probes Viruses, bacteria, protozoa of a lesion rather than at its center, particularly if there is necrosis. Nucleic acid-based tests have become routine methods for detecting or quantifying several pathogens. Molecular diagnostics have become particularly important in the care of people 40 infected with HIV.[ ] Quantification of the viral RNA is an important guide to antiretroviral therapy. The management of hepatitis B and C infections is similarly guided by nucleic acidbased viral quantification or typing to predict resistance to antiviral drugs. Nucleic acid amplification tests (NAATs), such as polymerase chain reaction (PCR) and transcription-mediated amplification, have become routine for diagnosis of gonorrhea, chlamydia, 41 42 tuberculosis, and herpes encephalitis. In some cases, molecular assays are much more sensitive than conventional testing.[ ] [ ] PCR testing of cerebrospinal fluid (CSF) for herpes simplex virus encephalitis has a sensitivity of about 80%, while viral culture of CSF has a sensitivity of less than 10%. Similarly, NAATs for genital chlamydia detect 10% to 30% more infections than does conventional chlamydia culture. In other cases, such as gonorrhea, the sensitivity of NAAT testing is similar to that of culture. SPECTRUM OF INFLAMMATORY RESPONSES TO INFECTION In contrast to the vast molecular diversity of microbes, the morphologic patterns of tissue responses to microbes are limited, as are the mechanisms directing these responses. At the microscopic level, therefore, many pathogens produce identical reaction patterns, and few features are unique or pathognomonic for a particular microorganism. Moreover, it is the interaction between the microorganism and the host that determines the histologic features of the inflammatory response. Thus, pyogenic bacteria, which normally evoke vigorous leukocyte responses, may cause rapid tissue necrosis with little leukocyte exudation in a profoundly neutropenic host. Similarly, in a normal patient, M. tuberculosis causes well-formed granulomas with few mycobacteria present, whereas in an AIDS patient, the same mycobacteria multiply profusely in macrophages, which fail to coalesce into granulomas. There are five major histologic patterns of tissue reaction in infections. Suppurative (Polymorphonuclear) Inflammation This pattern is the reaction to acute tissue damage, described in Chapter 2 , characterized by increased vascular permeability and leukocytic infiltration, predominantly of neutrophils ( Fig. 8-7 ). The neutrophils are attracted to the site of infection by release of chemoattractants from the "pyogenic" bacteria that evoke this response, mostly extracellular Gram-positive cocci and Gram-negative rods. Massing of neutrophils forms pus. The sizes of exudative lesions vary from tiny microabscesses formed in multiple organs during bacterial sepsis secondary to a colonized heart valve to diffuse involvement of entire lobes of the lung during pneumonia. How destructive the lesions are depends on their location and the organism involved. For example, pneumococci usually spare alveolar walls and cause lobar pneumonia that resolves 362 Figure 8-7 Pneumococcal pneumonia. Note the intra-alveolar polymorphonuclear exudate and intact alveolar septa. Figure 8-8 Secondary syphilis in the dermis with perivascular lymphoplasmacytic infiltrate and endothelial proliferation. Figure 8-9 Herpesvirus blister in mucosa. See Figure 8-13 for viral inclusions. Figure 8-10 Schistosoma haematobium infection of the bladder with numerous calcified eggs and extensive scarring. Figure 8-11 Measles giant cells in the lung. Note the glassy eosinophilic intranuclear inclusions. Figure 8-12 High-power view of cells from the blister in Figure 8-9 showing glassy intranuclear herpes simplex inclusion bodies. Figure 8-13 Cytomegalovirus: distinct nuclear and ill-defined cytoplasmic inclusions in the lung. Figure 8-14 Skin lesion of chickenpox (varicella zoster virus) with intraepithelial vesicle. Figure 8-15 Dorsal root ganglion with varicella zoster virus infection. Note the ganglion cell necrosis and associated inflammation. (Courtesy of Dr. James Morris, Radcliffe Infirmary, Oxford, England.) Figure 8-16 Pathways of transmission of the Epstein-Barr virus. In an individual with normal immune function, infection leads to mononucleosis. In the setting of cellular immunodeficiency, proliferation of infected B cells is uncontrolled and may cause B-cell neoplasms. One secondary genetic event that collaborates with Epstein-Barr virus (EBV) to cause B-cell transformation is a balanced 8;14 chromosomal translocation, which is seen in Burkitt lymphoma. EBV has also been implicated in the pathogenesis of nasopharyngeal carcinoma, Hodgkin disease, and certain other rare non-Hodgkin lymphomas. Figure 8-17 Atypical lymphocytes in infectious mononucleosis. Figure 8-18 The many consequences of staphylococcal infection. Figure 8-19 Staphylococcal abscess of the lung with extensive neutrophilic infiltrate and destruction of the alveoli (contrast with Figure 8-8 ). Figure 8-20 Streptococcal erysipelas. Figure 8-21 Membrane of diphtheria lying within a transverse bronchus (A) and forming a perfect cast (removed from the lung) of the branching respiratory tree (B). Figure 8-22 Mechanism of action of anthrax toxins. (Adapted from Mourez et al: 2001: a year of major advances in anthrax toxin research. Trends Microbiol 10(6):287, 2002.) Figure 8-23 B. anthracis in the subcapsular sinus of a hilar lymph node of a patient who died of inhalational anthrax. (Courtesy of Dr. Lev Grinberg, Department of Pathology, Hospital 40, Ekaterinburg, Russia and Dr. David Walker, UTMB Center for Biodefense and Emerging Infectious Diseases, Galveston, TX.) Figure 8-24 Nocardia asteroides in a Gram-stained sputum sample. Note the beaded, branched Gram-positive organisms and leukocytes. (Courtesy of Dr. Ellen Jo Baron, Stanford University Medical Center, Stanford, CA.) Figure 8-25 Gonococcal culture showing pili, as seen by scanning microscopy (A), and in clusters, as seen by transmission electron microscopy (B). (Courtesy of Dr. John Swanson, Rocky Mountain Laboratories, Hamilton, MT.) Figure 8-26 Whooping cough showing a haze of bacilli (arrows) etangled with the cilia of bronchial epithelial cells. Figure 8-27 Pseudomonas vasculitis in which masses of organisms form a perivascular blue haze. Figure 8-28 The sequence of events in primary pulmonary tuberculosis, commencing with inhalation of virulent M. tuberculosis and culminating with the development of cell-mediated immunity to the organism. A, Events occurring in the first 3 weeks after exposure. B, events thereafter. The development of resistance to the organism is accompanied by the appearance of a positive tuberculin test. Cells and bacteria are not drawn to scale. iNOS, inducible nitric oxide synthase; MHC, major histocompatibility complex; MTB, M. tuberculosis; NRAMP1, natural resistance-associated macrophage protein. Figure 8-29 The natural history and spectrum of tuberculosis. (Adapted from a sketch provided by Dr. R. K. Kumar, The University of New South Wales, School of Pathology, Sydney, Australia.) Figure 8-30 Primary pulmonary tuberculosis, Ghon complex. The gray-white parenchymal focus is under the pleura in the lower part of the upper lobe. Hilar lymph nodes with caseation are seen on the left. Figure 8-31 The morphologic spectrum of tuberculosis. A characteristic tubercle at low magnification (A) and in detail (B) illustrates central caseation surrounded by epithelioid and multinucleated giant cells. This is the usual response seen in patients who have developed cell mediated immunity to the organism. Occasionally, even in immunocompetent individuals, tubercular granulomas might not show central caseation (C); hence, irrespective of the presence or absence of caseous necrosis, special stains for acid-fast organisms need to be performed when granulomas are present in histologic section. In immunosuppressed individuals, tuberculosis may not elicit a granulomatous response ("nonreactive tuberculosis"); instead, sheets of foamy histiocytes are seen, packed with mycobacteria that are demonstrable with acid-fast stains (D). (D, Courtesy of Dr. Dominick Cavuoti, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 8-32 Secondary pulmonary tuberculosis. The upper parts of both lungs are riddled with gray-white areas of caseation and multiple areas of softening and cavitation. Figure 8-33 Miliary tuberculosis of the spleen. The cut surface shows numerous gray-white granulomas. Figure 8-34 Mycobacterium avium infection in a patient with AIDS, showing massive infection with acid-fast organisms. Figure 8-35 Leprosy. A, Peripheral nerve. Note the inflammatory cell infiltrates in the endoneural and epineural compartments. B, Cells within the endoneurium contain acid-fast positive lepra bacilli. (Courtesy of E.P. Richardson, Jr. and U. De Girolami, Harvard Medical School.) Figure 8-36 Lepromatous leprosy. Acid-fast bacilli ("red snappers") within macrophages. Figure 8-37 Treponema pallidum (dark-field microscopy) showing several spirochetes in scrapings from the base of a chancre. (Courtesy of Dr. Paul Southern, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 8-38 Protean manifestations of syphilis. Figure 8-39 Syphilitic chancre in the scrotum (see Figure 8-8 for the histopathology of syphilis). (Courtesy of Dr. Richard Johnson, Beth Israel-Deaconess Hospital, Boston, MA.) Figure 8-40 Trichrome stain of liver shows liver gumma (scar), stained blue, which is caused by tertiary syphilis (also known as hepar lobatum). Compare with nodules of alcoholic cirrhosis ( Chapter 18 ). Figure 8-41 Tiny deer tick (bottom), which transmits Lyme disease and Babesia and Ehrlichia organisms, contrasted with a larger dog tick (top), which is not thought to transmit human infections. (Courtesy of Dr. F.R. Matuschka, Free University of Berlin, Germany.) Figure 8-42 Clinical stages of Lyme disease. Figure 8-43 Boxcar-shaped Gram-positive Clostridium perfringens in gangrenous tissue. Figure 8-44 Peripheral blood granulocyte (band neutrophil) containing an Ehrlichia inclusion (arrow). (Courtesy of Dr. Stephen Dumler, Johns Hopkins Medical Institutions, Baltimore, MD.) TABLE 8-10 -- Rickettsial Diseases and Pathogens Typhus Group (No Eschar) Organism Disease Geography Transmission Distinctive Features R. prowazekii Epidemic typhus BrillZinsser disease Worldwide (war, famine) Louse feces Endothelial infection; centrifugal rash; reactivation with mild disease R. typhi Murine typhus Worldwide (rat related) Rat flea feces Similar to epidemic typhus, but mortality is lower Spotted Fever Group Organism Disease Geography Transmission Distinctive Features R. rickettsii Rocky Mountain spotted fever North and South America Tick bite Endothelia and vascular smooth muscle infected; centripetal rash, eschar rare R. conorii Boutonneuse fever Africa, Southern Europe, India Tick bite Prominent eschar, tache noire R. africae Africa tick fever Africa, Caribbean Tick bite Multiple eschars R. sibirica North Asia tick typhus Eurasia Tick bite Typical spotted fever with eschar R. japonica Japanese spotted fever Japan Tick bite Typical spotted fever with eschar R. australis Queensland tick typhus Eastern Australia Tick bite Typical spotted fever with eschar R. akari Rickettsialpox United States, Ukraine, Korea, Croatia Mite bite Mild spotted fever with eschar R. felis Similar to murine typhus United States Opossum flea Similar to murine typhus Orientia tsutsugamushi Scrub typhus Eastern Asia and Western Pacific region Chigger bite Eschar common, insects present in scrub vegetation Ehrlichiosis Group Organism Disease Geography Transmission Distinctive Features Ehrlichia chaffeensis Monocytic ehrlichiosis United States, Europe Tick bite Fever, lymphadenopathy, no eschar, rash in 40% Anaplasma phagocytophilum and E. ewingii Granulocytic ehrlichiosis United States, Europe Tick bite Fever, lymphadenopathy, no eschar or rash The innate immune response to rickettsial infection is mounted by natural killer cells, which produce γ-interferon, reducing bacterial proliferation. Cytotoxic T-lymphocyte responses are critical for elimination of rickettsial infections. IFN-γ and TNF, from activated natural killer cells, CD4+, and CD8+ T lymphocytes, stimulate the production of bactericidal nitric oxide. Cytotoxic T lymphocytes lyse infected cells, reducing bacterial proliferation. Rickettsial infections are diagnosed by immunostaining of organisms or by detection of antirickettsial antibodies in the serum. Morphology Typhus Fever. In mild cases, the gross changes are limited to a rash and small hemorrhages due to the vascular lesions. In more severe cases, there may be areas of necrosis of the skin with gangrene of the tips of the fingers, nose, earlobes, scrotum, penis, and vulva. In such cases, irregular ecchymotic hemorrhages may be found internally, principally in the brain, heart muscle, testes, serosal membrane, lungs, and kidneys. The most prominent microscopic changes are the small-vessel lesions that underlie the rash and the focal areas of hemorrhage and inflammation in the various organs and tissues affected. Endothelial swelling in the capillaries, arterioles, and venules may narrow the lumina of these vessels. A cuff of mononuclear inflammatory cells usually surrounds the affected vessel. The vascular lumina are sometimes thrombosed, but necrosis of the vessel wall is unusual in typhus compared with RMSF. Vascular thromboses lead to the gangrenous necroses of the skin and other structures in a minority of cases. In the brain, characteristic typhus nodules are composed of focal microglial proliferations with an infiltrate of mixed T lymphocytes and macrophages ( Fig. 8-45 ). Scrub typhus, or mite-borne infection, is usually a milder version of typhus fever. The rash is usually transitory or might not appear. Vascular necrosis or thrombosis is rare, but there may be a prominent inflammatory lymphadenopathy. Rocky Mountain spotted fever. A hemorrhagic rash that extends over the entire body, including the palms of the hands and soles of the feet, is the hallmark 397 Figure 8-45 Typhus nodule in the brain. Figure 8-46 Rocky Mountain spotted fever with a thrombosed vessel and vasculitis. Figure 8-47 The morphology of Candida infections. A, Severe candidiasis of the distal esophagus. B, Silver stain of esophageal candidiasis reveals the dense mat of Candida. C, Characteristic pseudohyphae and blastoconidia (budding yeast) of Candida. (C, Courtesy of Dr. Dominick Cuvuoti, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Figure 8-48 Mucicarmine stain of cryptococci (staining red) in a Virchow-Robin perivascular space of the brain (soap-bubble lesion). Figure 8-49 Aspergillus morphology. A, Invasive aspergillosis of the lung in a bone marrow transplant patient. B, Histologic sections from this case, stained with Gomori methenaminesilver (GMS) stain, show septate hyphae with acute-angle branching, features consistent with Aspergillus. Occasionally, Aspergillus may demonstrate fruiting bodies (inset) when it grows in areas that are well aerated (such as the upper respiratory tract). Figure 8-50 PAS stain of mucormycosis showing hyphae, which have an irregular width and right-angle branching, invading an artery wall. Figure 8-51 Life cycle of Plasmodium falciparum. (Drawn by Dr. Jeffrey Joseph, Beth Israel-Deaconess Hospital, Boston, MA.) Figure 8-52 P. falciparum-infected red cells marginating within a vein in cerebral malaria. Figure 8-53 Erythrocytes with Babesia, including the distinctive Maltese cross form. (Courtesy of Lynne Garcia, LSG and Associates, Santa Monica, CA.) Figure 8-54 Leishmania donovani parasites within the macrophages of a lymph node in visceral leishmaniasis (kala-azar). Figure 8-55 Slender bloodstream parasites of African trypanosomiasis. Figure 8-56 Strongyloides hyperinfection in a patient treated with high-dose cortisone. A female, her eggs and rhabditoid larvae are in the duodenal crypts; filariform larvae are entering the blood vessels and muscularis mucosa. (Courtesy of Dr. Franz C. Von Lichtenberg, Brigham and Women's Hospital, Boston, MA.) Figure 8-57 Portion of a cysticercus cyst. Figure 8-58 Coiled Trichinella spiralis larva within a skeletal muscle cell. Figure 8-59 Schistosome life cycle. Figure 8-60 Schistosoma mansoni granuloma with a miracidium-containing egg (center) and numerous, adjacent, scattered eosinophils. Figure 8-61 Pipe-stem fibrosis of the liver due to chronic Schistosoma japonicum infection. Figure 8-62 Massive edema and elephantiasis caused by filariasis of the leg. 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Kristensson K, Mhlanga JD, Bentivoglio M: Parasites and the brain: neuroinvasion, immunopathogenesis and neuronal dysfunctions. Curr Top Microbiol Immunol 265:227, 2002. 160. White AJ: Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management. Annu Rev Med 51:187, 2000. 161. McManus DP: The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease. Trans R Soc Trop Med Hyg 96 (Suppl 1):S151, 2002. 162. Polvere RI, Kabbash CA, Capo VA, Kadan I, Despommier DD: Trichinella spiralis: synthesis of type IV and type VI collagen during nurse cell formation. Exp Parasitol 86:191, 1997. 163. Ortega-Pierres MG, Yepez-Mulia L, Homan W, et al: Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite. Parasite Immunol 18:273, 1996. 164. Urban JF, Jr, Noben-Trauth N, Schopf L, Madden KB, Finkelman FD: Cutting edge: IL-4 receptor expression by non-bone marrow-derived cells is required to expel gastrointestinal nematode parasites. J Immunol 167:6078, 2001. 165. Akiho H, Blennerhassett P, Deng Y, Collins SM: Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 282:G226, 2002. 166. Ross AG, Bartley PB, Sleigh AC, et al: Schistosomiasis. N Engl J Med 346:1212, 2002. 167. Pearce EJ, MacDonald AS: The immunobiology of schistosomiasis. Nat Rev Immunol 2:499, 2002. 168. Allen JE, Loke P: Divergent roles for macrophages in lymphatic filariasis. Parasite Immunol 23:345, 2001. 169. King CL: Transmission intensity and human immune responses to lymphatic filariasis. Parasite Immunol 23:363, 2001. 170. Maizels RM, Blaxter ML, Scott AL: Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity. Parasite Immunol 23:327, 2001. 171. Lawrence RA, Devaney E: Lymphatic filariasis: parallels between the immunology of infection in humans and mice. Parasite Immunol 23:353, 2001. 172. Maizels RM, Gomez-Escobar N, Gregory WF, Murray J, Zang X: Immune evasion genes from filarial nematodes. Int J Parasitol 31:889, 2001. 173. Taylor MJ, Cross HF, Ford L, Makunde WH, Prasad GB, Bilo K: Wolbachia bacteria in filarial immunity and disease. Parasite Immunol 23:401, 2001. 174. Hoerauf A, Buttner DW, Adjei O, Pearlman E: Onchocerciasis. BMJ 326:207, 2003. 175. Hoerauf A, Mand S, Adjei O, Fleischer B, Buttner DW: Depletion of wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357:1415, 2001. 415 Chapter 9 - Environmental and Nutritional Pathology Agnes B. Kane MD, PhD Vinay Kumar MD Environment and Disease Environmental and occupational health encompasses the diagnosis, treatment, and prevention of injuries and illnesses resulting from exposure to exogenous chemical or physical agents. Such exposure may occur in the workplace, or people may voluntarily expose themselves to these hazards, for example, by abusing drugs or ethanol and smoking cigarettes. These personal habits may lead to involuntary exposure of fetuses and infants to drugs, ethanol, or environmental tobacco smoke. People are often confused about the magnitude of the adverse health effects of exogenous physical and chemical agents. There is widespread concern about the potential chronic or delayed effects of exposure to low levels of contaminants in air, water, and food, and hence patients frequently seek advice and information from their health care 416 professionals about the risk of disease associated with specific environmental and occupational exposures. This chapter provides a basic foundation in the most important diseases associated with environmental and occupational exposures, emphasizing the mechanisms leading to these diseases. This framework will help physicians to recognize and treat injuries and illness resulting from environmental and occupational exposures and to educate their patients about the risks of these exposures.[ 1] RECOGNITION OF OCCUPATIONAL AND ENVIRONMENTAL DISEASES Accidents, illness, and premature deaths threaten the health of 130 million workers in the United States. Occupational health risks are even greater in developing countries, where children and women constitute a larger proportion of the work force. In the United States, the annual rate of occupational injuries is 7400 per 100,000 workers. The overall fatality rate is 4.8 per 100,000 workers; the highest rates occur in the mining, agricultural, construction, transportation, and public utility industries. In addition to physical injury, occupational exposures contribute to a wide range of illnesses that may lead to premature death ( Table 9-1 ). The magnitude of occupational diseases is most likely underestimated because workers and their employers fear economic or legal pressures, physicians may not recognize that an illness is work related, and there may be a long latent period between exposure and the development of clinical illness. Nevertheless, occupational diseases are preventable if there is adequate surveillance by state and federal governments, responsible leadership in industry, and access to health professionals trained in occupational safety and health.[ 1] The magnitude and extent of illness related to environmental exposures are difficult to ascertain. The Environmental Protection Agency estimates that more than 80,000 chemicals are currently used in the United States; approximately 1500 are pesticides and 5500 are food additives that affect our water and food supplies. Although only 600 of these chemicals have been 2 tested, 10% have produced cancer in at least one rodent species.[ ] Industrial chemicals, production TABLE 9-1 -- Reported Occupational Diseases in the United States in 1997 Disease Number of Workers Repeated trauma Percentage 276,600 64 Skin disorders 57,900 13 Lung conditions due to toxic exposures 20,300 5 Physical injury 16,600 4 Poisoning 5100 1 Lung disease due to dusts 2900 1 50,600 12 430,000 100 All other illnesses Total Data from Levy BS, Wegman DH: Occupational health — an overview. In Levy BS, et al. (eds): Occupational Health. Recognizing and Preventing Work-Related Disease and Injury, fourth ed. Philadelphia, Lippincott Williams & Wilkins, 2000, p. 3; and Bureau of Labor Statistics, U.S. Department of Labor, www.hls.gov. byproducts, and metals are commonly detected at hazardous waste sites ( Table 9-2 ). There are currently 11,300 Superfund-designated waste sites in the United States. The potential human health hazards associated with exposure to chemical mixtures is a major concern.[ 2] There is considerable difference in the magnitudes of exposure in the occupational and environmental settings. Occupational exposures affect a defined cohort of workers who are exposed to chemicals in the range of parts per million (ppm); by contrast, environmental exposures to these same chemicals in the air, water, or hazardous waste sites may be in the parts per billion (ppb) or parts per trillion (ppt) range. The health effects of such chronic, low-level exposures are unknown. In the United States, four regulatory agencies determine exposure limits for environmental and occupational hazards: the Environmental Protection Agency, the Food and Drug Administration (FDA), the Occupational Safety and Health Administration, and the Consumer Products Safety Commission. The Environmental Protection Agency regulates exposure to pesticides, toxic chemicals, water and air pollutants, and hazardous wastes. The FDA regulates drugs, medical devices, food additives, and cosmetics. The Occupational Safety and Health Administration mandates that employers (including hospitals and physicians) provide safe working conditions for employees. All other products sold for use in homes, schools, or recreation are regulated by the Consumer Products Safety Commission. Physicians should be familiar with current approaches used by regulatory agencies in the United States and be prepared to explain the strengths and limitations of the scientific evidence in nontechnical terms. Health care providers must be prepared to counsel patients about the primary prevention of disease related to occupational and environmental exposures, taking into account potential synergistic effects of mixed exposures and individual genetic susceptibility. Prevention of tobacco smoking would prevent 80% to 90% of lung cancers; however, this objective has been difficult to achieve, especially in teenagers. Strategies for secondary prevention of lung cancer in former or current smokers (e.g., chemoprevention) have been 3 disappointing so far.[ ] Prevention of occupationally TABLE 9-2 -- Common Chemicals at Hazardous Waste Sites Acetone DDT, DDE, DDD Aldrin/Dieldrin 1,1 and 1,2-Dichloroethane Arsenic Lead Barium Mercury Benzene Methylene chloride 2-Butanone Nickel Cadmium Pentachlorophenol Carbon tetrachloride Polychlorinated biphenyls Chlordane Tri- and Tetrachloroethylene Chloroform Toluene Chromium Vinyl Chloride Cyanide Zinc Data from U.S. Environmental Protection Agency, www.epa.gov/superfund/resources/chemicals.htm. 417 related diseases rests on defining and enforcing safe exposure levels, developing new technologies to reduce industrial exposures, and identifying less toxic substitutes for industrial and chemical agents. These strategies require a basic understanding of biochemical and molecular mechanisms of toxicity. MECHANISMS OF TOXICITY Toxicology is the scientific discipline that studies the detection, effects, and mechanisms of action of poisons and toxic chemicals. Toxicity is a relative phenomenon that depends on the inherent structure and properties of a chemical and on its dose. Dose-response curves are typically generated in laboratory animals exposed to various amounts of a chemical. A typical dose-response curve for acute toxicity is illustrated in Figure 9-1 . In this example, a measurable response occurs at a dose of 0.1 mg/kg; this is defined as the threshold dose. To the left of this dose, at subthreshold levels, there is no measurable response. For this chemical, this is the no observed effect level and can be considered a safe dose. This information is used to establish a daily or annual threshhold limit value or permissible exposure level for occupational exposures. Frequently, a plateau is reached at higher doses; this is defined as the ceiling effect. It is uncertain whether carcinogens show a threshold effect or whether the dose-response curve should be extrapolated linearly to zero.[ 4] Despite the inherent limitations of toxicity testing in animals, several important toxicologic principles have been established by this experimental approach. Exogenous chemicals are absorbed after ingestion, inhalation, or skin contact, and then distributed to various organs ( Fig. 9-2 ). Chemicals are frequently metabolized, often by multiple enzymatic pathways, to 5 products that may be more toxic or less toxic than the parent chemical. One or more of these products then interacts with the target macromolecule, resulting in a toxic effect.[ ] The site of toxicity is frequently the site where metabolism or excretion of toxic metabolites occurs. The dose administered (external dose) may not be the same as the biologic effective dose delivered to the target organ and target macromolecule. Figure 9-1 The dose-response curve for acute chemical toxicity. Th, threshold dose; STh, subthreshold levels. (Data from Hughes WW: Essentials of Environmental Toxicology: The Effects of Environmentally Hazardous Substances on Human Health. Washington, DC, Taylor & Francis, 1996, p. 33.) Figure 9-2 Absorption and distribution of toxicants. (From Hodgson E, Levi PE: Absorption and distribution of toxicants. In Hodgson E, Levi PE [eds]: A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 52.) Figure 9-3 Biotransformation of lipophilic toxicants to hydrophilic metabolites. (Adapted from Hodgson E: Metabolism of toxicants. In Hodgson E, Levi PE [eds]: A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 57.) TABLE 9-3 -- Organ-Specific Carcinogens in Tobacco Smoke Organ Lung, larynx Carcinogen Polycyclic aromatic hydrocarbons 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-buta-none (NNK) Polonium 210 Esophagus N'-Nitrosonornicotine (NNN) Pancreas NNK (?) Bladder 4-Aminobiphenyl, 2-naphthylamine Oral cavity (smoking) Polycyclic aromatic hydrocarbons, NNK, NNN Oral cavity (snuff) NNK, NNN, polonium 210 Data from Szczesny LB, Holbrook JH: Cigarette smoking. In Rom WH (ed): Environmental and Occupational Medicine, 2nd ed. Boston, Little, Brown, 1992, p. 1211. such synergism is the increase in risk of lung cancer in cigarette smokers exposed to asbestos.[ 13] Mainstream cigarette smoke inhaled by the smoker is composed of a particulate phase and a gas phase; tar is the total particulate phase without water or nicotine. There are 0.3 to 3.3 billion particles per milliliter of mainstream smoke and more than 4000 constituents, including 43 known carcinogens. Examples of the organ-specific carcinogens found in tobacco smoke and snuff are listed in Table 9-3 . In addition to these chemical carcinogens, cigarette smoke contains carcinogenic metals such as arsenic, nickel, cadmium, and chromium; potential promoters such as acetaldehyde and phenol; irritants such as nitrogen dioxide and formaldehyde; cilia toxins such as hydrogen cyanide; and carbon monoxide. Carbon monoxide is a colorless, odorless gas produced during incomplete combustion of fossil fuels or tobacco. It has 200 times higher affinity for hemoglobin than oxygen does and it impairs release of oxygen from hemoglobin. Thus, carbon monoxide exposure decreases the delivery of oxygen to peripheral tissues. Carbon monoxide also binds to other heme-containing proteins such as myoglobin and cytochrome oxidase. Nicotine is an important constituent of cigarette smoke. It is an alkaloid that readily crosses the blood-brain barrier and stimulates nicotine receptors in the brain. It is also responsible for the acute pharmacologic effects associated with tobacco use that are most likely mediated by catecholamines: increased heart rate and blood pressure, increased coronary artery blood flow, increased contractility and cardiac output, and mobilization of free fatty acids. Nicotine is responsible for tobacco addiction. The inhaled agents in cigarette smoke may act directly on the mucous membranes, may be swallowed in saliva, or may be absorbed into the bloodstream from the abundant alveolar capillary bed. By various routes of delivery, the constituents of cigarette smoke act on distant target organs and cause a variety of systemic diseases, listed in Table 9-4 . The greatest numbers of deaths attributable to cigarette smoking are due to lung cancer, ischemic heart disease, and chronic obstructive lung disease. Lung cancer is caused by multiple carcinogens and promoters in cigarette smoke. As described in Chapter 15 , specific preneoplastic changes are found in the tracheobronchial lining of cigarette smokers. These cellular changes 420 Figure 9-4a A, Xenobiotic metabolism: phase I reactions. FMO, flavin-containing monooxygenase; PHS, prostaglandin-H synthases. Figure 9-4b B, Xenobiotic metabolism: phase II reactions (see text for details). (Adapted from Parkinson A: Biotransformation of xenobiotics. In Klaasen CD [ed]: Casarett and Doull's Toxicology: The Basic Science of Poisons, 5th ed. New York, McGraw-Hill, 1996, pp. 113–186; and Hodgson E, Levi PE [(eds]: A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, pp. 57, 95.) TABLE 9-4 -- Deaths per Year Attributable to Cigarette Smoking in the United States Number of Deaths Cause of Death Cancer Cardiovascular disease Men Women 102,812 54,664 90,906 57,699 Respiratory disease 53,713 44,429 Residential fires 589 377 Perinatal deaths 598 407 15,517 22,536 264,135 80,112 Lung cancer and heart disease attributable to passive smoking Total Data from CDC. Annual smoking-attributable mortality, years of potential life lost, and economic costs—United States, 1995–1999. MMWR 51:300, 2002. exposure to silica, coal dust, grain dust, cotton dust, and welding fumes. Tobacco use also increases the prevalence of peptic ulcers; smoking impairs healing of ulcers and increases the likelihood of recurrence. Smoking may also increase pyloric reflux and decrease bicarbonate secretion from the pancreas. In addition to the health hazards of mainstream tobacco smoke, there are risks associated with exposure to sidestream smoke, also called passive smoking or environmental tobacco smoke (ETS). In 1986, two reports issued by the National Research Council and the Surgeon General concluded that ETS increases the risk of lung cancer, ischemic heart disease, and acute 14 myocardial infarction.[ ] The Environmental Protection Agency classified ETS as a known human carcinogen in 1992. ETS is especially hazardous for infants and young children. Maternal smoking increases the incidence of sudden infant death syndrome. Young children in households of cigarette smokers suffer from an increased incidence of respiratory and ear infections and exacerbation of asthma. Alcohol Abuse Ethanol is the most widely used and abused agent throughout the world. There are 15 to 20 million alcoholics in the United States; approximately 100,000 deaths in the United States are 15] attributed to alcohol abuse per year, with an economic cost of $100 to $130 billion.[ concentration of 80 to 100 mg/dL is the legal Ethanol is ingested in alcoholic beverages such as beer, wine, and distilled spirits. A blood alcohol 422 definition for driving under the influence of alcohol in many states. Approximately 3 ounces (44 ml) of ethanol are required to produce this blood alcohol level in a 70-kg person. This is equivalent to 12 ounces of fortified wine, 8 bottles of beer (12 ounces each), or 6 ounces of 100-proof whiskey. In occasional drinkers, a blood alcohol level of 200 mg/dL produces inebriation, with coma, death, and respiratory arrest at 300 to 400 mg/dL. Habitual drinkers can tolerate blood alcohol levels up to 700 mg/dL. This metabolic tolerance is partially explained by a fivefold to tenfold induction of the cytochrome P-450 xenobiotic-metabolizing enzyme CYP2E1. Such induction increases the metabolism of ethanol as well as that of other drugs and chemicals, including cocaine and acetaminophen. Although no specific receptor for ethanol has been identified, chronic use results in psychologic and physical dependence. The biologic basis for ethanol addiction is unknown, although genetic factors may be involved. Ethanol is metabolized to acetaldehyde by alcohol dehydrogenase in the gastric mucosa and liver, and by cytochrome P-450 (CYP2E1) and catalase in the liver ( Fig. 9-5 ). Acetaldehyde is converted to acetic acid by aldehyde dehydrogenase. There are genetic polymorphisms in aldehyde dehydrogenase that affect ethanol metabolism; approximately 50% of Chinese, Vietnamese, and Japanese people have reduced activity of this enzyme due to a point mutation that converts glutamine to lysine at amino acid 487. These ethnic groups also rapidly convert ethanol to acetaldehyde, which builds up and triggers a facial flushing syndrome. Women have lower levels of gastric alcohol dehydrogenase activity than men do; therefore, they may develop higher blood alcohol levels than men after drinking the same quantity of ethanol.[ 15] The metabolism of ethanol is directly responsible for most of its toxic effects. In addition to its acute action as a Figure 9-5 Metabolism of ethanol. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase. (From Parkinson A: Biotransformation of xenobiotics. In Klassen CD [ed]: Casarett and Doull's Toxicology: The Basic Science of Poisons, 5th ed. New York, McGraw-Hill, 1996, p. 128.) TABLE 9-5 -- Mechanisms of Disease Caused by Ethanol Abuse Organ System Liver Lesion Fatty change Acute hepatitis Alcoholic cirrhosis Mechanism Toxicity Nervous system Wernicke syndrome Thiamine deficiency Korsakoff syndrome Toxicity and thiamine deficiency Cerebellar degeneration Nutritional deficiency Peripheral neuropathy Thiamine deficiency Cardiomyopathy Toxicity Hypertension Vasopressor Gastritis Toxicity Pancreatitis Toxicity Skeletal muscle Rhabdomyolysis Toxicity Reproductive system Testicular atrophy ? Spontaneous abortion ? Growth retardation Toxicity Cardiovascular system Gastrointestinal tract Fetal alcohol syndrome Mental retardation Birth defects Data from Rubin E: Alcohol abuse. In Craighead JE (ed): Pathology of Environmental and Occupational Disease. St. Louis, Mosby-Year Book, 1996, p. 249; and Lewis DD, Woods SE: Fetal alcohol syndrome. Am Fam Physician 50:1025, 1994. central nervous system depressant, chronic ethanol use can cause a wide range of systemic effects ( Table 9-5 ). Some of these chronic effects can be attributed to specific vitamin deficiencies; for example, damage to the peripheral and central nervous systems is related to thiamine deficiency, whereas 423 other systemic effects result from direct toxicity. The effects of ethanol on various organ systems are discussed next. Liver. Ethanol can cause fatty change, acute alcoholic hepatitis, and cirrhosis. Fatty change is an acute, reversible manifestation of ethanol ingestion. In chronic alcoholism, fat accumulation can cause massive enlargement of the liver. The biochemical mechanisms responsible for fat accumulation in hepatocytes are the following: • Catabolism of fat by peripheral tissues is increased, and there is increased delivery of free fatty acids to the liver. • Metabolism of ethanol in the cytosol and of its derivative, acetaldehyde, in the mitochondria converts the oxidized form of nicotinamide adenine dinucleotide (NAD+ ) to the reduced form (NADH); an excess of NADH over NAD stimulates lipid biosynthesis. • Oxidation of fatty acids by mitochondria is decreased. • Acetaldehyde forms adducts with tubulin and impairs function of microtubules, resulting in decreased transport of lipoproteins from the liver. Acute alcoholic hepatitis is another potentially reversible form of liver injury ( Chapter 18 ). Although fatty change is asymptomatic except for liver enlargement, alcoholic hepatitis can produce fever, liver tenderness, and jaundice. On histologic examination, there are focal areas of hepatocyte necrosis and cell injury manifest by fat accumulation and alcoholic hyalin, or Mallory bodies. Neutrophils accumulate around foci of necrosis ( Fig. 9-6 ). Ethanol and its metabolites are directly toxic to hepatocytes; this toxicity is believed to be mediated by 16 glutathione depletion, mitochondrial injury, altered metabolism of methionine, and cytokine release from Kupffer cells.[ ] Hepatocellular necrosis, as well as fibrosis, begins around the central vein, suggesting that hypoxia may contribute to this injury. With chronic ethanol use, 10% to 15% of alcoholics develop irreversible liver damage, or alcoholic cirrhosis. This is characterized by a hard, shrunken liver with formation of micronodules of regenerating hepatocytes surrounded by dense bands of collagen ( Fig. 9-7 ). Alcoholic cirrhosis is a serious, potentially fatal disease accompanied by weakness, muscle wasting, ascites, gastrointestinal hemorrhage, and coma. Perisinusoidal fibrosis occurs initially, with Figure 9-6 Acute alcoholic hepatitis. The liver cells show cytoplasmic accumulation of fat and hyalin (arrow). A scattered inflammatory infiltrate is present. (MEDCOM © 1976.) Figure 9-7 Micronodular cirrhosis is a late complication of chronic alcoholism. The liver architecture is distorted by regenerating nodules of hepatocytes surrounded by dense bands of fibrous tissue that stain blue (Masson trichrome stain). (Courtesy of Dr. Steve Kroft, Department of Pathology, Southwestern Medical School, Dallas, TX.) TABLE 9-6 -- Common Drugs of Abuse Class Opioid narcotics Molecular Target Mu opioid receptor (agonist) Example Heroin, hydromorphone (Dilaudid) Oxycodone (Percodan, Percocet, Oxycontin) Methadone (Dolophine) Meperidine (Demerol) Sedative-hypnotics GABAA receptor (agonist) Barbiturates Ethanol Methaqualone (Quaalude) Glutethimide (Doriden) Ethchlorvynol (Placidyl) Psychomotor stimulants Dopamine transporter (antagonist) Cocaine Amphetamine Phencyclidine-like drugs Serotonin receptors (toxicity) 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) NMDA glutamate receptor channel (antagonist) Phencyclidine (PCP, angel dust) Ketamine Cannabinoids CBI cannabinoid receptors (agonist) Marijuana Hashish Nicotine Nicotine acetylcholine receptor (agonist) Tobacco products Hallucinogens Serotonin 5-HT2 receptors (agonist) Lysergic acid diethylamide (LSD) Mescaline Psilocybin Data from Hyman SE: A 28-year-old man addicted to cocaine. JAMA 286:2586, 2001. more slowly than ethanol, resulting in initial symptoms of intoxication, followed by toxic effects after several hours or days. Methanol is metabolized to formaldehyde and formic acid, resulting in metabolic acidosis, dizziness, vomiting, blurred vision or blindness, and respiratory depression. Methanol has been proposed as a gasoline additive or substitute, but there is concern that chronic inhalation of methanol-containing fumes may cause central nervous system depression. The lethal dose of ethylene glycol is only 1.4 mL/kg; it is metabolized by alcohol dehydrogenase to aldehydes, glycolate, oxalate, and lactate. If a person survives the initial toxicity, acute renal failure may occur several days later because of obstruction of the kidney tubules by calcium oxalate crystals. Acute methanol or ethylene glycol poisoning is treated by administration of ethanol, which slows the production of toxic metabolites. Drug Abuse Drug abuse, addiction, and overdose are serious public health problems. In a recent survey, 8% to 23% of teenagers reported marijuana use, and 2% reported cocaine use during the previous month. A National Comorbidity Survey conducted in 1995 discovered that 7.5% of US residents 15 to 54 years old had a history of drug dependence. Risk factors for drug use 21 include family history, male sex, psychiatric disorders, ethanol abuse, easy access to drugs, and peer pressure.[ ] The molecular targets of many commonly abused drugs have recently been identified, as summarized in Table 9-6 . Identification of specific neurotransmitter pathways that may activate reward circuits in the brain, as diagrammed in Figure 9-8 , may lead to more effective therapies for drug abuse and addiction. [ Sedative-Hypnotics. 19] Ethanol is the most widely abused central nervous system depressant, as discussed before. Barbiturates are circulated illegally and are known as downers. They 425 Figure 9-8 Effects of cocaine on neurotransmitters. Cocaine inhibits the reuptake of the neurotransmitters dopamine and norepinephrine in the central and peripheral nervous systems. TABLE 9-7 -- Mechanisms of Adverse Drug Reactions Mechanism Toxicity due to overdose Example Acetaminophen Adverse Effect Liver necrosis and failure Predictable reaction based on pharmacologic mechanism Nonselective, nonsteroidal anti-inflammatory drugs Peptic ulcer ••Thiopurine S-methyltransferase deficiency Azathioprine Bone marrow failure ••Cytochrome P-450 CYP2C9 variants Oral anticoagulants Bleeding ••Cytochrome P-450 CYP2D6 variants Some antipsychotic drugs Excessive sedation; parkinsonism ••N-acetyltransferase, slow acetylator phenotype Hydralazine Lupus Idiopathic Chloramphenicol Aplastic anemia Altered drug metabolism related to: and numbness. PCP characteristically induces nystagmus. High doses can induce coma lasting a few hours up to 10 days. Lysergic acid diethylamide (LSD) is a potent synthetic drug usually taken orally. It is absorbed rapidly and produces psychic effects, visual illusions, and altered perception for up to 12 hours. In high doses, LSD can cause death. THERAPEUTIC DRUGS An adverse drug reaction is defined as a toxic or undesired response to a drug used at therapeutic doses to prevent, diagnose, or treat disease. It is estimated that approximately 2 million hospitalized patients suffered from serious adverse drug reactions in 1994, resulting in 106,000 deaths. These estimates are conservative because they do not include errors in drug dosage 24 or administration or patient noncompliance.[ ] In Table 9-7 , adverse drug reactions are classified on the basis of their underlying mechanisms. Predictable reactions are based on the known toxicity or mechanism of action of a drug; these reactions are usually related to dose. Individual variations or polymorphisms in drug-metabolizing enzymes contribute to variable responses to drug therapy and an increased incidence of side effects. At least 5% of commonly prescribed drugs are metabolized by the cytochrome P-450 CYP1A2 pathway; approximately 12% of Caucasians carry variant alleles that reduce drug metabolism by this pathway. [ 25] Pharmacogenomics is a new field that uses genotyping to predict and prevent adverse drug reactions; for example, children with leukemia are screened for thiopurine methyltransferase variants to determine the optimal dose of azathiopurine,[ 26] and genotyping for 27 effects.[ ] the cytochrome P-450 CYP2D6 enzyme will help individualize doses of antipsychotic drugs to reduce side In contrast to these predictable types of adverse drug reactions, idiopathic or idiosyncratic reactions are rare and unpredictable, although the consequences may be severe or even fatal. Herbal medicines are widely used in the United States and throughout the world; although many of these preparations have been shown to be effective in short-term trials, there is lack of quality control in this industry and few long-term studies of effectiveness and safety. As summarized in Table 9-8 , the most commonly used herbal medicines in the United States can produce adverse effects, including allergic or hypersensitivity reactions, and potentially serious interactions with prescription drugs.[ 28] 427 Oral Contraceptives and Hormone Replacement Therapy Estrogens, alone or in combination with progestin, have been widely used for over 35 years as oral contraceptives or as hormone replacement therapy by perimenopausal and postmenopausal women. Most oral contraceptives combine synthetic ethinyl estradiol or mestranol with a progestin or use progestin alone; hormone replacement therapy uses natural estrogens alone or in combination with progesterone. Recent epidemiologic evidence has clarified the potential benefits and risks of these widely used drugs. Oral Contraceptives. There has been considerable concern and controversy about the safety of oral contraceptives, especially in relation to breast cancer. Two population-based, case-control studies, the Cancer and Steroid Hormone Study published in 1986 and the Women's Contraceptive and Reproductive Experiences Study published in 2002, explored the association between past or current 29 use of oral contraceptives and breast cancer. [ ] The most recent study included women between ages 35 and 64 diagnosed with breast cancer between 1994 and 1998. Potential effects of duration, formulations containing a high dose of estrogen, and family history of breast cancer were compared in women diagnosed with breast cancer or in control women without cancer. 30 Past or current use of oral contraceptives was not found to be associated with an increased risk of breast cancer in white or black women in the United States.[ ] Previous studies of other hormone-responsive cancers have also shown no increased risk of cancer; in fact, oral contraceptive use was found to decrease the risk of endometrial and ovarian cancers. In contrast, women infected with human papillomavirus have an increased risk of developing cervical cancer if they use oral contraceptives, although this risk may be related to other lifestyle factors ( Chapter 22 ). Uncommon adverse effects of oral contraceptives include: 26 • Venous thrombosis and pulmonary embolism. Oral contraceptives increase the risk of thrombosis; this risk is higher in carriers of mutations in factor V or prothrombin,[ ] as described in Chapter 4 . The older, high-dose preparations incurred a greater risk, but a smaller risk persists even with the low-estrogen-containing oral contraceptives. The newer, third-generation oral contraceptives that combine low-dose estrogen with synthetic progestins confer an even higher risk. • Cardiovascular disease. Estrogens and progestins have opposing effects on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels. The overall effect on lipoproteins depends on the preparations used, especially the dose of progestin in the formulation. Recent epidemiologic evidence suggests that nonsmoking healthy women younger than age 45 who use the newer low-estrogen formulations do not have an increased risk of atherosclerosis or myocardial infarction. However, the risk of myocardial infarction is increased in women older than age 35 who smoke. The risk of ischemic stroke is also increased, regardless of age or smoking history. • Liver tumors. Benign hepatic adenomas may occur, especially in older women who have used oral contraceptives for prolonged periods. These tumors may rupture and cause intra-abdominal bleeding. TABLE 9-8 -- Adverse Effects of Herbal Medicines Example Adverse Effects Drug Interactions Echinacea Allergic reactions None described Ginkgo Headache, nausea Potentiates anticoagulants Ginseng Headache, insomnia, euphoria, diarrhea Interacts with monamine oxidase inhibitors, hypoglycemic drugs, anticoagulants Saw palmetto Constipation, decreased libido, urine retention None described St. John's wort Allergic reactions, nausea, photosensitivity Accelerated drug metabolism (oral contraceptives, anticoagulants) Data from Ernst E: The risk-benefit profile of commonly used herbal therapies: ginkgo, St. John's wort, ginseng, echinacea, saw palmetto, and kava. Ann Intern Med 136:42, 2002. Hormone Replacement Therapy. In the United States, approximately one third of perimenopausal and postmenopausal women use hormone replacement therapy (HRT), either estrogen in combination with a progestin or a natural estrogen alone. There has been recent controversy about the risks and benefits of HRT. Short-term benefits include reduction in symptoms that accompany menopause, including hot flashes, vaginal dryness, and sleep disturbances. Long-term benefits include maintenance of bone mineral density and prevention of osteoporotic fractures. The major controversies surrounding HRT are the potential increased risk of cancer versus the potential benefits associated with prevention of ischemic heart disease and dementia. Recent results from the Women's Health Initiative and the Heart and Estrogen/Progestin Replacement Study have provided new information about the risks and benefits of HRT:[ 31] • Cancer. Unopposed estrogen therapy greatly increases the risk of endometrial hyperplasia and cancer; therefore, most postmenopausal women now use estrogen in combination with a progestin. This combination drastically reduces or eliminates the risk of endometrial cancer. The risk of colon cancer was reduced in women who used HRT in some studies, but not in the Heart and Estrogen/Progestin Replacement Study. Recent results from the Women's Health Initiative indicate an increased risk of breast cancer in women who used HRT combined therapy for 5 years. • Venous thrombosis and pulmonary embolism. The risk of thromboembolic events, including deep vein thrombosis, 428 pulmonary embolism, stroke, and retinal thrombosis, is elevated approximated twofold in HRT users, especially within the first 2 years. • Cardiovascular disease. The recent Women's Health Initiative reported an approximate 29% increased risk of myocardial infarction, especially during the first year of combined HRT use. This is in contrast to earlier studies in which either no effect or slight protection against cardiovascular diseases was reported. Methodologic differences probably 32 underlie these divergent results. [ ] • Cholecystitis. There is an increased risk of gallbladder disease in HRT users that increases with time. • Dementia. The current studies are not adequate to evaluate whether HRT use prevents dementia. Overall, the risks and benefits associated with the use of oral contraceptives and HRT must be evaluated for each individual patient in the context of her overall health, individual risk factors, and family history. Acetaminophen When taken in large doses, this widely used nonprescription analgesic and antipyretic causes hepatic necrosis. The window between the usual therapeutic dose (0.5 gm) and the toxic dose (15 to 25 gm) is large, however, and the drug is ordinarily safe in adults. Doses should be reduced for infants and children, especially in the setting of fever, reduced food intake, or 33 dehydration, since these conditions may predispose to liver injury.[ ] Toxicity begins with nausea, vomiting, diarrhea, and sometimes shock, followed in a few days by evidence of jaundice; with serious overdosage, liver failure ensues, with centrilobular necrosis that may extend to the entire lobule. Some patients show evidence of concurrent renal and myocardial damage. Aspirin (Acetylsalicylic Acid) Overdose may result from accidental ingestion by young children; in adults, overdose is frequently suicidal. The major untoward consequences are metabolic with few morphologic changes. At first respiratory alkalosis develops, followed by metabolic acidosis that often proves fatal before anatomic changes can appear. Ingestion of as little as 2 to 4 gm by children or 10 to 30 gm by adults may be fatal, but survival has been reported after doses five times larger. TABLE 9-9 -- National Ambient Air Quality Standards: Sources and Number of People at Risk Pollutant Primary Standard Tons Emitted (Millions) People at Risk (Millions) Not applicable 143•• Ozone 0.08 ppm 8 hr average Nitrogen oxides 0.053 ppm annual arithmetic mean 25 Not available Sulfur dioxide 0.03 ppm annual arithmetic mean 19 0.3 Particulates (PM10 ) 50 µg/µL annual arithmetic mean 24 8.7 Carbon monoxide 9 ppm 8 hr average 97 31•• Lead 1.5 µg/µL quarterly average 30 2.5 Data from U.S. Environmental Protection Agency: epa.gov/oar/oaqps, www.scorecard.org/env-releases, the American Lung Association: www.lungusa.org/air, and Goldman LR: Environmental health and its relationship to occupational health. In Levy BS, et al. (eds): Occupational Health. Recognizing and Preventing Work-Related Disease and Injury, fourth ed. Philadelphia, Lippincott Williams & Wilkins, 2000, p. 51. Chronic aspirin toxicity (salicylism) may develop in persons who take 3 gm or more daily, the dose required to treat chronic inflammatory conditions. Chronic salicylism is manifested by headache, dizziness, ringing in the ears (tinnitus), difficulty in hearing, mental confusion, drowsiness, nausea, vomiting, and diarrhea. The central nervous system changes may progress to convulsions and coma. The morphologic consequences of chronic salicylism are varied. Most often there is an acute erosive gastritis ( Chapter 17 ), which may produce overt or covert gastrointestinal bleeding and lead to gastric ulceration. A bleeding tendency may appear concurrently with chronic toxicity, because aspirin acetylates platelet cyclooxygenase and blocks the ability to make thromboxane A2 , an activator of platelet aggregation. Petechial hemorrhages may appear in the skin and internal viscera, and bleeding from gastric ulcerations may be exaggerated. Proprietary analgesic mixtures of aspirin and phenacetin or its active metabolite, acetaminophen, when taken for a span of years, have caused renal papillary necrosis, referred to as analgesic nephropathy ( Chapter 20 ). OUTDOOR AIR POLLUTION Air pollution is a serious problem in the United States and many other industrialized countries. In the United States, the Environmental Protection Agency is charged with identification and regulation of pollutants in the ambient air that may cause adverse health effects. The current National Ambient Air Quality Standards for the six major pollutants are listed in Table 9-9 . Despite federal and state regulations, many cities and regions in the United States currently do not meet these primary standards. Epidemiologic research, human clinical studies, and animal toxicologic studies continue to provide evidence for adverse health effects of ambient air pollutants, even at exposure levels below the current standards. The major sources of ambient air pollutants are: • Combustion of fossil fuels. These are divided into mobile sources such as motor vehicles, stationary sources such as power plants and factories, and other sources such as barbecues and fireplaces. Tailpipe emissions from motor vehicles are a complex mixture of carbon monoxide, oxides of nitrogen, hydrocarbons, diesel exhaust particles, and other particulates including lead oxide from tetraethyl lead contained in leaded gasoline. 429 • Photochemical reactions. Oxides of nitrogen and volatile hydrocarbons interact in the atmosphere to produce ozone (O3 ) as a secondary pollutant. • Power plants. These release sulfur dioxide (SO2 ) and particulates into the atmosphere. Coal and oil contain sulfur, leading to atmospheric formation of sulfates. Automobiles release oxides of nitrogen, leading to atmospheric formation of nitrates. Aerosolized acid sulfates contribute to acid rain. • Waste incinerators, industry, smelters. These point sources release acid aerosols, metals, mercury vapor, and organic compounds that may be hazardous for human health. One example of the numerous hazardous chemicals emitted by these sources is methyl isocyanate that was accidentally released at Bhopal in India in 1984, resulting in 3000 deaths due 34] to pulmonary edema. Some of the air toxins, such as polycyclic aromatic hydrocarbons, are known carcinogens.[ Lungs are the major target of common outdoor air pollutants; especially vulnerable are children, asthmatics, and people with chronic lung or heart disease, as summarized in Table 9-10 . The serious toxicity associated with lead exposure is discussed subsequently under Industrial Exposures. The major air pollutants and the mechanisms responsible for their adverse health effects are summarized briefly.[ 34] Ozone. Ozone is a major component of smog that accompanies summer heat waves over much of the United States. Exposure of exercising children and adults to as little as 0.08 ppm produces cough, chest discomfort, and inflammation in the lungs. Asthmatics are especially sensitive and require more frequent visits to emergency rooms and more hospitalizations during smog episodes. It is not known whether these acute changes lead to chronic, irreversible lung injury. Ozone is highly reactive and oxidizes polyunsaturated lipids to hydrogen peroxide and lipid aldehydes. These products act as irritants and induce release of inflammatory mediators, TABLE 9-10 -- Health Effects of Outdoor Air Pollutants Pollutant Ozone Populations at Risk Healthy adults and children Effects Decreased lung function Increased airway reactivity Lung inflammation Nitrogen dioxide Sulfur dioxide Athletes, outdoor workers Decreased exercise capacity Asthmatics Increased hospitalizations Healthy adults Increased airway reactivity Asthmatics Decreased lung function Children Increased respiratory infections Healthy adults Increased respiratory symptoms Patients with chronic lung disease Increased mortality Increased hospitalization Acid aerosols Asthmatics Decreased lung function Healthy adults Altered mucociliary clearance Children Increased respiratory infections Asthmatics Decreased lung function Increased hospitalizations Particulates Children Increased respiratory infections Decreased lung function Patients with chronic lung or heart disease Excess mortality Asthmatics Increased attacks Data from Bascom R, et al: Health effects of outdoor air pollution, Am J Respir Crit Care Med 153:3, 477, 1996. cause increased epithelial permeability and reactivity of the airways, and decrease ciliary clearance. The highest inhaled dose is delivered at the bronchoalveolar junction; however, ozone also causes inflammation of the upper respiratory tract. Nitrogen Dioxide. Oxides of nitrogen include NO and NO2 . These have lower reactivity than ozone. Nitrogen dioxide dissolves in water in the airways to form nitric and nitrous acids, which damage the airway epithelial lining. Children and patients with asthma have increased susceptibility to nitrogen dioxide; there is a wide variation in individual responses to this pollutant. Sulfur Dioxide. This pollutant is highly soluble in water; it is absorbed in the upper and lower airways, where it releases H+ , HSO3 - (bisulfite), and SO3 - (sulfite), which cause local irritation. Acid Aerosols. Primary combustion products of fossil fuels are emitted by tall smoke stacks at high altitudes and are transported by air. In the atmosphere, sulfur and nitrogen dioxide are oxidized to sulfuric acid and nitric acid, respectively, which are dissolved in water droplets or adsorbed to particulates. These acid aerosols are irritants to the airway epithelium and alter mucociliary clearance. Asthmatics have decreased lung function and increased hospitalizations when exposed to acid aerosols, although there is a wide variation in airway responses. Particulates. As discussed in Chapter 15 , the deposition and clearance of particulates inhaled into the lungs depend on their size. Ambient particulates are highly heterogeneous in size and in chemical composition. It is uncertain which characteristics of ambient particulates contribute to their adverse health effects. Recent epidemiologic and toxicologic studies suggest that ultrafine particles (less than 0.1 µm in aerodynamic diameter) are more hazardous. They contribute to increased morbidity and mortality, especially among infants, the elderly, and people with chronic cardiopulmonary disease. The mechanisms responsible for these adverse health effects are suspected to involve: (1) systemic cytokine release 430 associated with pulmonary inflammation; (2) increased blood viscosity; and (3) autonomic changes associated with variable heart rates and arrhythmias.[ 35] INDOOR AIR POLLUTION Rising energy costs during the past 30 years have led to increased insulation and decreased ventilation of homes, which elevates the level of indoor air pollutants. The health hazards of environmental tobacco smoke have already been discussed. Other sources of indoor air pollutants are gas cooking stoves and furnaces, wood stoves, construction materials, furniture, radon, allergens associated with pets, dust mites, and fungal spores and bacteria. The major categories of indoor air pollutants and their health effects are summarized in Table 9-11 and discussed briefly next.[ 36] Carbon Monoxide. This odorless, colorless gas is a byproduct of combustion produced from burning gasoline, oil, coal, wood, and natural gas. It is also a major pollutant in tobacco smoke, and its untoward effects were discussed earlier along with cigarette smoking. Here we should note that carbon monoxide levels in ambient air should not exceed 9 ppm; however, indoor levels of 2 to 4 ppm have been measured in homes during the winter. Such carbon monoxide pollution of indoor air can reduce exercise capacity and aggravate myocardial ischemia. Higher levels can cause poisoning manifested as headaches, dizziness, loss of motor control, and coma. Approximately 900 accidental deaths due to asphyxia are caused by indoor carbon monoxide pollution each year in the United States. Nitrogen Dioxide. Gas stoves and kerosene space heaters can raise indoor levels of nitrogen dioxide to 20 to 40 ppm in homes; this is several orders of magnitude higher than outdoor air levels. Children are more susceptible to the untoward TABLE 9-11 -- Health Effects of Indoor Air Pollutants Pollutant Populations at Risk Effects Carbon monoxide Adults and children Acute poisoning Nitrogen dioxide Children Increased respiratory infections Wood smoke Children Increased respiratory infections Formaldehyde Adults and children Eye and nose irritation, asthma Radon Adults and children Lung cancer Asbestos fibers Maintenance and abatement workers Lung cancer, mesothelioma Manufactured mineral fibers Maintenance and construction workers Skin and airway irritation Bioaerosols Adults and children Allergic rhinitis, asthma Data from Lambert WE, Samet JM: Indoor air pollution. In Harber P, et al (eds): Occupational and Environmental Respiratory Disease. St. Louis, Mosby-Year Book, 1996, p. 784; and Menzies D, Bourbeau J: Building-related illnesses. N Engl J Med 337:1524, 1997. effects of nitrogen dioxide. It impairs lung defenses and is hence associated with increased respiratory infections. Wood Smoke. This is a complex mixture of nitrogen oxides, particulates, and polycyclic aromatic hydrocarbons. High concentrations of wood smoke in poorly ventilated homes can increase the incidence of respiratory infections in children. Formaldehyde. This highly soluble, volatile chemical has been used in the manufacture of many consumer products, including textiles, pressed wood, furniture, and urea formaldehyde foam insulation. Although indoor levels are usually less than 1 ppm, it can cause acute irritation of the eyes and upper respiratory tract and exacerbation of asthma. Formaldehyde is frequently emitted with acrolein and acetaldehyde, which may have additive or synergistic irritant effects. Additional volatile organic compounds that may be present at low levels in indoor air include benzene, tetrachloroethylene, polycyclic aromatic hydrocarbons, and chloroform. The potential for toxicity or carcinogenicity at these exposure levels is low, although occupational exposure to these volatile compounds can be hazardous. Formaldehyde at high doses (6 to 14 ppm) has produced nasal tumors in rats.[ Radon. 37] Radon, a radioactive gas, is a decay product of uranium widely distributed in the soil. Radon gas emanating from the earth is prevalent in homes. Indoor levels of radon average around 1.5 pCi/L; approximately 4% of homes have an annual average level greater than 4 pCi/L. Radon gas is inhaled into the lungs; its decay products emit alpha radiation, which has been associated with lung cancer in miners. According to some estimates, the low levels found in indoor air account for 10,000 lung cancers per year in the United States.[ 38] Asbestos Fibers. Homes and public buildings built before the 1970s in the United States contain asbestos insulation, pipe covers, ceiling tiles, and flooring. If these materials are non-friable and undisturbed, low levels of fibers can be measured in indoor air. Maintenance and abatement workers who repair or remove asbestos-containing materials are at risk for lung cancer and mesothelioma if they do not use respirators. [ 39] [40] Manufactured Mineral Fibers. Fiberglass has been widely used as an asbestos substitute for home insulation. Low levels of these fibers can be measured in indoor air. Maintenance and construction workers can develop skin and lung irritation when using these materials. [ 41] Bioaerosols. Aerosolization of bacteria responsible for Legionella pneumonia has been associated with contaminated heating and cooling systems in public buildings ( Chapter 8 ). More common hazards in indoor air are allergens associated with pets, dust mites, cockroaches, fungi, and molds. These allergens cause allergic rhinitis and exacerbate asthma. [ 41] The etiology of the so-called sick building syndrome, or multiple chemical sensitivity syndrome, is less clear. In some cases, high levels of one or more of these indoor air pollutants may be responsible. In most cases, poor ventilation is at fault.[ 41] INDUSTRIAL EXPOSURES For centuries, physicians have recognized that occupational exposures contribute to human disease. The spectrum of human diseases associated with occupational exposures is summarized in Table 9-12 . Almost all organ systems can be affected, resulting in acute toxicity or irritation, hypersensitivity 431 reactions, chronic toxicity, fibrosis, and cancer. The chronic effects of occupational exposures are complex; they include degenerative changes in the nervous system, reproductive dysfunction, lung fibrosis, and cancer. The mechanisms responsible for these effects are not well understood. Some examples of acute and chronic diseases resulting from occupational exposures and potential hazards of environmental exposures are discussed in the following sections. Volatile Organic Compounds Large volumes of organic solvents and vapors are used in industry and in homes. These chemicals are known as volatile organic compounds (VOCs). They are used in manufacturing, degreasing, and dry cleaning and as components of paint removers and aerosol sprays. VOCs and petroleum products such as kerosene, mineral oil, and turpentine are stored in underground tanks. Surface spills and leakage from storage tanks can cause contamination of underground water supplies. In general, high levels of exposure encountered in industry cause headache, dizziness, and liver or kidney toxicity. At lower levels of exposure, there is concern about potential carcinogenicity and adverse reproductive effects. Some VOCs and their adverse effects are described next. Aliphatic Hydrocarbons. These compounds are the most widely used industrial solvents and dry-cleaning agents. All of these chemicals are readily absorbed through the lungs, skin, and gastrointestinal tract. In addition to acute central nervous system depression, they can cause liver and kidney toxicity. Common examples of these chemicals are chloroform and carbon tetrachloride; both are carcinogenic in rodents. Methylene chloride, another such chemical, is used in paint TABLE 9-12 -- Human Diseases Associated with Occupational Exposures Organ Effect Toxicant Cardiovascular system Heart disease Carbon monoxide, lead, solvents, cobalt, cadmium Respiratory system Nasal cancer Isopropyl alcohol, wood dust Lung cancer Radon, asbestos, silica, bis(chloromethyl)ether, nickel, arsenic, chromium, mustard gas Chronic obstructive lung disease Grain dust, coal dust, cadmium Hypersensitivity Beryllium, isocyanates Irritation Ammonia, sulfur oxides, formaldehyde Fibrosis Silica, asbestos, cobalt Peripheral neuropathies Solvents, acrylamide, methyl chloride, mercury, lead, arsenic, DDT Ataxic gait Chlordane, toluene, acrylamide, mercury Central nervous system depression Alcohols, ketones, aldehydes, solvents Cataracts Ultraviolet radiation Toxicity Mercury, lead, glycol ethers, solvents Bladder cancer Naphthylamines, 4-aminobiphenyl, benzidine, rubber products Male infertility Lead, phthalate plasticizers Female infertility Cadmium, lead Teratogenesis Mercury, polychlorinated biphenyls Hematopoietic system Leukemia Benzene, radon, uranium Skin Folliculitis and acneiform dermatosis Polychlorinated biphenyls, dioxins, herbicides Cancer Ultraviolet radiation Liver angiosarcoma Vinyl chloride Nervous system Urinary system Reproductive system Gastrointestinal tract Data from Leigh JP, et al: Occupational injury and illness in the United States. Estimates of costs, morbidity, and mortality. Arch Intern Med 157:1557, 1997; Mitchell FL: Hazardous waste. In Rom WN (ed): Environmental and Occupational Medicine, 2nd ed. Boston, Little, Brown, 1992, p. 1275; and Levi PE: Classes of toxic chemicals. In Hodgson E, Levi PE (eds): A Textbook of Modern Toxicology, Stamford, CT, Appleton & Lange, 1997, p. 229. removers and aerosols. In enclosed areas, high concentrations of methylene chloride can be reached because it is highly volatile. Methylene chloride is metabolized by cytochrome P-450 to carbon dioxide and carbon monoxide. Carbon monoxide can form carboxyhemoglobin, causing respiratory depression and death. Perchloroethylene and related compounds are widely used in the dry-cleaning industry. Acute exposure causes central nervous system depression, confusion, dizziness, impaired gait, and nausea. Repeated exposures may cause dermatitis. Perchloroethylene is a potential human carcinogen. Petroleum Products. Gasoline, kerosene, mineral oil, and turpentine are highly volatile and are a common cause of poisoning in children. Inhalation of these vapors causes dizziness, incoordination, and central nervous system depression. Aromatic Hydrocarbons. Benzene, toluene, and xylene are widely used solvents in the rubber and shoe industries and in printing and paper-coating. Although toluene and xylene are not carcinogenic, inhalation of benzene is hazardous because it can cause bone marrow toxicity, aplastic anemia, and acute leukemia. Benzene is metabolized by the cytochrome P-450 system in liver, producing benzoquinone and muconaldehyde. These metabolic products are believed to cause bone marrow toxicity. Polycyclic Aromatic Hydrocarbons Polycyclic aromatic hydrocarbons are among the most potent chemical carcinogens ( Chapter 7 ). The carcinogenicity of these compounds was recognized in 1775, with the description of scrotal cancer in English chimney sweeps exposed to soot. A variety of polycyclic aromatic hydrocarbons characterized 432 by three or more fused benzene rings are produced by combustion of fossil fuels; high-temperature processing of coke, coal, and crude oil; and iron and steel foundries. Benzo[a]pyrene is the prototype of polycyclic aromatic hydrocarbons. As described earlier (see Fig. 9-4A ), it is metabolized by cytochrome P-450, prostaglandin H synthetase, and epoxide hydrolase, an inducible microsomal enzyme in the liver. Activated epoxide intermediates bind to DNA; these adducts have been used as markers of polycyclic aromatic hydrocarbon exposure. 42 Occupational exposure to polycyclic aromatic hydrocarbons is associated with an increased risk of lung and bladder cancers.[ ] Cigarette smoking is another important source of benzo[a] pyrene. Mutations in the p53 tumor-suppressor gene found in lung cancers associated with cigarette smoking are most commonly G:C→T:A transversions. This mutational spectrum is consistent with metabolism of benzo[a]pyrene to reactive intermediates that attack deoxyguanines on the nontranscribed DNA strand.[ 7] Plastics, Rubber, and Polymers Millions of tons of synthetic plastics, rubber, and polymers are produced throughout the world. These products are then fabricated into latex fabrics, pipe, cables, flooring, home and recreational products, medical products, and containers. In 1974, occupational exposure to vinyl chloride monomers used to produce polyvinyl chloride resins was found to be associated with angiosarcoma of the liver. Vinyl chloride is a colorless gas that is flammable and explosive. Before the polymerization step in the manufacturing of polyvinyl chloride, it can be absorbed through the skin or lungs. Vinyl chloride is metabolized by the cytochrome P-450 system in the liver to chloroacetaldehyde. This metabolite covalently binds to DNA and is mutagenic. Exposure of rubber workers to 1,3-butadiene TABLE 9-13 -- Toxic and Carcinogenic Metals Metal Lead Disease Renal toxicity Occupation Battery and ammunition workers, foundry workers, spray painting, radiator repair Anemia, colic Peripheral neuropathy Insomnia, fatigue Cognitive deficits Mercury Renal toxicity Chlorine-alkali industry Muscle tremors, dementia Cerebral palsy Mental retardation Arsenic Cancer of skin, lung, liver Miners, smelters, oil refinery workers, farm workers Beryllium Acute lung irritant Beryllium refining, aerospace manufacturing, ceramics Chronic lung hypersensitivity ? Lung cancer Cobalt and tungsten carbide Lung fibrosis Toolmakers, grinders, diamond polishers Asthma Cadmium Renal toxicity Battery workers, smelters, welders, soldering ? Prostate cancer Chromium Cancer of lung and nasal cavity Pigment workers, smelters, steel workers Nickel Cancer of lung and nasal sinuses Smelters, steel workers, electroplating Data from Levi PE: Classes of toxic chemicals. In Hodgson E, Levi PE (eds): A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 229; and Sprince NL: Hard metal disease. In Rom WN (eds): Environmental and Occupational Medicine, 2nd ed. Boston, Little, Brown, 1992, p. 791. has been shown to be associated with an increased risk of leukemia. Plastics are widely used in consumer products, including food and beverage containers. Public exposure to plasticizers, such as phthalate esters, and to additives such as bisphenol-A raises concern about potential adverse reproductive effects of these synthetic chemicals. Phthalate esters have been shown to induce testicular injury in rats, and bisphenol-A mimics the proliferative effects of estrogen. Metals Occupational exposure to metals in mining and manufacturing is associated with acute and chronic toxicity, as well as carcinogenicity, as summarized in Table 9-13 .[ 43] Occupational as well as environmental exposure to lead continues to be a serious public health problem. Agricultural exposure to arsenic-containing pesticides is discussed subsequently. The pulmonary effects of beryllium are described in Chapter 15 . The health effects of inorganic and organic mercury were discussed earlier in this chapter under "Mechanisms of Toxicity." The untoward effects of some of the remaining metals listed in Table 9-13 are described here. Lead. More than 4 million tons of lead are produced each year for use in batteries, alloys, exterior red lead paint, and ammunition. Workers employed in these industries as well as in mining, smelting, spray painting, recycling, and radiator repair are exposed to lead. In some countries, tetraethyl lead is still used as a gasoline additive, thus polluting the air. Inhalation is the most important route of occupational exposure. Environmental sources of lead are urban air due to use of leaded gasoline, soil contaminated with exterior lead paint, the water supply due to lead plumbing, and house dust in homes with interior lead paint. Consumers may be exposed to lead-glazed ceramics, lead solder in food and soft drink cans, and illegally 433 produced alcoholic beverages (moonshine). Lead ingested in this manner is absorbed through the gastrointestinal tract. Intestinal absorption of lead is enhanced by calcium, iron, or zinc deficiency; compared with adults, the absorption is greater in children and infants and hence they are particularly vulnerable to lead toxicity. Absorbed lead is mainly (80% to 85%) taken up by bone and developing teeth in children; the blood accumulates 5% to 10%, and the remainder is distributed throughout the soft tissues. Lead clears rapidly from blood, but that deposited in bones has a half-life of 30 years. Thus, the presence of lead in blood indicates recent exposure, and it does not allow the determination of total body burden. The toxicity of lead is related to its multiple biochemical effects: • High affinity for sulfhydryl groups. The most important enzymes inhibited by lead due to this mechanism are involved in heme biosynthesis: δ-aminolevulinic acid dehydratase and ferroketolase. These enzymes catalyze the incorporation of iron into the heme molecule, and hence patients develop hypochromic anemia. • Competition with calcium ions. As a divalent cation, lead competes with calcium and is stored in bone. It also interferes with nerve transmission and brain development. • Inhibition of membrane-associated enzymes. Lead inhibits 5'-nucleotidase activity and sodium-potassium ion pumps, leading to decreased survival of red blood cells (hemolysis), renal damage, and hypertension. • Impaired production of 1,25-dihydroxyvitamin D, the active metabolite of vitamin D. Lead contributes to multiple chronic health effects, illustrated in Figure 9-9 . Injury to the central and peripheral nervous systems causes headache, dizziness, memory deficits, and decreased nerve conduction velocity. Blood changes occur early and are characteristic. Because lead interferes with heme biosynthesis, it causes a microcytic hypochromic anemia; punctate basophilic stippling of erythrocytes is characteristic. There is also an element of hemolysis because lead inhibits membrane-associated red cell enzymes. Because lead inhibits incorporation of iron into heme, the iron is displaced, and zinc protoporphyrin is formed. Thus, an elevated blood level of zinc protoporphyrin or its product, free erythrocyte protoporphyrin, is an important indicator of lead poisoning. Gastrointestinal symptoms include colic and anorexia. The kidneys are a major route of excretion of lead. Acutely, there is damage to the proximal tubules, with intranuclear lead inclusions and clinical evidence of renal tubule dysfunction. Chronically, lead can cause diffuse interstitial fibrosis, gout, and renal failure. Even in the absence of overt clinical symptoms of kidney damage, lead causes hypertension. Lead can cause infertility in men due to testicular injury; failure of implantation of the 44] fertilized ovum can occur in women.[ Infants and children are especially vulnerable to lead toxicity. It is estimated by the CDC that in the year 2000 approximately 454,000 children in the United States had blood lead levels greater than 10 µg/dL. A recent study indicates that even below this level there is an inverse correlation between blood lead concentration and IQ scores. Very slightly elevated blood levels (∼3 µg/dL) in young females have also been reported to delay puberty.[ pregnancy and it readily crosses the placental barrier. Hence 45] Thus, lead toxicity continues to be a matter of concern. Lead may be mobilized from the maternal skeleton during Figure 9-9 Consequences of lead exposure. TABLE 9-14 -- Health Effects of Agricultural Pesticides Category Insecticides Example Organochlorines Effects and Disease Associations Neurotoxicity; hepatotoxicity ••DDT ••Chlordane ••Lindane ••Methoxychlor Organophosphates Neurotoxicity; delayed neuropathy ••Parathion ••Diazinon ••Malathion Carbamates Neurotoxicity (reversible) ••Aldicarb ••Carbaryl Botanical agents Paresthesia; lung irritant; allergic dermatitis ••Nicotine ••Pyrethrins ••Rotenone Herbicides Arsenic compounds Hyperpigmentation; gangrene; anemia; sensory neuropathy; cancer Dinitrophenols Hyperthermia; sweating Chlorophenoxy herbicides Fungicides ••2,4-D and 2,4,5-T ? Lymphoma; sarcoma ••TCDD Fetotoxicity; immunotoxicity; cancer Paraquat Acute lung injury Atrazine ? Cancer Alachlor ? Cancer Captan ? Reproductive toxicity Maneb Benomyl Rodenticides Fumigants Fluoroacetate Cardiac and respiratory failure Warfarin Hemorrhage Strychnine Respiratory failure Carbon disulfide Cardiac toxicity Ethylene dibromide Neurotoxicity Phosphine Lung edema; brain damage Chloropicrin Eye irritation; lung edema; arrhythmias Data from Hodgson E: Introduction to toxicology. In Hodgson E, Levi PE (eds): A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 1; and Levi PE: Classes of toxic chemicals. In Hodgson E, Levi PE (eds): A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 229. soil and water supplies. Environmental contamination is a threat to wildlife; some pesticides undergo bioaccumulation and persist in wildlife and humans for decades. Bioaccumulation and biopersistence are characteristic of organochlorines, such as DDT (dichlorodiphenyltrichloroethane), and dioxins, such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). There is considerable controversy about the adverse health effects of these persistent pesticides and their metabolites, especially concerning their relationship to breast cancer,[ 49 abnormalities,[ ] and cognitive 48] reproductive 50 deficits.[ ] Agricultural pesticides are divided into five categories, depending on the target pest: insecticides, herbicides, fungicides, rodenticides, and fumigants ( Table 9-14 ). All pesticides are toxic to some plant or rodent species; at higher doses, they can also be toxic to farm animals, pets, and humans. In general, herbicides used to control weeds have low acute toxicity for mammals; fungicides are characterized as moderately toxic. Acute toxicity of insecticides for mammals ranges from low to high. For example, DDT was widely used as an insecticide in the 1940s and 1950s because it has low acute toxicity for humans. However, DDT persists in the environment and accumulates in the food chain. Birds that ingested DDT-contaminated insects and fish suffered reproductive defects. DDT and its major metabolite, DDE 435 (1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene), accumulate in fat tissue and have been detected in human milk. Organochlorines, as well as industrial chemicals such as polychlorinated 48 biphenyls (PCBs), are weakly estrogenic. Some of these chemicals are carcinogenic in rodents and cause reproductive dysfunction in amphibians, birds, and fish.[ ] Although several epidemiologic studies have not found increased levels of DDE or PCBs in women with breast cancer compared with matched control subjects, there is still concern that these persistent organochlorines, other potentially estrogenic pesticides, and natural phytoestrogens in plants such as soybeans may have adverse reproductive effects in humans. The mechanisms of action 48] [49] of these xenoestrogens, alone or in combination, in the development of cancer and in reproductive dysfunction are unknown.[ The major health effects of the most common agricultural pesticides are summarized in Table 9-14 . Selected examples are discussed here. • Organochlorines, such as DDT, have low acute toxicity for humans; however, they bioaccumulate and persist in the environment and in fat tissue. These chemicals are absorbed through the skin, gastrointestinal tract, and lungs. As alluded to earlier, the role of DDT and its metabolites as an endocrine-disrupting agent is controversial. Chlordane is representative of cyclodienes that are used to control termites and other soil insects. Acute toxicity causes hypothermia, tremor, and convulsions. Chlordane also causes immune dysfunction and may act as a nongenotoxic carcinogen. These effects may contribute to the increased incidence of lymphoma observed in some farm workers. Lindane is an isomer of benzene hexachloride that is used to control lice and scabies, as a wood preservative, and as a household fumigant. It has been reported to cause immune dysfunction and reproductive problems in women. • Organophosphates are irreversible inhibitors of cholinesterases resulting in abnormal transmission at peripheral and central nerve endings. These chemicals are absorbed through the skin, gastrointestinal tract, and lungs. Up to 40% of farm workers in the United States show measurable inhibition of red blood cell or plasma cholinesterase activity; fatalities have been reported from organophosphate exposure. Carbamates are reversible inhibitors of cholinesterase that produce acute neurotoxic effects similar to those of organophosphate insecticides. Carbaryl (Sevin) is potentially mutagenic and teratogenic because it poisons the mitotic spindle. • Herbicides like the dioxin TCDD has received much attention. During the Vietnam War, the defoliant Agent Orange was contaminated with TCDD. A chemical factory explosion in Seveso, Italy, in 1976 caused local environmental contamination and human exposure to TCDD, resulting in chloracne and an increased incidence of leukemia, lymphoma, and sarcomas. TCDD and structurally similar dioxins are also produced in the paper pulp industry using chlorine bleach and by waste incinerators. Low doses of dioxin are present in our food, soil, and water. In some laboratory animals, TCDD is highly toxic, immunosuppressive, teratogenic, and carcinogenic. The sensitivity of some strains of laboratory mice to dioxin is linked to the aryl hydrocarbon hydroxylase receptor. TCDD can induce liver cytochrome P-450 enzyme activity, increase estrogen metabolism, and interfere with development of the male reproductive tract. TCDD also decreases thyroxine levels in adult rats. Extrapolation of these multiple adverse effects observed in laboratory animals to 51 low-dose exposure of humans is difficult.[ ] • Rodenticides are highly toxic chemicals with restricted use. The major health threat is death from suicidal or accidental ingestion. TABLE 9-15 -- Natural Toxins Category Mycotoxins Phytotoxins Animal toxins Example Source Effects and Associated Diseases Ergot alkaloids Claviceps fungi Gangrene, convulsions, abortion Aflatoxins Aspergillus flavus Liver cancer Tricothecenes Fusarium, Trichoderma Diarrhea, ataxia Cycasin Cycad flour Amyotrophic lateral sclerosis Monocrotaline Senecio plants Hepatitis Safrole Black pepper; oil of Sassafras Cancer Solanine Solanaceae plants (potato) Neurotoxin Venoms Snakes Cardiotoxin, neurotoxin Bees Direct toxicity, cardiotoxin Saxitoxin Dinoflagellates Neurotoxin, paralysis Ciguatoxin Dinoflagellates Paresthesia, paresis, vomiting, diarrhea Tetrodotoxin Puffer fish Neurotoxin, shock Data from Hodgson E: Introduction to toxicology. In Hodgson E, Levi PE (eds): A Textbook of Modern Toxicology. Stamford, CT, Appleton & Lange, 1997, p. 1. NATURAL TOXINS In addition to manufactured pesticides, potent toxins and carcinogens are present in the natural environment, as summarized in Table 9-15 . These mycotoxins and phytotoxins may contaminate foods. For example, cycad flour is used in arid climates. This plant contains the toxin cycasin (methylazoxymethanol β-glucoside). If the plant and seeds are cut into small pieces, soaked in water, and dried, the toxin is leached. However, if these precautions are not followed, a degenerative neurologic disorder (amyotrophic lateral sclerosis) is produced by ingestion of cycasin. Animal toxins can be ingested by eating fish, snails, or mollusks. The most common poisoning results from eating tropical fish and snails that have ingested dinoflagellates containing ciguatoxin. Ciguatera 436 poisoning can be severe and occurs in the South Pacific and the Caribbean. Paralytic shellfish poisoning occurs in North America after eating mollusks that have ingested dinoflagellates that contain saxitoxin. Aflatoxin B1 is produced by fungi that contaminate peanuts, corn, and cottonseed. It is a potent carcinogen that contributes to the high incidence of liver cancer in some regions of Africa and the Far East ( Chapter 7 and Chapter 18 ). RADIATION INJURY Radiation is energy distributed across the electromagnetic spectrum as waves (long wavelengths, low frequency) or particles (short wavelengths, high frequency). The types, frequencies, and biologic effects of electromagnetic radiation are summarized in Table 9-16 . Approximately 80% of radiation is derived from natural sources, including cosmic radiation, ultraviolet light, and natural radioisotopes, especially radon gas. The remaining 20% is derived from manufactured sources that include instruments used in medicine and dentistry, consumer products that emit radio waves or microwaves, and nuclear power plants. The potentially catastrophic effects of radiation are most vividly illustrated by the effects of nuclear explosions. The atomic bombs dropped on Hiroshima and Nagasaki in 1945 not only caused acute injury and death but also increased incidence of various cancers among the survivors. Numerous historical incidents document the deleterious effects of therapeutic radiation. For example, early in the 20th century, American radiologists experienced an increased incidence of aplastic anemia and neoplasms of the skin, brain, and hematopoietic system. Children who were treated with radiation for an enlarged thymus or benign skin lesions between 1910 and 1959 suffered from an increased incidence of thyroid abnormalities, thyroid tumors, and leukemias and lymphomas. Exposure of the fetus to radiation can produce mental retardation, congenital anomalies, leukemia, and solid tumors. Investigation of these deliberate or accidental exposures to radiation led to an understanding of the relationship between the dose and timing of radiation and the acute and chronic health effects. However, in general, these historical exposures were higher than radiation currently received by the general population from natural and manufactured sources, TABLE 9-16 -- Ionizing and Nonionizing Electromagnetic Radiation Frequency (Hz) 1–50 Radiation Biologic Effects Electric power ? 106 –1011 Radio waves and radar Thermal effects, cataracts 109 –1010 Microwaves Lens opacities 1011 –1014 Infrared Cataracts Visible light Retinal burns (lasers) 1015 –1018 Ultraviolet light Skin burns, cancer 1018 –1020 X-rays and gamma rays Acute and delayed injury; cancer Cosmic radiation ? 1015 1027 by patients undergoing diagnostic procedures such as mammography or chest radiography, and by nuclear power plant workers. Unfortunately, fear of widespread radiation exposure following a terrorist attack reinforces the importance of understanding the mechanisms and clinical manifestations of radiation injury.[ 52] Despite our understanding of the health effects of high doses of radiation, the potential adverse effects of low doses are controversial. Furthermore, accidents at nuclear power plants in Windscale, England, in 1957, at Three Mile Island in Pennsylvania in 1979, and at Chernobyl in the former Soviet Union in 1986 perpetuate public anxiety about excess cancers associated with the medical, commercial, and military uses of radioactivity.[ 53] Electromagnetic radiation characterized by long wavelengths and low frequencies is described as nonionizing radiation. Electric power, radio waves and microwaves, infrared, and ultraviolet light are examples of nonionizing radiation. They produce vibration and rotation of atoms in biologic molecules. Radiation energy of short wavelengths and high frequency can ionize biologic target molecules and eject electrons. X-rays, gamma rays, and cosmic rays are forms of ionizing radiation. Ionizing radiation can be in the form of electromagnetic waves, such as x-rays produced by a roentgen tube or gamma rays emitted from natural sources, or particles that are released by natural decay of radioisotopes or by artificial acceleration of subatomic particles. Particulate radiation is classified by the type of particles emitted: alpha particles, beta particles or electrons, protons, neutrons, mesons, or deuterons. The energy of these particles is measured in million electron volts (MeV). Radioisotopes decay by emission of alpha or beta particles or by capture of electrons. In the case of radon gas, unstable daughter nuclei are produced that subsequently disintegrate, releasing alpha particles. Alpha particles consist of two neutrons and two protons; they have strong ionizing power but low penetration because of their large size. In contrast, beta particles are electrons emitted from the nucleus of an atom; these have weaker ionizing power but higher penetration than alpha particles. The decay of radioisotopes is expressed by the curie (Ci), 3.7 × 1010 disintegrations per second, or the becquerel (Bq), 1 disintegration per second. The rate of decay of radioisotopes is usually expressed as the half-life (t1/2 ) and ranges from a few seconds to centuries. Internal deposition of radioisotopes with long half-lives is especially dangerous because it results in continuous release of radioactive particles and gamma rays. For example, radium was used to paint watch dials and treat cancer in the first half of the 20th century; its long halflife of 1638 years and ability to be concentrated in the skeleton result in delayed appearance of bone tumors. Ionizing Radiation The dose of ionizing radiation is measured in several units: • roentgen: unit of charge produced by x-rays or gamma rays that ionize a specific volume of air • rad: the dose of radiation that will produce absorption of 100 ergs of energy per gram of tissue; 1 gm of tissue exposed to 1 roentgen of gamma rays is equal to 93 ergs • gray (Gy): the dose of radiation that will produce absorption of 1 joule of energy per kilogram of tissue; 1 Gy corresponds to 100 rad 437 • rem: the dose of radiation that causes a biologic effect equivalent to 1 rad of x-rays or gamma rays • sievert (Sv): the dose of radiation that causes a biologic effect equivalent to 1 Gy of x-rays or gamma rays; 1 Sv corresponds to 100 rem.[ 53] These measurements do not directly quantify energy transferred per unit of tissue and therefore do not predict the biologic effects of radiation. The following terms provide a better approximation of such information. • Linear energy transfer (LET) expresses energy loss per unit of distance traveled as electron volts per micrometer. This value depends on the type of ionizing radiation. LET is high for alpha particles, less so for beta particles, and even less for gamma rays and x-rays. Thus, alpha and beta particles penetrate short distances and interact with many molecules within that short distance. Gamma rays and x-rays penetrate deeply but interact with relatively few molecules per unit distance. It should be evident that if equivalent amounts of energy entered the body in the form of alpha and gamma radiation, the alpha particles would induce heavy damage in a restricted area, whereas gamma rays would dissipate energy over a longer course and produce considerably less damage per unit of tissue. • Relative biologic effectiveness (RBE) is simply a ratio that represents the relationship of the LETs of various forms of irradiation to cobalt gamma rays and megavolt x-rays, both of which have an RBE of unity (1). In addition to the physical properties of the radioactive material and the dose, the biologic effects of ionizing radiation depend on several factors: • Dose rate: a single dose can cause greater injury than divided or fractionated doses that allow time for cellular repair. • Since DNA is the most important subcellular target of ionizing radiation, rapidly dividing cells are more radiosensitive than are quiescent cells. Hematopoietic cells, germ cells, gastrointestinal epithelium, squamous epithelium, endothelial cells, and lymphocytes are highly susceptible to radiation injury; bone, cartilage, muscle, and peripheral nerves are more resistant. • A single dose of external radiation administered to the whole body is more lethal than regional doses with shielding. For example, the median lethal dose (LD50 ) of ionizing radiation is 2.5 to 4.0 Gy (250 to 400 rad), whereas doses of 40 to 70 Gy (4000 to 7000 rad) can be delivered in a fractionated manner during several weeks for cancer therapy. • Cells in the G2 and mitotic phases of the cell cycle are most sensitive to ionizing radiation. • Different cell types differ in the extent of their adaptive and reparative responses. • Since ionizing radiation produces oxygen-derived radicals from the radiolytic cleavage of water ( Chapter 1 ), cell injury induced by x-rays and gamma rays is enhanced by hyperbaric oxygen. Halogenated pyrimidines can also increase radiosensitivity to tumor cells. Conversely, free radical scavengers and antioxidants protect against radiation injury. Cellular Mechanisms of Radiation Injury. The acute effects of ionizing radiation range from overt necrosis at high doses (>10 Gy), killing of proliferating cells at intermediate doses (1 to 2 Gy), and no histopathologic effect at doses less than 0.5 Gy. Subcellular damage does occur at these lower doses, primarily targeting DNA; however, most cells show adaptive and reparative responses to low doses of ionizing radiation. If cells undergo extensive DNA damage or if they are unable to repair this damage, they undergo apoptosis ( Chapter 7 ). Surviving cells may show delayed effects of radiation injury: mutations, chromosome aberrations, and genetic instability. These genetically damaged cells may become malignant; tissues with rapidly proliferating cell populations are especially susceptible to the carcinogenic effects of ionizing radiation. Most cancers induced by ionizing radiation have occurred after doses greater than 0.5 Gy. Acute cell death, especially of vascular endothelial cells, can cause delayed organ dysfunction several months or years after radiation exposure. In general, this delayed injury is caused by a combination of atrophy of parenchymal cells, ischemia due to vascular damage, and fibrosis.[ described next. 53] Acute and delayed effects of ionizing radiation are listed in Table 9-17 , and their mechanisms are Acute Effects. Ionizing radiation can produce a variety of lesions in DNA, including DNA-protein cross-links, cross-linking of DNA strands, oxidation and degradation of bases, cleavage of sugarphosphate bonds, and single-stranded or double-stranded DNA breaks. This damage may be produced directly by particulate radiation, x-rays, or gamma rays or indirectly by oxygen54 derived free radicals or soluble products derived from peroxidized lipids.[ ] Even relatively low doses of ionizing radiation (less than 0.5 Gy) induce alterations in gene expression in some target cell populations. Free radicals generated directly or indirectly by exposure to ionizing radiation may produce oxidant stress that activates transcription factors (such as NF-κB) that 55 increase gene expression.[ ] DNA damage itself stimulates the expression of several genes involved in DNA repair, cell-cycle arrest, and apoptosis. As discussed in Chapter 7 , the tumorsuppressor gene p53 is activated after many different forms of DNA damage. The end-points resulting from activation of this p53-mediated DNA damage response are discussed in Chapter 7 . Briefly, activation of p53 induces cell-cycle arrest, DNA repair and, in some cases, apoptosis. Apoptosis of microvascular endothelial cells may be the primary target of acute radiation in the GI tract, resulting in secondary damage to intestinal crypt stem cells[ 56] and the GI syndrome (see Table 9-18 ). Fibrosis. An important delayed complication of ionizing radiation, usually at doses used for cancer therapy, is replacement of normal parenchymal tissue by fibrosis, resulting in scarring and loss of 57 function. These fibrotic changes may be secondary to ischemic injury caused by vascular damage, death of parenchymal cells, or deletion of stem cells.[ ] The mechanisms responsible for fibrosis have been explored in a murine model of radiation-induced pulmonary fibrosis using microarray analysis of gene expression. Up-regulation of chemokines that recruit inflammatory cells to the lungs as well as cytokines and growth factors involved in fibroblast activation and collagen deposition are central components of radiation-induced fibrosis.[ described in Chapter 3 , these chemokines, cytokines, and growth factors also play important roles in wound healing. 58] As 438 TABLE 9-17 -- Acute Injury and Delayed Complications Caused by Ionizing Radiation Organ Acute Injury Delayed Complications Bone marrow Atrophy Hypoplasia, leukemia Skin Erythema Atrophy of epidermis and fibrosis of dermis; cancer Heart — Interstitial fibrosis Lung Edema, endothelial and epithelial cell death Interstitial and intra-alveolar fibrosis; cancer Gastrointestinal tract Edema, mucosal ulcers Ulcers; fibrosis; strictures; adhesions; cancer Liver Veno-occlusive disease Cirrhosis; liver tumors Kidney Vasodilation Cortical atrophy, interstitial fibrosis Urinary bladder Mucosal erosion Submucosal fibrosis; cancer Brain Edema, necrosis Necrosis of white matter, gliosis; brain cancer Testis Necrosis Tubular atrophy Ovary Atresia of follicles Stromal fibrosis Thyroid — Hypothyroidism; cancer Breast — Fibrosis; cancer Thymus, lymph nodes Atrophy Lymphoma Carcinogenesis. Occupational or accidental exposures to ionizing radiation produce an increased incidence of various types of cancer, including skin cancers, leukemia, osteogenic sarcomas, and lung cancer. There is usually a latent period of 10 to 20 years before appearance of these cancers. In survivors of the atomic blasts at Hiroshima and Nagasaki, all types of leukemias were especially common, with the exception of chronic lymphocytic leukemia. Exposure of children to irradiation causes an increased incidence of breast and thyroid cancers as well as gastrointestinal and urinary tract tumors. The nuclear power accident at Chernobyl in 1986 caused more than 50 deaths, with estimated exposures of 50 to 300 rad. More than 20,000 people were exposed to up to 40 rem. As early as 1990, an increased incidence of thyroid cancer was seen in exposed children. Approximately 2 million people living near Three Mile Island were exposed to low doses of 100 mrem in 1979; no adverse effects have yet been reported. Workers in the nuclear energy industry and in health care and research are exposed annually to doses ranging from 1 to 9 mSv. The annual maximal permissible exposure level for TABLE 9-18 -- Clinical Features of the Acute Radiation Syndrome Category Whole-Body Dose (rem) Symptoms Prognosis Subclinical ••<200 Mild nausea and vomiting 100% survival Lymphocytes <1500/µL Hematopoietic 200–600 Intermittent nausea and vomiting Infections Petechiae, hemorrhage May require bone marrow transplant Maximum neutrophil and platelet depression in 2 wk Lymphocytes <1000/µL Gastrointestinal 600–1000 Nausea, vomiting, diarrhea Shock and death in 10–14 days even with replacement therapy Hemorrhage and infection in 1–3 wk Severe neutrophil and platelet depression Lymphocytes <500/µL Central nervous system ••>1000 Intractable nausea and vomiting Death in 14–36 hr Confusion, somnolence, convulsions Coma in 15 min–3 hr Lymphocytes absent these workers is 50 mSv or 1 rem. There is uncertainty about the potential carcinogenic risk at these low exposures because the shape of the dose-response curve is unknown. The mechanisms responsible for the delayed carcinogenic effects of ionizing radiation are not completely understood. The latent period between acute exposure to ionizing radiation and the delayed appearance of cancer may be due to a phenomenon called induced genetic instability. Quantitative analysis of mutation rates in irradiated cells in culture shows that mutations continue to be expressed in surviving cells after several generations. Accumulation of these delayed mutations may be the result of persistent DNA lesions that are not repaired or due to an epigenetic mechanism, such as altered methylation at CpG sites or shortening of telomeres. Delayed chromosome aberrations are also observed after exposure to ionizing radiation, especially in human lymphocytes.[ 59] These mechanisms may be responsible for induction of secondary cancers, especially leukemias, in cancer patients treated with radiation therapy. 439 Clinical Manifestations of Exposure to Ionizing Radiation The clinical effects of ionizing radiation depend on the dose, duration, and mode of exposure. These are described next. Acute, Whole-Body Exposure. Whole-body irradiation is potentially lethal; the clinical manifestations are dose dependent and described as the acute radiation syndrome or radiation sickness. On the basis of calculated doses delivered in nuclear reactor accidents or the atomic bombing of Japan, the LD50 at 60 days for humans exposed to a single dose of x-rays or gamma radiation is 2.5 to 4.0 Gy (250 to 400 rad). Depending on the dose, four clinical syndromes are produced: a subclinical or prodromal syndrome, hematopoietic syndrome, gastrointestinal syndrome, or central nervous system syndrome. These are summarized in Table 9-18 . The acute symptoms are manifestations of the high sensitivity of rapidly proliferating tissues, such as the lymphohematopoietic cells and gastrointestinal epithelium, to acute radiation-induced necrosis or apoptosis ( Fig. 9-10 ). If the patient survives the acute radiation syndrome, sublethally injured cells may repair the radiation damage, and the necrotic or apoptotic cells may be replaced by the progeny of more radioresistant stem cells. Effects of Radiation Therapy. External radiation is delivered to malignant neoplasms at fractionated doses up to 40 to 70 Gy (4000 to 7000 rad), with shielding of adjacent normal tissues. Radiation therapy, especially when it is delivered to the chest or abdomen, can cause acute radiation sickness and neutrophil and platelet depression. These patients may experience transient fatigue, vomiting, and anorexia that may require reduction of the dose. Acutely, radiation therapy may shrink the tumor mass and relieve pain or compression of adjacent tissues. Unfortunately, cancer patients treated with radiation therapy may develop sterility, a secondary malignant neoplasm, or delayed radiation injury (described later).[ 53] Effects on Growth and Development. The developing fetus and young children are highly sensitive to growth and developmental abnormalities induced by ionizing radiation. Four susceptible phases can be defined: Figure 9-10 Atrophy of the thymus gland after exposure to ionizing radiation. The right panel shows a normal thymus with deeply staining cortex and pale staining medulla; the left panel shows depletion of lymphocytes with preservation of (pink, concentric) Hassall corpuscles. (American Registry of Pathology © 1990.) Figure 9-11 Acute vascular injury with fibrinoid necrosis and edema after exposure to ionizing radiation. (American Registry of Pathology © 1990.) Figure 9-12 Chronic vascular injury with subintimal fibrosis occluding the lumen. (American Registry of Pathology © 1990.) Figure 9-13 Chronic radiation dermatitis with atrophy of the epidermis, dermal fibrosis, and telangiectasia of the subcutaneous blood vessels. (American Registry of Pathology © 1990.) Figure 9-14 Extensive mediastinal fibrosis after radiotherapy for carcinoma of the lung. Note the markedly thickened pericardium. (From the teaching collection of the Department of Pathology, Southwestern Medical School, Dallas, TX.) Figure 9-15 Radiation fibrosis of the breast stroma after radiotherapy for infiltrating ductal carcinoma. The nests of remaining tumor cells are pleomorphic and multinucleated. (American Registry of Pathology © 1990.) TABLE 9-19 -- Acute and Delayed Effects of Ultraviolet Radiation Radiation UVA Wavelength (nm) 320–400 Acute Effects Delayed Effects Erythema 8–48 hr Tanning Depletion of Langerhans cells ? Skin cancer Pigment darkening Dermal inflammation UVB 290–320 Erythema 3–24 hr Tanning Apoptosis of keratinocytes Solar elastosis Depletion of Langerhans cells Premature aging Actinic keratosis Skin cancer UVC 200–290 ? Skin cancer Data from Rosen CF: Ultraviolet radiation. In Craighead JE (ed): Pathology of Environmental and Occupational Disease. St. Louis, Mosby-Year Book, 1996, p. 193. Ultraviolet Radiation Solar radiation spans the spectrum of wavelengths between 200 and 4000 nm, including ultraviolet, visible, and infrared radiation. Ultraviolet radiation is divided into ultraviolet A (UVA), ultraviolet B (UVB), and ultraviolet C (UVC); 3% to 5% of the total solar radiation that penetrates the earth's surface is ultraviolet radiation. Ozone in the atmosphere is an important protective agent against ultraviolet radiation because it completely absorbs all UVC and partially absorbs UVB. Chlorofluorocarbons, used commercially as propellants, as solvents, and in refrigerators and air conditioners, interact with and deplete ozone. Such depletion is predicted to contribute to an increase in UVB and possibly UVC exposure, thus triggering a 2% to 4% increase in the incidence of skin cancers. Some protection from the effects of UV light is afforded by window glasses: they absorb UVB radiation, but they transmit UVA radiation. Sunblocks and sunscreens offer greater protection because they absorb or block UVB and UVA to variable degrees. There are two major health effects of ultraviolet radiation: premature aging of the skin and skin cancer ( Table 9-19 ). The carcinogenic effects of ultraviolet light are discussed in Chapter 7 . Here we focus on other effects of ultraviolet radiation. The acute effects of UVA and UVB are short-lived and reversible. They include erythema, pigmentation, and injury to Langerhans cells and keratinocytes in the epidermis. The kinetics and chemical mediators of these reactions differ in response to UVA and UVB. Depending on the intensity and length of exposure, erythema, edema, and acute inflammation are mediated by release of histamine from mast cells in the dermis, synthesis of arachidonic acid metabolites, and the production of pro-inflammatory cytokines like IL-1. UVA produces oxidation of melanin with transient, immediate 442 darkening, especially in individuals with darker skin. Tanning induced by UVA and UVB is due to a delayed increase in the number of melanocytes, elongation and extension of dendritic processes, and transfer of melanin to keratinocytes. Tanning induced by UVB is protective against subsequent exposures; tanning induced by UVA provides limited protection. Both UVA and UVB deplete Langerhans cells and thus reduce the processing of antigens introduced through the epidermis. UVB causes apoptosis of keratinocytes in the epidermis, resulting in dyskeratotic, sunburn cells. Repeated exposures to ultraviolet radiation give rise to changes in the skin that are characteristic of premature aging (e.g., wrinkling, solar elastosis, and irregularities in pigmentation). In contrast to ionizing radiation that increases deposition of collagen in the dermis, ultraviolet radiation causes degenerative changes in elastin and collagen, leading to wrinkling, increased laxity, and a leathery appearance. These connective tissue alterations accumulate over time and are largely irreversible. They are caused by increased expression of the elastin gene, increased expression of matrix metalloproteinases that degrade collagen, and induction of a tissue inhibitor of matrix metalloproteinase. The end result of these changes in connective tissue enzymes is degradation of type I collagen fibrils and disorganization and degeneration of the dermal connective tissue[ 61] ( Fig. 9-16 ). Skin damage induced by UVB is believed to be caused by the generation of reactive oxygen species and by damage to endogenous chromophores such as melanin. Ultraviolet radiation also damages DNA, resulting in the formation of pyrimidine dimers between adjacent pyrimidines on the same DNA strand. Other forms of DNA damage, for example, formation of pyrimidine-pyrimidone (6-4) photoproducts, single-stranded breaks, and DNA-protein cross-links, are also noted.[ and malignant skin lesions in humans, 61] A unique spectrum of mutations has been identified in premalignant Figure 9-16 Solar elastosis with basophilic degeneration of the connective tissue in the upper layer of the dermis. (American Registry of Pathology © 1990.) TABLE 9-20 -- Adult Mortality Rates in the United States, Ages 25–44, in 1998 Rate per 100,000 population Cause Hispanic Black White Unintentional injuries 33.4 40.1 31.6 Cancer 16.8 38.0 25.3 Homicide 13.1 36.2 4.7 Human immunodeficiency virus 12.1 43.3 4.8 Heart disease 10.3 43.5 18.3 Suicide 7.8 Total 130.2 — 17.0 303.7 139.4 Data from CDC Fact Book, 2000/2001, Department of Health and Human Services, Centers for Disease Control and Prevention. abuse, is a major concern in the United States. Hence, firearm safety and access are important matters of public health.[ 66] Injuries caused by the physical environment, resulting from human activities as well as from external forces, can be divided into four categories: mechanical force, heat and cold, electrical injuries, and high altitudes. Mechanical Force Mechanical force may inflict soft-tissue injuries, bone injuries, and head injuries. Injuries of the bones and of the head are considered in Chapter 28 . Soft-tissue injuries can be superficial, involving mainly the skin, or deep, associated with visceral damage. The skin injuries can be further described as follows. Abrasion. This type of skin injury represents basically a scrape, in which the superficial epidermis is torn off by friction or force ( Fig. 9-17 ). Regeneration without scarring usually occurs promptly unless infection complicates the process. Figure 9-17 Abrasion. Note the superficial tears in the epidermis. There is bleeding under the skin as well. (From the teaching collection of the Department of Pathology, Southwestern Medical School, Dallas, TX.) Figure 9-18 Laceration of the scalp. The bridging strands of the fibrous tissue are evident. (From the teaching collection of the Department of Pathology, Southwestern Medical School, Dallas, TX.) Figure 9-19 Contusion resulting from blunt trauma. The skin is intact, but there is hemorrhage in subcutaneous vessels, producing extensive discoloration. (From the teaching collection of the Department of Pathology, Southwestern Medical School, Dallas, TX.) Figure 9-20 A, Gunshot wound of entry from a long distance. (From the teaching collection of the Department of Pathology, Southwestern Medical School, Dallas, TX.) B, An entry gunshot wound at close range revealing the prominent black discoloration produced by unburned powder, heat, and smoke as well as the more peripheral stippling resulting from larger particles of unburned powder. (Courtesy of George Katsas, MD, Forensic Pathologist, Boston, MA.) Figure 9-21 Kwashiorkor. The infant shows generalized edema, seen in the form of puffiness of the face, arms, and legs. TABLE 9-21 -- Comparison of Severe Marasmus-Like and Kwashiorkor-Like Secondary Protein-Energy Malnutrition Syndrome Marasmus-like protein-energy malnutrition Clinical Setting Chronic illness (e.g., chronic lung disease, cancer) Time Course Months Clinical Features History of weight loss Muscle wasting Laboratory Findings Prognosis Normal or mildly reduced serum proteins Variable; depends on underlying disease Absent subcutaneous fat Kwashiorkor-like protein-energy malnutrition Acute, catabolic illness (e.g., severe trauma, burns, sepsis) Weeks Normal fat and muscle Edema Easily pluckable hair Data from Bennett JC, Plum F (eds): Cecil Textbook of Medicine, 20th ed. Philadelphia, WB Saunders, 1996, p. 1156. Serum albumin <2.8 gm/ Poor dL weak and bedridden may show physical signs of protein and energy malnutrition: (1) depletion of subcutaneous fat in the arms, chest wall, shoulders, or metacarpal regions; (2) wasting of the quadriceps femoris and deltoid muscles; and (3) ankle or sacral edema. 68 Bedridden or hospitalized malnourished patients have an increased risk of infection, sepsis, impaired wound healing, and death after surgery.[ ] The biochemical mechanisms responsible for secondary PEM in patients with cachexia are complex. In contrast to patients with anorexia nervosa, described next, patients with cachexia show loss of fat as well as muscle mass, which may occur before a decrease in appetite. Cachectic patients show increased expenditure of resting energy; in contrast, in chronic starvation, the basal metabolic rate is decreased. Cytokines produced by the host during sepsis, for example, or by tumors have been postulated to be involved in cachexia: tumor necrosis factor, interleukin-1, interleukin-6, and interferon69] γ. In addition, as discussed in Chapter 7 , lipid- and protein-mobilizing factors have been isolated from animals and people with cancer cachexia.[ Morphology. The central anatomic changes in PEM are (1) growth failure; (2) peripheral edema in kwashiorkor; and (3) loss of body fat and atrophy of muscle, more marked in marasmus. The liver in kwashiorkor, but not in marasmus, is enlarged and fatty; superimposed cirrhosis is rare. In kwashiorkor (rarely in marasmus), the small bowel shows a decrease in the mitotic index in the crypts of the glands, associated with mucosal atrophy and loss of villi and microvilli. In such cases, concurrent loss of small intestinal enzymes occurs, most often manifested as disaccharidase deficiency. Hence, infants with kwashiorkor initially may not respond well to a fullstrength, milk-based diet. With treatment, the mucosal changes are reversible. The bone marrow in both kwashiorkor and marasmus may be hypoplastic, mainly because of decreased numbers of red cell precursors. How much of this derangement is due to a deficiency of protein and folates or to reduced synthesis of transferrin and ceruloplasmin is uncertain. Thus, anemia is usually present, most often hypochromic microcytic anemia, but a concurrent deficiency of folates may lead to a mixed microcytic-macrocytic anemia. The brain in infants who are born to malnourished mothers and who suffer PEM during the first 1 or 2 years of life has been reported by some observers to show cerebral atrophy, a reduced number of neurons, and impaired myelinization of the white matter, but there is no universal agreement on the validity of these findings. Many other changes may be present, including (1) thymic and lymphoid atrophy (more marked in kwashiorkor than in marasmus); (2) anatomic alterations induced by intercurrent infections, particularly with all manner of endemic worms and other parasites; and (3) deficiencies of other required nutrients, such as iodine and vitamins. Anorexia Nervosa and Bulimia Anorexia nervosa is self-induced starvation, resulting in marked weight loss; bulimia is a condition in which the patient binges on food and then induces vomiting. These eating disorders occur primarily in previously healthy young women who have developed an obsession with attaining thinness. The clinical findings in anorexia nervosa are generally similar to those in severe PEM. In addition, effects on the endocrine system are prominent. Amenorrhea, resulting from decreased secretion of gonadotropin-releasing hormone (and subsequent decreased secretion of luteinizing hormone and follicle-stimulating hormone), is so common that its presence is a diagnostic feature for the disorder. Other common findings, related to decreased thyroid hormone release, include cold intolerance, bradycardia, constipation, and changes in the skin and hair. The skin becomes dry and scaly and may be yellow because of excess carotene in the blood. Body hair may be increased but is usually fine and pale (lanugo). Bone density is decreased, most likely owing to low estrogen levels, which mimic the postmenopausal acceleration of osteoporosis. As expected with severe PEM, anemia, lymphopenia, and hypoalbuminemia may be present. A major complication of anorexia nervosa is an increased susceptibility to cardiac arrhythmia and sudden death, resulting in all likelihood from hypokalemia. In bulimia, binge eating is the norm. Huge amounts of food, principally carbohydrates, are ingested, only to be followed by induced vomiting. Although menstrual irregularities are common, amenorrhea occurs in less than 50% of bulimia patients, probably because weight and gonadotropin levels are maintained near normal. The major medical complications relate to continual induced vomiting and include (1) electrolyte imbalances (hypokalemia), which predispose the patient to cardiac arrhythmias; (2) pulmonary aspiration of gastric contents; and (3) esophageal and cardiac rupture. 450 Vitamin Deficiencies Thirteen vitamins are necessary for health; four—A, D, E, and K—are fat-soluble, and the remainder are water-soluble. The distinction between fat- and water-soluble vitamins is important, because although fat-soluble vitamins are more readily stored in the body, they are likely to be poorly absorbed in gastrointestinal disorders of fat malabsorption ( Chapter 17 ). Small amounts of some vitamins can be synthesized endogenously—vitamin D from precursor steroids; vitamin K and biotin by the intestinal microflora; and niacin from tryptophan, an essential amino acid—but the rest must be supplied in the diet. A deficiency of vitamins may be primary (dietary in origin) or secondary (because of disturbances in intestinal absorption, transport in the blood, tissue storage, or metabolic conversion). In the following sections, the major vitamins, together with their well-defined deficiency states, are discussed individually (with the exception of vitamin B12 and folate, which are discussed in Chapter 13 ) beginning with the fat-soluble vitamins. However, deficiencies of a single vitamin are uncommon, and the expression of a deficiency of a combination of vitamins may be submerged in concurrent PEM. A summary of all the essential vitamins, along with their functions and deficiency syndromes, is presented in Table 9-22 . Vitamin A. Vitamin A is actually a group of related natural and synthetic chemicals that exert a hormone-like activity or function. The relationship of some important members of this TABLE 9-22 -- Vitamins: Major Functions and Deficiency Syndromes Vitamin Functions Deficiency Syndromes Fat-Soluble Vitamin A Vitamin D A component of visual pigment Night blindness, xerophthalmia, blindness Maintenance of specialized epithelia Squamous metaplasia Maintenance of resistance to infection Vulnerability to infection, particularly measles Facilitates intestinal absorption of calcium and phosphorus and mineralization of bone Rickets in children Osteomalacia in adults Vitamin E Major antioxidant; scavenges free radicals Spinocerebellar degeneration Vitamin K Cofactor in hepatic carboxylation of procoagulants—factors II (prothrombin), VII, IX, and X; and protein C and protein S Bleeding diathesis As pyrophosphate, is coenzyme in decarboxylation reactions Dry and wet beriberi, Wernicke syndrome, ?Korsakoff syndrome Water-Soluble Vitamin B1 (thiamine) Vitamin B2 (riboflavin) Converted to coenzymes flavin mononucleotide and flavin adenine dinucleotide, cofactors for many enzymes in intermediary metabolism Ariboflavinosis, cheilosis, stomatitis, glossitis, dermatitis, corneal vascularization Niacin Incorporated into nicotinamide adenine dinucleotide (NAD) and NAD phosphate, involved in a variety of redox reactions Pellagra—three "D's": dementia, dermatitis, diarrhea Vitamin B6 (pyridoxine) Derivatives serve as coenzymes in many intermediary reactions Cheilosis, glossitis, dermatitis, peripheral neuropathy Vitamin B12 Required for normal folate metabolism and DNA synthesis Combined system disease (megaloblastic pernicious anemia and degeneration of posterolateral spinal cord tracts) Maintenance of myelinization of spinal cord tracts Vitamin C Serves in many oxidation-reduction (redox) reactions and hydroxylation of collagen Scurvy Folate Essential for transfer and use of 1-carbon units in DNA synthesis Megaloblastic anemia, neural tube defects Pantothenic acid Incorporated in coenzyme A No nonexperimental syndrome recognized Biotin Cofactor in carboxylation reactions No clearly defined clinical syndrome group is presented in Figure 9-22 . Retinol, perhaps the most important form of vitamin A, is the transport form and, as the retinol ester, also the storage form. It is oxidized in vivo to the aldehyde retinal (the form used in visual pigment) and the acid retinoic acid. Important dietary sources of vitamin A are animal derived (e.g., liver, fish, eggs, milk, butter). Yellow and leafy green vegetables such as carrots, squash, and spinach supply large amounts of carotenoids, many of which are provitamins that can be metabolized to active vitamin A in vivo; the most important of these is beta-carotene. A widely used term, retinoids, refers to both natural and synthetic chemicals that are structurally related to vitamin A but do not necessarily have vitamin A activity. As with all fats, the digestion and absorption of carotenes and retinoids require bile, pancreatic enzymes, and some level of antioxidant activity in the food. Retinol, whether derived from ingested esters or from beta-carotene (through an intermediate oxidation step involving retinal), is transported in chylomicrons to the liver for esterification and storage. More than 90% of the body's vitamin A reserves are stored in the liver, predominantly in the perisinusoidal stellate (Ito) cells. In normal persons who consume an adequate diet, these reserves are sufficient for at least 6 months' deprivation. Retinoic acid, on the other hand, can be absorbed unchanged; it represents a small fraction of vitamin A in the blood and is active in epithelial differentiation and growth but not in the maintenance of vision. 451 Figure 9-22 Interrelationships of retinoids and their major functions. Figure 9-23 Vitamin A deficiency: its major consequences in the eye and in the production of keratinizing metaplasia of specialized epithelial surfaces, and its possible role in potentiating neoplasia. Figure 9-24 A, Schema of normal vitamin D metabolism. B, Vitamin D deficiency. There is inadequate substrate for the renal hydroxylase (1), yielding a deficiency of 1,25(OH)2 D (2), and deficient absorption of calcium and phosphorus from the gut (3), with consequent depressed serum levels of both (4). The hypocalcemia activates the parathyroid glands (5), causing mobilization of calcium and phosphorus from bone (6a). Simultaneously, the parathyroid hormone (PTH) induces wasting of phosphate in the urine (6b) and calcium retention. Consequently, the serum levels of calcium are normal or nearly normal, but the phosphate is low; hence, mineralization is impaired (7). TABLE 9-23 -- Predisposing Conditions for Rickets or Osteomalacia Inadequate Synthesis or Dietary Deficiency of Vitamin D Inadequate exposure to sunlight Limited dietary intake of fortified foods Poor maternal nutrition Dark skin pigmentation Decreased Absorption of Fat-Soluble Vitamin D Cholestatic liver disease Pancreatic insufficiency Biliary tract obstruction Celiac sprue Extensive small-bowel disease Derangements in Vitamin D Metabolism Increased degradation of vitamin D and 25(OH)D ••Induction of cytochrome P-450 enzymes (phenytoin, phenobarbital, rifampin) Impaired synthesis of 25(OH)D ••Diffuse liver disease Decreased synthesis of 1,25(OH)2 D ••Advanced renal disease ••Inherited deficiency of renal α1 -hydroxylase (vitamin D-dependent rickets type I) End-Organ Resistance to 1,25(OH)2 D Inherited absence of or defective receptors for acute metabolite of vitamin D (vitamin D-dependent rickets type II) Phosphate Depletion Poor absorption of phosphate due to chronic use of antacids—binding by aluminum hydroxide Excess renal tubule excretion of phosphate (X-linked hypophosphatemic rickets) Figure 9-25 A, Detail of a rachitic costochondral junction. The palisade of cartilage is lost. Some of the trabeculae are old, well-formed bone, but the paler ones consist of uncalcified osteoid. B, For comparison, normal costochondral function from a young child demonstrates the orderly transition from cartilage to new bone formation. Figure 9-26 Rickets. The bowing of legs in a toddler due to the formation of poorly mineralized bones is evident. Figure 9-27 A, The flabby, four-chambered, dilated heart of wet beriberi. B, The peripheral neuropathy with myelin degeneration leading to footdrop, wristdrop, and sensory changes in dry beriberi. C, Hemorrhages into the mamillary bodies in the Wernicke-Korsakoff syndrome. Figure 9-28 The sharply demarcated, characteristic scaling dermatitis of pellagra. Figure 9-29 A, Longitudinal section of a scorbutic costochondral junction with widening of the epiphyseal cartilage and projection of masses of cartilage into the adjacent bone. B, Detail of a scorbutic costochondral junction. The orderly palisade is totally destroyed. There is dense mineralization of the spicules but no evidence of newly formed osteoid. Figure 9-30 The major consequences of vitamin C deficiency. TABLE 9-24 -- Functions of Trace Metals and Deficiency Syndromes Nutrient Functions Deficiency Syndromes Iron Essential component of hemoglobin as well as a number of iron-containing metalloenzymes Hypochromic microcytic anemia Zinc Component of enzymes, principally oxidases Acrodermatitis enteropathica, growth retardation, infertility Iodine Component of thyroid hormone Goiter and hypothyroidism Selenium Component of glutathione peroxidase Myopathy, rarely cardiomyopathy Copper Component of cytochrome c oxidase, dopamine β-hydroxylase, tyrosinase, lysyl oxidase, and unknown enzyme involved in cross-linking keratin Muscle weakness, neurologic defects, hypopigmentation, abnormal collagen cross-linking Manganese Component of metalloenzymes, including oxidoreductases, hydrolases, and lipases No well-defined deficiency syndrome Fluoride Mechanism unknown Dental caries Figure 9-31 Zinc deficiency with hemorrhagic dermatitis around the mouth and eyes. TABLE 9-25 -- Body Mass Index Associated Disease Risk Obesity Class BMI (kg/m2 ) Underweight <18.5 Risk Increased Normal 18.5–24.9 Normal Overweight 25.0–29.9 Increased I 30.0–34.9 High II 35.0–39.9 Very high III ≥40.0 Obesity Extreme Obesity Extremely high Data from National Institutes of Health, National Heart, Lung, and Blood Institute. Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults —The evidence report. Obes Res 6 (suppl 2):515, 1998. 80] These three components are described next.[ autonomic intermediaries. Not shown in Figure 9-32 is that energy expenditure occurs through a variety of hormonal (e.g., thyrotropin-releasing hormone) and Among the afferent signals, insulin and leptin exert long-term control over the energy cycle by activating catabolic circuits and inhibiting anabolic pathways, as discussed in greater detail below. By contrast, ghrelin is predominately a short-term mediator. Produced in the stomach, ghrelin levels rise sharply before every meal and fall promptly when the stomach is "filled." In fact, it is thought that the success of gastric bypass surgery in massively obese individuals may relate more to the associated suppression of ghrelin levels than to an anatomic reduction in stomach capacity. Whereas both insulin and leptin influence the energy cycle, available data suggest that leptin has a more important role than insulin in the central nervous system control of energy 81] [81A] homeostasis.[ Hence, our discussion will be focused on leptin, recognizing that leptin and insulin share some of their actions. It is now established that adipocytes communicate with the hypothalamic centers that control appetite and energy expenditure by secreting leptin, a member of the cytokine family. When there is an abundance of stored energy in the form of adipose tissue, the resultant high levels of leptin cross the blood-brain barrier, binding to leptin receptors. Leptin receptor signaling has two effects: it inhibits anabolic circuits that normally promote food intake and inhibit energy expenditure, and, through a distinct set of neurons, leptin triggers catabolic circuits ( Fig. 932 ). The net effect of leptin, therefore, is to reduce food intake and promote energy expenditure. Hence, over a period of time, energy stores (adipocytes) are reduced, and weight is lost. This in turn reduces the circulating levels of leptin, and a new equilibrium is reached. This cycle is reversed when adipose tissue is lost and leptin levels are reduced below a threshold. Equilibrium is again reached, since with low leptin levels, the anabolic circuits are relieved of inhibition and catabolic circuits are not activated, resulting in net gain of weight. The molecular basis of leptin action is extremely complex and not yet fully unraveled. For the most part, leptin exerts its function through a series of integrated neural pathways referred to as the leptin-melanocortin circuit, described in Box 9-1 and illustrated in Figure 9-33 . The understanding of this circuitry is important since obesity is a serious public health problem, and development of antiobesity drugs will depend on a full understanding of these pathways. 79 Obesity, particularly central obesity, increases the risk for a number of conditions,[ ] including diabetes, hypertension, osteoarthritis, pancreatitis, and many others, listed in Table 9-26 . Only some of these complications are discussed here. The mechanisms underlying these associations are complex and likely to be interrelated. Obesity, for instance, is associated with insulin resistance and hyperinsulinemia, important features of non-insulin-dependent, or type II, diabetes, and weight loss is associated with improvement. It has been speculated that excess insulin, in turn, may play a role in the retention of sodium, expansion of blood volume, production of 463 Figure 9-32 A simplified schema of the circuitry that regulates energy balance. When sufficient energy is stored in adipose tissue and the individual is well fed, afferent adiposity signals (insulin, leptin, ghrelin) are delivered to the central neuronal processing units, in the hypothalamus. Here the adiposity signals inhibit anabolic circuits and activate catabolic circuits. The effector arms of these central circuits then impact on energy balance by inhibiting food intake and promoting energy expenditure. This in turn reduces the energy stores and the adiposity signals are obtunded. Conversely, when energy stores are low, the available anabolic circuits take over at the expense of catabolic circuits to generate energy stores in the form of adipose tissue, thus generating an equilibrium. Figure 9-33 The neurohumoral circuits in the hypothalamus that regulate energy balance. Details are in the text. Box 9-1. Genetics of Obesity Obesity is a disorder with a multifactorial etiology. Only rarely does it result from single gene disorders. Evidence supporting an important role for genes in weight control includes familial clustering of obesity and higher concordance of body mass index (BMI) among monozygotic twins (74%) versus dizygotic twins (32%) living in the same environment. Although monogenic forms of obesity in humans are rare, studies of these genetic forms of obesity and their murine counterparts have significantly advanced our understanding of the molecular basis of obesity. Some of these are discussed below. In recent years many "obesity" genes have been identified. As might be expected, they encode the molecular components of the neuroendocrine system that regulates energy balance. Leptin, the key player in energy homeostasis, is the product of the OB gene. Its role as an antiobesity factor is buttressed by the observation that mice homozygous for mutations in the leptin gene (OB/OB) do not secrete leptin, are massively obese, and are "cured" by the administration of exogenous leptin. Mice with mutations in the leptin receptor (db/db) are also obese, but, unlike the case with ob/ob mice, their obesity cannot be ameliorated by the administration of leptin. In these mice, obesity occurs because the leptin-mediated afferent signals impinging on the hypothalamus fail to regulate appetite and energy expenditure. Although leptin receptors are expressed at several sites in the brain, those most critical for regulation of the leptin-melanocortin circuit are expressed in the arcuate nucleus of the hypothalamus. There are two major types of neurons in this locale that bear leptin receptors: one set (oraxogenic) produces appetite-stimulating neurotransmitters called neuropeptide Y (NPY) and agouti-related peptide (AgRP). These are appropriately called NPY/AgRP neurons (see Fig. 9-33 ). As can be surmised from the discussion in the text, leptin reduces the expression of NPY and AgRP. The other set of leptin-sensitive neurons, the so-called POMC/CART neurons, transcribe two anorexigenic neuropeptides—α-melanocyte-stimulating hormone (α-MSH) and cocaine and amphetamine-related transcript (CART). Both of these peptides are products of proopiomelanocortin (POMC). When the POMC/CART neurons are activated by leptin signals, they exert catabolic effects mainly through the secretion of α-MSH. As indicated in Figure 9-33 , the NPY/AgRP and POMC/CART neurons are referred to as first-order neurons of the leptin-melanocortin circuit, since they are the initial targets of leptin action. The neurotransmitters produced by them (NPY, AgRP, and α-MSH) then interact through their own specific receptors with second-order neurons that trigger the efferent systems with peripheral actions. The effects of these neurotransmitters are described next. In the anabolic pathway, the first-order NPY/AgRP neurons make monosynaptic connections to second-order neurons, which express oraxogenic peptides melanin-concentrating hormone (MCH) and oraxins A and B. As illustrated in Figure 9-33 , NPY released from first-order neurons binds to its receptor on second-order neurons and thus transmits feeding signals. Such signals are attenuated when leptin is in excess and are activated by low levels of leptin. AgRP, like NPY, exerts anabolic effects but by a somewhat distinct mechanism. α-MSH produced by the POMC/CART neurons exerts its catabolic effects by binding to a set of second-order neurons (in the paraventricular nucleus) that express the melanocortin 4 receptor (MC4R). Catabolic output from the MC4R neurons is relayed to the periphery via the endocrine and autonomic systems. This reduces feeding and increases energy expenditure. The energy-consuming actions of MC4R neurons are mediated in part by the release of thyrotropin-releasing hormone (TRH), which activates the thyroxine axis through the anterior pituitary; TRH not only increases thermogenesis via secretion of thyroxine, but it is also an appetite suppressant. Corticotropin-releasing hormone (CRH) is another product of MC4R neurons. It induces anorexia and also activates the sympathetic nervous system. A subset of MC4R neurons projects to sympathetic motor output areas. Fibers from these areas innervate brown adipose tissue, rich in β3 -adrenergic receptors. When these receptors are stimulated, they cause fatty acid hydrolysis and also uncouple energy production from storage. Thus, the fats are literally burned, and energy so produced is dissipated as heat. It is noteworthy that each of the six single gene defects that give rise to human obesity involves proteins in the leptin-melanocortin pathway. Four of these are autosomal recessive and affect the leptin receptor, POMC, and PC1. (The last mentioned is a prohormone convertase that cleaves POMC). In all these cases, there is profound hyperphagia and childhood-onset massive obesity. While these four forms of genetic obesity are quite rare, those caused by mutations in the melanocortin receptor, MC4R, are by comparison quite common. In a recent 83 study, 5% to 8% of a cohort of 500 obese individuals had functionally important mutations in the MC4R gene.[ ] In these patients, despite abundant fat stores and leptin, energy consumption cannot be stimulated. The sixth monogenic form of human obesity results from mutation in a transcription factor (SIM1) that is essential for the formation of second-order leptin neurons. Despite the remarkable advances in our understanding of genetic control of pathways that regulate energy balance, the genetic basis of the most common forms of human obesity remains mysterious. As a multifactorial disorder, one might expect mutations or polymorphisms in several genes of small effect that give rise to obesity in concert with environmental factors. It is interesting to note that blood leptin levels are elevated in most humans with obesity. Clearly, the high levels of leptin are unable to down-regulate the anabolic pathways or activate the catabolic pathways. The basis of such leptin resistance is unclear but it may be contributed to by a decrease in the ability of leptin to cross the blood-brain barrier, possibly due to defective transport across endothelial cells. The fact that in some obese individuals leptin levels in the cerebrospinal fluid are lower than in the plasma supports this hypothesis. 465 TABLE 9-26 -- Medical Complications Associated with Obesity Gastrointestinal Gallstones, pancreatitis, abdominal hernia, NAFLD (steatosis, steatohepatitis, and cirrhosis), and possibly GERD Endocrine/metabolic Metabolic syndrome, insulin resistance, impaired glucose tolerance, type II diabetes mellitus, dyslipidemia, polycystic ovary syndrome Cardiovascular Hypertension, coronary artery disease, congestive heart failure, arrhythmias, pulmonary hypertension, ischemic stroke, venous stasis, deep vein thrombosis, pulmonary embolus Respiratory Abnormal pulmonary function, obstructive sleep apnea, obesity hypoventilation syndrome Musculoskeletal Osteoarthritis, gout, low back pain Gynecologic Abnormal menses, infertility Genitourinary Urinary stress incontinence Ophthalmologic Cataracts Neurologic Idiopathic intracranial hypertension (pseudotumor cerebri) Cancer Esophagus, colon, gallbladder, prostate, breast, uterus, cervix, kidney Postoperative events Atelectasis, pneumonia, deep vein thrombosis, pulmonary embolus Data from Klein S, Wadden T, Sugerman HJ: AGA technical review on obesity. Gastroenterol 123:882, 2002. NAFLD, non-alcoholic fatty liver disease; GERD, gastroesophageal reflux disease. excess norepinephrine, and smooth muscle proliferation that are the hallmarks of hypertension. Regardless of whether these pathogenic mechanisms are actually operative, the risk of developing hypertension among previously normotensive persons increases proportionately with weight. Obesity is also associated with a somewhat distinctive metabolic syndrome, the socalled syndrome X, which is characterized by abdominal obesity, insulin resistance, hypertriglyceridemia, low serum HDL, hypertension, and increased risk for coronary artery disease.[ 82] Obese persons are likely to have hypertriglyceridemia and a low HDL cholesterol value, and these factors may increase the risk of coronary artery disease. The association between obesity and heart disease is not straightforward, and the linkage may be related to the associated diabetes and hypertension rather than to weight. Nevertheless, the American Heart Association has recently added obesity to its list of major risk factors.[ 79] Nonalcoholic steatohepatitis occurs in adolescents and adults who are obese and have type II diabetes. Fatty change accompanied by liver cell injury and inflammation may progress to fibrosis or regress following weight loss. Cholelithiasis (gallstones) is six times more common in obese than in lean subjects. The mechanism is mainly an increase in total body cholesterol, increased cholesterol turnover, and augmented biliary excretion of cholesterol in the bile, which in turn predisposes to the formation of cholesterol-rich gallstones ( Chapter 18 ). Hypoventilation syndrome is a constellation of respiratory abnormalities in very obese persons. It has been called the pickwickian syndrome, after the fat lad who was constantly falling asleep in Charles Dickens' Pickwick Papers. Hypersomnolence, both at night and during the day, is characteristic and is often associated with apneic pauses during sleep, polycythemia, and eventual right-sided heart failure. Marked adiposity predisposes to the development of degenerative joint disease (osteoarthritis). This form of arthritis, which typically appears in older persons, is attributed in large part to the cumulative effects of wear and tear on joints. It is reasonable to assume that the greater the body burden of fat, the greater the trauma to joints with passage of time. Obesity increases the risk of ischemic stroke in both men and women. Abdominal obesity is associated with increased risk of venous thrombosis. Somewhat controversial is the association between obesity and cancer. A recent large prospective study has revealed an association between increasing BMI and mortality from many 84 forms of cancer, including cancers of the esophagus, colon, rectum, liver, and non-Hodgkin lymphoma.[ ] The basis of this association is difficult to discern. With hormone-dependent cancers, such as those arising in the endometrium, the blame can be placed on hormonal imbalance since obesity is known to raise estrogen levels, but for others we remain in the dark. DIET AND SYSTEMIC DISEASES The problems of undernutrition and overnutrition, as well as specific nutrient deficiencies, have been discussed; however, the composition of the diet, even in the absence of any of these problems, may make a significant contribution to the causation and progression of a number of diseases. A few examples suffice here. Currently one of the most important and controversial issues is the contribution of diet to atherogenesis. The central question is, Can dietary modification prevent or retard the development of atherosclerosis (most importantly, coronary artery disease)? The average adult in the United States consumes an inordinate amount of fat and cholesterol daily, with a ratio of saturated fatty acids to polyunsaturated fatty acids of about 3:1. Vegetable oils (e.g., corn and safflower oils) and fish oils contain polyunsaturated fatty acids and are good sources of cholesterollowering lipids. Fish oil fatty acids belonging to the omega-3, or n-3, family have more double bonds than do the omega-6, or n-6, fatty acids found in vegetable oils. A recent metaanalysis of 11 studies with over 16,000 patients revealed that a diet enriched in omega-3 fatty acids (vs. placebo) significantly reduced the incidence of fatal myocardial infarction and 85] sudden cardiac death.[ There are other examples of the effect of diet on disease: 466 • Hypertension is beneficially affected by restricting sodium intake. • Dietary fiber, or roughage, resulting in increased fecal bulk, has a preventive effect against diverticulosis of the colon. • People who consume diets that contain abundant fresh fruits and vegetables with limited intake of meats and processed foods have a lower risk of myocardial infarction. One mechanism that may explain these epidemiologic observations is the association of hyperhomocysteinemia with increased intake of meats and decreased intake of vitamin B6 , vitamin B12 , and folate. Excess levels of homocysteine are hypothesized to contribute to atherosclerosis ( Chapter 11 ). • Calorie restriction has been convincingly demonstrated to increase life span in experimental animals. The basis of this striking observation is not entirely clear ( Chapter 1 ). • Even lowly garlic has been touted to protect against heart disease (and also, alas, kisses), although research has yet to prove the effect on heart disease unequivocally. CHEMOPREVENTION OF CANCER Epidemiologic studies have provided evidence that populations who consume large quantities of fruits and vegetables in their diets have a lower risk of cancer. It is hypothesized that carotenoids that are converted to vitamin A in the liver and intestine may be important in the primary chemoprevention of cancer. [ anticarcinogenic effects of carotenoids and retinoids: 86] The following mechanisms are proposed for the • Retinoic acid promotes differentiation of mucus-secreting epithelial tissues. Supplementation of the diet with beta-carotene and retinol is hypothesized to reverse squamous metaplasia and preneoplastic lesions in the respiratory tract of cigarette smokers and workers exposed to asbestos. • Fruits and vegetables provide antioxidants such as betacarotene, vitamins C and E, and selenium that prevent oxidative damage to DNA. • Vitamin A can enhance immune responses; other retinoids may modulate inflammatory reactions that are potential sources of reactive oxygen and nitrogen intermediates. Notwithstanding such theoretical considerations, clinical studies on the role of vitamin A supplementation and cancer risk have failed to provide clear answers. Clinical trials using betacarotene and retinyl palmitate as primary preventive agents against lung cancer were terminated because the participants showed an excess of lung cancers and increased mortality. On the other hand, 13-cis-retinoic acid was effective in prevention of secondary squamous cell carcinomas of the head and neck region. These apparently conflicting results are not easily explained; however, there are multiple chemical forms of retinoids that alter gene expression, cell proliferation, differentiation, and apoptosis by binding to six different nuclear receptors. Some retinoids are associated with significant toxicity, including dry skin, conjunctivitis, and hypertriglyceridemia. Until the biochemical and molecular mechanisms of action of individual retinoids and other antioxidants are understood, it is unwise to recommend dietary supplements for the primary chemoprevention of cancer. However, a diet rich in fruits, vegetables, and unprocessed grains that is low in fat and animal protein has been associated with a decreased risk of cardiovascular disease and some types of cancer.[ 84] High animal fat intake combined with low fiber intake has been implicated in the causation of colon cancer. The most convincing explanation for these associations is as follows: high fat intake increases the level of bile acids in the gut, which in turn modifies intestinal flora, favoring the growth of microaerophilic bacteria. The bile acids or bile acid metabolites produced by these bacteria might serve as carcinogens or promoters. The protective effect of a high-fiber diet might relate to (1) increased stool bulk and decreased transit time, which decrease the exposure of mucosa to putative offenders, and (2) the capacity of certain fibers to bind carcinogens and thereby protect the mucosa. 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Moline JM, et al: Exposure to hazardous substances and male reproductive health: a research framework. Environ Health Perspect 108:803, 2000. 50. Schantz SL, Widholm JJ: Cognitive effects of endocrine-disrupting chemicals in animals. Environ Health Perspect 109:1197, 2001. 51. Huff J, et al: Carcinogenicity of TCDD: experimental, mechanistic, and epidemiologic evidence. Annu Rev Pharmacol Toxicol 34:343, 1994. 52. Mettler FA, Voelz GL: Major radiation exposure—what to expect and how to respond. N Engl J Med 346:1554, 2002. 53. Upton AC: Ionizing radiation. In Craighead JE (ed): Pathology of Environmental and Occupational Disease. St. Louis, Mosby, 1996, pp 205–214. 54. Karanjawla ZE et al.: Supplementary oxygen metabolism causes chromosome breaks and is associated with neuronal apoptosis observed in double stranded DNA strand repair mutants. Curr Biol 12:397, 2002. 55. Smith ML, Fornace AJ Jr: Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. 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Science 278:1073, 1997. 469 Chapter 10 - Diseases of Infancy and Childhood * Anirban Maitra MBBS Vinay Kumar MD Children are not merely little adults, and the diseases they get are not merely variants of adult diseases. Many childhood conditions are unique to, or at least take distinctive forms in, this stage of life and so are discussed separately in this chapter. Diseases originating in the perinatal period are important in that they account for significant morbidity and mortality. As would be expected, the chances for survival of live-born infants improve with each passing week. This differential represents, at least in part, a triumph of improved medical care. Better prenatal care, more effective methods of monitoring the condition of the fetus, and judicious resort to cesarean section before term when there is evidence of fetal distress all contribute to bringing into this "mortal coil" live-born infants who in past years might have been stillborn. These infants represent an increased number of high-risk infants. Nonetheless, the infant mortality rate 1 in the United States has shown a decline from a level of 20.0 deaths per 1000 live births in 1970 to about 6.9 deaths in 2000.[ ] Although the death rate has continued * The contributions of Dr. Deborah Scofield to this chapter in earlier editions are gratefully acknowledged. 470 to decline for all infants, American blacks continue to have an infant mortality rate more than twice (13.9 deaths per 1000 live births) that of American whites (6.0 deaths). Worldwide, the infant mortality rates vary widely, from as low as 3 deaths per 1,000 live births in Sweden, to as high as 82 deaths in the Indian subcontinent. Each stage of development of the infant and child is prey to a somewhat different group of disorders. The data available permit a survey of four time spans: (1) the neonatal period (the first 4 weeks of life), (2) infancy (the first year of life), (3) age 1 to 4 years, and (4) age 5 to 14 years. The major causes of death in infancy and childhood are cited in Table 10-1 . Congenital anomalies, disorders relating to short gestation (prematurity) and low birth weight, and sudden infant death syndrome (SIDS) represent the leading causes of death in the first 12 months of life. Once the infant survives the first year of life, the outlook brightens measurably. In the next two age groups—1 to 4 years and 5 to 14 years—injuries resulting from accidents have become the leading cause of death (see Table 10-1 ). Among the natural diseases, in order of importance, congenital anomalies and malignant neoplasms assume major significance. It would appear then that, in a sense, life is an obstacle course. For the great majority, the obstacles are surmounted or, even better, bypassed. We now take a closer look at the specific conditions encountered during the various stages of infant and child development. Congenital Anomalies Congenital anomalies are morphologic defects that are present at birth, but some, such as cardiac defects and renal anomalies, may not become clinically apparent until years later. The term congenital does not imply or exclude a genetic basis for the birth defect. It is estimated that about 3% of newborns have a major anomaly, defined as an anomaly having either cosmetic or functional significance. As indicated in Table 10-1 , they are the most common cause of mortality in the first year of life and contribute significantly to morbidity and mortality throughout the early years of life. In a real sense, anomalies found in live-born infants represent the less serious developmental failures in embryogenesis that are compatible with live birth. Perhaps 20% of fertilized ova are so anomalous that they are blighted from the outset. Others may be compatible with early fetal development, only to lead to spontaneous abortion. Less severe anomalies allow more prolonged intrauterine survival, with some disorders terminating in still-birth and those still less significant permitting live birth despite the handicaps imposed. DEFINITIONS Before proceeding, we define some of the terms used for various kinds of errors in morphogenesis—malformations, disruptions, deformations, sequences, and syndromes. • Malformations represent primary errors of morphogenesis, in other words there is an intrinsically abnormal developmental process ( Fig. 10-1 ). They are usually multifactorial rather than the result of a single gene or chromosomal defect. Malformations may present in several patterns. Some, such as congenital heart defects and anencephaly (absence of brain), involve single body systems, whereas in other cases multiple malformations involving many organs may coexist. • Disruptions result from secondary destruction of an organ or body region that was previously normal in development; thus, in contrast to malformations, disruptions arise from an extrinsic disturbance in morphogenesis. Amniotic 471 bands, denoting rupture of amnion with resultant formation of "bands"that encircle, compress, or attach to parts of the developing fetus, are the classic example of a disruption ( Fig. 10-2). A variety of environmental agents may cause disruptions (see below). Understandably, disruptions are not heritable and hence are not associated with risk of recurrence in subsequent pregnancies. • Deformations, like disruptions, also represent an extrinsic disturbance of development rather than an intrinsic error of morphogenesis. Deformations are common problems, affecting approximately 2% of newborn infants to varying degrees. Fundamental to the pathogenesis of deformations is localized or generalized compression of the growing fetus by abnormal biomechanical forces, leading eventually to a variety of structural abnormalities. The most common underlying factor responsible for deformations is uterine constraint. Between the 35th and 38th weeks of gestation, rapid increase in the size of the fetus outpaces the growth of the uterus, and the relative amount of amniotic fluid (which normally acts as a cushion) also decreases. Thus, even the normal fetus is subjected to some form of uterine constraint. Several factors increase the likelihood of excessive compression of the fetus resulting in deformations. Maternal factors include first pregnancy, small uterus, malformed (bicornuate) uterus, and leiomyomas. Fetal or placental factors include oligohydramnios, multiple fetuses, and abnormal fetal presentation. An example of a deformation is clubfeet, often a component of Potter sequence, described later. • A sequence is a pattern of cascade anomalies. Approximately half the time, congenital anomalies occur singly; in the remaining cases, multiple congenital anomalies are recognized. In some instances, the constellation of anomalies may be explained by a single, localized aberration in organogenesis (malformation, disruption, or deformation) leading to secondary effects in other organs. A good example of a sequence is the oligohydramnios (or Potter) sequence ( Fig. 10-3 ). Oligohydramnios (decreased amniotic fluid) may be caused by a variety of unrelated maternal, placental, or fetal abnormalities. Chronic leakage of amniotic fluid because of rupture of the amnion, uteroplacental 472 insufficiency resulting from maternal hypertension or severe toxemia, and renal agenesis in the fetus (as fetal urine is a major constituent of amniotic fluid) are all causes of oligohydramnios. The fetal compression associated with significant oligohydramnios, in turn, results in a classic phenotype in the newborn infant, including flattened facies and positional abnormalities of the hands and feet ( Fig. 10-4). The hips may be dislocated. Growth of the chest wall and the contained lungs is also compromised so that the lungs are frequently hypoplastic, occasionally to the degree that they are the cause of fetal demise. Nodules in the amnion (amnion nodosum)are frequently present. • A syndrome is a constellation of congenital anomalies, believed to be pathologically related, that, in contrast to a sequence, cannot be explained on the basis of a single, localized, initiating defect. Syndromes are most often caused by a single etiologic agent, such as a viral infection or specific chromosomal abnormality, which simultaneously affects several tissues. TABLE 10-1 -- Cause of Death Related with Age Causes * Under 1 Year: All Causes Congenital malformations, deformations, and chromosomal anomalies Disorders related to short gestation and low birth weight Sudden infant death syndrome (SIDS) Newborn affected by maternal complications of pregnancy Rate † 727.4 Newborn affected by complications of placenta, cord, and membranes Respiratory distress of newborn Accidents (unintentional injuries) Bacterial sepsis of newborn Intrauterine hypoxia and birth asphyxia Diseases of the circulatory system 1–4 Years: All Causes 32.6 Accidents and adverse effects Congenital malformations, deformations, and chromosomal abnormalities Malignant neoplasms Homicide and legal intervention Diseases of the heart ‡ Influenza and pneumonia 5–14 Years: All Causes 18.5 Accidents and adverse effects Malignant neoplasms Homicide and legal intervention Congenital malformations, deformations, and chromosomal abnormalities Suicide Diseases of the heart 15–24 Years: All Causes 80.7 Accidents and adverse effects Homicide Suicide Malignant neoplasms Diseases of the heart *Causes are listed in decreasing order of frequency. All causes and rates are preliminary 2000 statistics. (Minino AM, Smith BL. Deaths: Preliminary data for 2000. National Vital Statistics Report, 49:12, 2001). †Rates are expressed per 100,000 population. ‡Excludes congenital heart disease. Figure 10-1 Malformations. Human malformations can range in severity from the incidental to the lethal. Polydactyly (one or more extra digits) and syndactyly (fusion of digits), both of which are illustrated in A, have little functional consequence when they occur in isolation. Similarly, cleft lip (B), with or without associated cleft palate, is compatible with life when it occurs as an isolated anomaly; in the present case, however, this child had an underlying malformation syndrome (trisomy 13) and expired because of severe cardiac defects. The stillbirth illustrated in C represents a severe and essentially lethal malformation, where the midface structures are fused or ill-formed; in almost all cases, this degree of external dysmorphogenesis is associated with severe internal anomalies such as maldevelopment of the brain and cardiac defects. (Pictures A and C courtesy of Dr. Reade Quinton, and B courtesy of Dr. Beverly Rogers, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Figure 10-2 Disruption. Disruptions occur in a normally developing organ because of an extrinsic abnormality that interferes with normal morphogenesis. Amniotic bands are a frequent cause of disruptions. In the illustrated example, note the placenta at the right of the diagram and the band of amnion extending from the top portion of the amniotic sac to encircle the leg of the fetus. (Courtesy of Dr. Theonia Boyd, Children's Hospital of Boston, MA.) Figure 10-3 Schematic diagram of the pathogenesis of the oligohydramnios sequence. Figure 10-4 Infant with oligohydramnios sequence. Note the flattened facial features and deformed right foot (talipes equinovarus). TABLE 10-2 -- Causes of Congenital Anomalies in Humans Cause Frequency (%) Genetic Chromosomal aberrations 10–15 Mendelian inheritance •2–10 Environmental Maternal/placental infections ••Rubella •2–3 ••Toxoplasmosis ••Syphilis ••Cytomegalovirus ••Human immunodeficiency virus (HIV) Maternal disease states •6–8 ••Diabetes ••Phenylketonuria ••Endocrinopathies Drugs and chemicals •1 ••Alcohol ••Folic acid antagonists ••Androgens ••Phenytoin ••Thalidomide ••Warfarin ••13-cis-retinoic acid ••Others Irradiations •1 Multifactorial (Multiple Genes ? Environment) 20–25 Unknown 40–60 Adapted from Stevenson RE, et al (eds): Human Malformations and Related Anomalies. New York, Oxford University Press, 1993, p. 115. 2 Single gene mutations of large effect may underlie major congenital anomalies, which, as expected, follow mendelian patterns of inheritance.[ ] Of these, approximately 90% are inherited in an autosomal dominant or recessive pattern, while the remainder segregates in an X-linked pattern. Not surprisingly, many of the mutations that give rise to birth defects involve abrogation of function of genes involved in normal organogenesis and development. For example, holoprosencephaly is the most common developmental defect of the forebrain and midface in humans (see Chapter 28 ); mutations of sonic hedgehog, a gene involved in developmental patterning (see below), have been reported in a subset of patients with 3 holoprosencephaly.[ ] Similarly, mutations of a downstream target of sonic hedgehog signaling, GLI3, have been reported in patients with anomalies of digits, either conjoined digits (syndactyly) or supernumerary digits (polydactyly). Environmental Causes Environmental influences, such as viral infections, drugs, and irradiation, to which the mother was exposed during pregnancy may cause fetal malformations (the appellation of "malformation" is loosely used in this context, since technically, these anomalies represent disruptions). Viruses. Many viruses have been implicated in causing malformations, including the agents responsible for rubella, cytomegalic inclusion disease, herpes simplex, varicella-zoster infection, influenza, mumps, human immunodeficiency virus (HIV), and enterovirus infections. Among these, the rubella virus and cytomegalovirus are the most extensively investigated. With all viruses, the gestational age at which the infection occurs in the mother is critically important. The at-risk period for rubella infection extends from shortly before conception to the 16th 4 week of gestation, the hazard being greater in the first 8 weeks than in the second 8 weeks.[ ] The incidence of malformations is reduced from 50% to 20% to 7% if infection occurs in the first, second, or third month of gestation. The fetal defects are varied, but the major tetrad comprises cataracts, heart defects (persistent ductus arteriosus, pulmonary artery hypoplasia or stenosis, ventricular septal defect, tetralogy of Fallot), deafness, and mental retardation, referred to as rubella embryopathy. Intrauterine infection with cytomegalovirus, mostly asymptomatic, is the most common fetal viral infection. This viral disease is considered in detail in Chapter 8 ; the highest at-risk period is the second trimester of pregnancy. Because organogenesis is largely completed by the end of the first trimester, congenital malformations occur less frequently than in rubella; nevertheless, the effects of virus-induced injury on the formed organs are often severe. Involvement of the central nervous system is a major feature, and the most prominent clinical changes are mental retardation, microcephaly, deafness, and hepatosplenomegaly. Drugs and Other Chemicals. A variety of drugs and chemicals have been suspected to be teratogenic, but perhaps less than 1% of congenital malformations are caused by these agents. The list includes thalidomide, 5 folate antagonists, androgenic hormones, alcohol, anticonvulsants, warfarin (oral anticoagulant), and 13-cis-retinoic acid used in the treatment of severe acne.[ ] For example, thalidomide, 6 once used as a tranquilizer in Europe, caused an extremely high frequency (50% to 80%) of limb abnormalities in exposed fetuses.[ ] Alcohol, perhaps the most widely used agent today, is a teratogen. Affected infants show growth retardation, microcephaly, atrial septal defect, short palpebral fissures, maxillary hypoplasia, and several other minor anomalies. These together 7 are labeled the fetal alcohol syndrome.[ ] While cigarette smoke-derived nicotine has not been convincingly demonstrated to be a teratogen, there is a high incidence of spontaneous abortions, premature labor, and placental abnormalities in pregnant smokers; babies born to smoking mothers often have a low birth weight and may be prone to sudden infant death syndrome (see later). In light of these findings, it is best to avoid nicotine exposure altogether during pregnancy. Radiation. In addition to being mutagenic and carcinogenic, radiation is teratogenic. Exposure to heavy doses of radiation during the period of organogenesis leads to malformations, such as microcephaly, blindness, skull defects, spina bifida, and other deformities. Such exposure occurred in the past when radiation was used to treat cervical cancer. Maternal Diabetes. Among maternal conditions listed in Table 10-2 , diabetes mellitus is a common entity, and despite advances in antenatal obstetric monitoring and glucose 474 control, the incidence of major malformations in infants of diabetic mothers stands between 6% and 10% in most series. Maternal hyperglycemia-induced fetal hyperinsulinemia results in increased body fat, muscle mass, and organomegaly (fetal macrosomia); cardiac anomalies, neural tube defects, and other central nervous system malformations are some of the major 8] anomalies seen in diabetic embryopathy.[ Multifactorial Causes The genetic and environmental factors just discussed account for no more than half of human congenital anomalies. The causes of the vast majority of birth defects, including some relatively common disorders such as cleft lip and cleft palate, remain unknown. In these anomalies, it would appear that inheritance of a certain number of mutant genes and their interaction with the environment is required before the disorder is expressed. In the case of congenital dislocation of the hip, for example, depth of the acetabular socket and laxity of the ligaments are believed to be genetically determined, whereas a significant environmental factor is believed to be frank breech position in utero, with hips flexed and knees extended. The importance of environmental contribution to multifactorial inheritance is underscored by a dramatic reduction in the incidence of neural tube defects by periconceptional intake of folic acid 9 10 in the diet.[ ] [ ] The approximate frequency of some common congenital anomalies in the United States is presented in Table 10-3 . Both temporal and regional variability are common in the reporting of many malformations. For example, between 1979 and 1989, there was a mean annual percent decrease in the incidence of anencephaly of 6.4 and a mean annual increase in the incidence of atrial septal defect of 22.0.[ 11] PATHOGENESIS OF CONGENITAL ANOMALIES The pathogenesis of congenital anomalies is complex and still poorly understood, but certain general principles of TABLE 10-3 -- Approximate Frequency of the More Common Congenital Malformations in the United States Malformation Frequency per 10,000 Total Births Clubfoot without central nervous system anomalies 25.7 Patent ductus arteriosus 16.9 Ventricular septal defect 10.9 Cleft lip with or without cleft palate •9.1 Spina bifida without anencephalus •5.5 Congenital hydrocephalus without anencephalus •4.8 Anencephalus •3.9 Reduction deformity (musculoskeletal) •3.5 Rectal and intestinal atresia •3.4 Adapted from James LM: Maps of birth defects occurrence in the U.S., birth defects monitoring program (BDMP)/CPHA, 1970–1987. Teratology 48:551, 1993. developmental pathology are relevant regardless of the etiologic agent. The timing of the prenatal teratogenic insult has an important impact on the occurrence and the type of anomaly produced ( Fig. 10-5 ). The intrauterine development of humans can be divided into two phases: (1) the embryonic period occupying the first 9 weeks of pregnancy and (2) the fetal period terminating at birth. In the early embryonic period (first 3 weeks after fertilization), an injurious agent damages either enough cells to cause death and abortion or only a few cells, presumably allowing the embryo to recover without developing defects. Between the third and the ninth weeks, the embryo is extremely susceptible to teratogenesis, and the peak sensitivity during this period occurs between the fourth and the fifth weeks. During this period, organs are being crafted out of the germ cell layers. The fetal period that follows organogenesis is marked chiefly by the further growth and maturation of the organs, with greatly reduced susceptibility to teratogenic agents. Instead the fetus is susceptible to growth retardation or injury to already formed organs. It is therefore possible for a given agent to produce different anomalies if exposure occurs at different times of gestation. Teratogens and genetic defects may act at several steps involved in normal morphogenesis. These include the following: [ 12] • Proper cell migration to predetermined locations that influence the development of other structures • Cell proliferation, which determines the size and form of embryonic organs • Cellular interactions among tissues derived from different structures (e.g., ectoderm, mesoderm), which affect the differentiation of one or both of these tissues • Cell-matrix associations, which affect growth and differentiation • Programmed cell death (apoptosis), which, as we have seen, allows orderly organization of tissues and organs during embryogenesis ( Chapter 1 ) • Hormonal influences and mechanical forces, which affect morphogenesis at many levels. The complex interplay between environmental teratogens and intrinsic genetic defects is underscored by the fact that features of dysmorphogenesis caused by environmental insults can be recapitulated by certain genetic defects. This is exemplified in the relationship between the teratogen, retinoic acid (see below and Fig. 10-6 ), and two growth factors—transforming growth factor (TGF) and fibroblast growth factor (FGF)—both involved in morphogenesis. As discussed later, retinoic acid can induce defects in palatal development (cleft lip and cleft palate), possibly by impacting on multiple targets associated with secondary palatal development. In experimental models of retinoic acid teratogenesis, abnormal expression of TGF and FGF has been reported in the developing palate.[ 13] [14] Not unexpectedly, therefore, rare single gene mutations in one or more of these growth factors or their receptors may also cause palatal abnormalities. There is an association, for example, between rare mutations of the TGF-α gene and nonsyndromic cleft lip or cleft palate in humans;[ of the epidermal growth factor receptor, which acts as a receptor for TGF-α, can result in abnormal 16 palatogenesis.[ ] 15] in addition, loss of function Disruption of TGF-β3 in mice also results in cleft palate.[ 17] 475 Figure 10-5 Critical periods of development for various organ systems and the resultant malformations. (Modified and redrawn from Moore KL: The Developing Human, 5th ed. Philadelphia, WB Saunders, 1993, p. 156.) Figure 10-6 Schematic representation of the postulated role of retinoic acid in normal development, the general features of its deficiency (vitamin A deficiency) (left) and retinoic acid embryopathy (right). 1, Retinol in the maternal circulation is bound by retinol-binding protein (RBP), which is synthesized by the placenta and enters the fetal circulation. 2, Once in fetal cells, retinol is bound by cytoplasmic retinol-binding protein (CRBP), which (3) regulates the conversion to retinoic acid and metabolites. The retinoic acid either remains in the cytoplasm (bound to cytoplasmic/cellular retinoic acid-binding protein [CRABP]) or (4) enters the nucleus, where it is bound to nuclear retinoic acid receptors (RAR, RXR). The retinoic acid-receptor complex acts as a transcriptional regulator of various patterning genes (e.g., HOX) that have the appropriate retinoic acid response element (RARE). Expression of the binding proteins and receptors in various tissues and at various times during embryogenesis may be a mechanism of selectively modulating the action of retinoic acid. This differential expression may also explain the pattern of abnormalities seen in vitamin A deficiency and retinoic acid embryopathy. Figure 10-7 Diagrammatic representation of constitutional chromosomal mosaicism. A, Generalized. B, Confined to the placenta. C, Confined to the embryo. (Modified and redrawn from Kalousek DK: Confined placental mosaicism and intrauterine development. Pediatr Pathol 10:69, 1990.) Figure 10-8 Schematic diagrams of fetal lung maturation. TABLE 10-4 -- Evaluation of the Newborn Infant * Sign 0 1 2 Heart rate Absent Below 100 Over 100 Respiratory effort Absent Slow, irregular Good, crying Muscle tone Limp Some flexion of extremities Active motion Response to catheter in nostril (tested after oropharynx is clear) No response Grimace Cough or sneeze Color Blue, pale Body pink, extremities blue Completely pink Data from Apgar V: A proposal for a new method of evaluation of the newborn infant. Anesth Analg 32:260, 1953. *Sixty seconds after the complete birth of the infant (disregarding removal of the cord and placenta), the five objective signs are evaluated and each is given a score of 0, 1, or 2. A total score of 10 indicates an infant in the best possible condition. 480 risk for birth injury, in particular those involving the skeletal system and peripheral nerves. We briefly discuss only injuries involving the head because they are the most ominous. Intracranial hemorrhages are the most common important birth injury. These hemorrhages are generally related to excessive molding of the head or sudden pressure changes in its shape as it is subjected to the pressure of forceps or sudden precipitate expulsion. Prolonged labor, hypoxia, hemorrhagic disorders, or intracranial vascular anomalies are important predispositions. The hemorrhage may arise from tears in the dura or from rupture of vessels that traverse the brain. The substance of the brain may be torn or bruised, leading to intraventricular hemorrhages or bleeding into the brain substance. The consequences of intracranial hemorrhages are mentioned later under germinal matrix hemorrhage. Caput succedaneum and cephalhematoma are so common, even in normal uncomplicated births, that they hardly merit the designation birth injury. The first refers to progressive accumulation of interstitial fluid in the soft tissues of the scalp, giving rise to a usually circular area of edema, congestion, and swelling at the site where the head begins to enter the lower uterine canal. Hemorrhage may occur into the scalp, producing a cephalhematoma. Both forms of injury are of little clinical significance and are important only insofar as they must be differentiated from skull fractures with attendant hemorrhage and edema. In approximately 25% of cephalhematomas, there is an underlying skull fracture. Such skull fractures may occur in cases of precipitate delivery, inappropriate use of forceps, or prolonged labor with disproportion between the size of the fetal head and birth canal. Perinatal Infections Infections of the embryo, fetus, and neonate are manifested in a variety of ways and are mentioned as etiologic factors in numerous other sections within this chapter. Here we discuss only the general routes and timing of infections. In general, fetal and perinatal infections are acquired via one of two primary routes—transcervically (also referred to as ascending) or transplacentally (hematologic). Occasionally, infections occur by a combination of the two routes in that an ascending microorganism infects the endometrium and then the fetal bloodstream via the chorionic villi. TRANSCERVICAL (ASCENDING) INFECTIONS Most bacterial and a few viral (e.g., herpes simplex II) infections are acquired by the cervicovaginal route. Such infections may be acquired in utero or around the time of birth. In general, the fetus acquires the infection either by inhaling infected amniotic fluid into the lungs shortly before birth or by passing through an infected birth canal during delivery. As previously stated, preterm birth is often an unfortunate consequence and may be related either to damage and rupture of the amniotic sac as a direct consequence of the inflammation or to the induction of labor associated with a release of prostaglandins by the infiltrating neutrophils. Chorioamnionitis of the placental membranes and funisitis are usually demonstrable, although the presence or absence and severity of chorioamnionitis do not necessarily correlate with the severity of the fetal infection. In the fetus infected via inhalation of amniotic fluid, pneumonia, sepsis, and meningitis are the most common sequelae. TRANSPLACENTAL (HEMATOLOGIC) INFECTIONS Most parasitic (e.g., toxoplasma, malaria) and viral infections and a few bacterial infections (i.e., Listeria, Treponema) gain access to the fetal bloodstream transplacentally via the chorionic villi. This hematogenous transmission may occur at any time during gestation or occasionally, as may be the case with hepatitis B and HIV, at the time of delivery via maternalto-fetal transfusion. The clinical manifestations of these infections are highly variable, depending largely on the gestational timing and microorganism involved. 40 Some infections, such as those with parvovirus B19 (which causes fifth disease in the mother), may induce spontaneous abortion, stillbirth, hydrops fetalis, and congenital anemia.[ ] While the virus can bind to different cell types, replication occurs only in erythroid cells, and diagnostic viral cytopathic effect can be recognized in late erythroid progenitor cells of infected infants ( Fig. 10-9 ). The TORCH group of infections (see above) are grouped together because they may evoke similar clinical and pathologic manifestations, including fever, encephalitis, chorioretinitis, hepatosplenomegaly, pneumonitis, myocarditis, hemolytic anemia, and vesicular or hemorrhagic skin lesions. Such infections occurring early in gestation may also cause chronic sequelae in the child, including growth and mental retardation, cataracts, congenital cardiac anomalies, and bone defects. ONSET OF SEPSIS Perinatal infections can also be grouped clinically by whether they tend to result in early-onset (within the first 7 days of life) versus late-onset sepsis (from 7 days to 3 months). Most cases of early-onset sepsis are acquired at or shortly before birth and tend to result in clinical signs and symptoms of pneumonia, sepsis, and occasionally meningitis within 4 or 5 days of life. Group B streptococcus is the most common Figure 10-9 Bone marrow from an infant infected with parvovirus B19. The arrows point to two erythroid precursors with large homogeneous intranuclear inclusions and a surrounding peripheral rim of residual chromatin. Figure 10-10 Schematic outline of the pathophysiology of the respiratory distress syndrome (see text). Figure 10-11 Hyaline membrane disease. There is alternating atelectasis and dilation of the alveoli. Note the eosinophilic thick hyaline membranes lining the dilated alveoli. Figure 10-12 Necrotizing enterocolitis. A, Postmortem examination in a severe case of NEC shows the entire small bowel is markedly distended with a perilously thin wall (usually this implies impending perforation). B, The congested portion of the ileum corresponds to areas of hemorrhagic infarction and transmural necrosis microscopically. Submucosal gas bubbles (pneumatosis intestinalis) can be seen in several areas (arrows). Figure 10-13 Hydrops fetalis. There is generalized accumulation of fluid in the fetus. In B, fluid accumulation is particularly prominent in the soft tissues of the neck, and this condition has been termed cystic hygroma. Cystic hygromas are characteristically seen, but not limited to, constitutional chromosomal anomalies such as 45,X0 karyotypes. (Courtesy of Dr. Beverly Rogers, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) TABLE 10-5 -- Selected Causes of Hydrops Fetalis (in decreasing order of frequency) Cardiovascular Malformations Tachyarrhythmia High-output failure Chromosomal Turner syndrome Trisomy 21, trisomy 18 Thoracic Causes Cystic adenomatoid malformation Diaphragmatic hernia Fetal Anemia Homozygous alpha-thalassemia Parvovirus B19 Immune hydrops (Rh and ABO) Twin Gestation Twin-to-twin transfusion Infection (excluding parvovirus) Cytomegalovirus Syphilis Toxoplasmosis Major Malformations Tumors Metabolic disorders Note: The cause of fetal hydrops may be undetermined ("idiopathic") in up to 20% of cases. Data from Machin GA: Hydrops, cystic hygroma, hydrothorax, pericardial effusions, and fetal ascites, In Gilbert-Barness E (ed): Potter's Pathology of Fetus and Infant. St. Louis, Mosby-Year Book, 1997. 485 IMMUNE HYDROPS Immune hydrops is defined as a hemolytic disease in the newborn caused by blood-group incompatibility between mother and child. When the fetus inherits red cell antigenic determinants from the father that are foreign to the mother, a maternal immune reaction may occur, leading to hemolytic disease in the infant. Any of the numerous red cell antigenic systems may theoretically be involved, but the major antigens known to induce clinically significant immunologic disease are the ABO and certain of the Rh antigens. The incidence of immune hydrops in urban populations has declined remarkably, owing largely to the current methods of preventing Rh immunization in at-risk mothers. Successful prophylaxis of this disorder has resulted directly from an understanding of its pathogenesis. Etiology and Pathogenesis. The underlying basis of immune hydrops is the immunization of the mother by blood group antigens on fetal red cells and the free passage of antibodies from the mother through the placenta to the fetus ( Fig. 10-14 ). Fetal red cells may reach the maternal circulation during the last trimester of pregnancy, when the cytotrophoblast is no longer present as a barrier, or during childbirth itself. The mother thus becomes sensitized to the foreign antigen. Of the numerous antigens included in the Rh system, only the D antigen is the major cause of Rh incompatibility. Several Figure 10-14 Pathogenesis of immune hydrops fetalis (see text). Figure 10-15 Numerous islands of extramedullary hematopoiesis (small blue cells) are scattered among mature hepatocytes in this infant with nonimmune hydrops fetalis. Figure 10-16 Kernicterus. Severe hyperbilirubinemia in the neonatal period, for example, secondary to immune hemolysis, results in deposition of bilirubin pigment in the brain parenchyma. This occurs because the blood-brain barrier is less well developed in the neonatal period than it is in adulthood. Infants who survive develop long-term neurologic sequelae. TABLE 10-6 -- Abnormalities Suggesting Inborn Errors of Metabolism General Dysmorphic features Deafness Self-mutilation Abnormal hair Abnormal body or urine odor ("sweaty feet"; "mousy or musty"; "maple syrup") Hepatosplenomegaly; cardiomegaly Hydrops Neurologic Hypotonia or hypertonia Coma Persistent lethargy Seizures Gastrointestinal Poor feeding Recurrent vomiting Jaundice Eyes Cataract Cherry red macula Dislocated lens Glaucoma Muscle, Joints Myopathy Abnormal mobility Adapted from Barness LA and Gilbert-Barness E: Metabolic diseases, In Gilbert-Barness E (ed): Potter's Pathology of Fetus and Infant. St. Louis, Mosby-Year Book, 1997. Homozygotes with this autosomal recessive disorder classically have a severe deficiency of phenylalanine hydroxylase, leading to hyperphenylalaninemia and its pathologic consequences. Affected infants are normal at birth but within a few weeks develop a rising plasma phenylalanine level, which in some way impairs brain development. Usually by 6 months of life severe mental retardation becomes evident; fewer than 4% of untreated PKU children have intelligence quotient values greater than 50 or 60. About one third of these children are never able to walk, and two thirds cannot talk. Seizures, other neurologic abnormalities, decreased pigmentation of hair and skin, and eczema often accompany the mental retardation in untreated children. Hyperphenylalaninemia and the resultant mental retardation can be avoided by restriction of phenylalanine intake early in life. Hence, a number of screening procedures are routinely used for detection of PKU in the immediate postnatal period. Many clinically normal female PKU patients who are treated with dietary control early in life reach childbearing age. Most of them discontinue dietary treatment and have marked hyperphenylalaninemia. Between 75% and 90% of children born to such women are mentally retarded and 488 microcephalic, and 15% have congenital heart disease, even though the infants themselves are heterozygotes. This syndrome, termed maternal PKU, results from the teratogenic effects of 62 phenylalanine or its metabolites that cross the placenta and affect specific fetal organs during development.[ ] The presence and severity of the fetal anomalies directly correlate with the maternal phenylalanine level, so it is imperative that maternal dietary restriction of phenylalanine is initiated before conception and continues throughout the pregnancy. The biochemical abnormality in PKU is an inability to convert phenylalanine into tyrosine. In normal children, less than 50% of the dietary intake of phenylalanine is necessary for protein synthesis. The rest is irreversibly converted to tyrosine by a complex hepatic phenylalanine hydroxylase system ( Fig. 10-17 ), which, in addition to the enzyme phenylalanine hydroxylase, has two other components: the cofactor tetrahydrobiopterin (BH4 ) and the enzyme dihydropteridine reductase, which regenerates BH4 . Although neonatal hyperphenylalaninemia can be caused by deficiencies in any of these components, 98% to 99% of cases are attributable to abnormalities in phenylalanine hydroxylase. With a block in phenylalanine metabolism owing to lack of phenylalanine hydroxylase, minor shunt pathways come into play, yielding phenylpyruvic acid, phenyllactic acid, phenylacetic acid, and o-hydroxyphenylacetic acid, which are excreted in large amounts in the urine in PKU. Some of these abnormal metabolites are excreted in the sweat, and phenylacetic acid in particular imparts a strong musty or mousy odor to affected infants. It is believed that excess phenylalanine or its metabolites contribute to the brain damage in PKU. At the molecular level, several mutant alleles of the phenylalanine hydroxylase gene have been identified. Each mutation induces a particular alteration in the enzyme resulting in a corresponding quantitative effect on residual enzyme activity ranging from complete absence to 50% of normal values. The degree of hyperphenylalaninemia and clinical phenotype is inversely related to the amount of residual enzyme activity. Infants with mutations resulting in a lack of phenylalanine hydroxylase activity present with the classic features of PKU, while those with up to 6% residual activity present with milder disease. Moreover, some mutations result in only modest elevations of phenylalanine levels, and the affected children have no neurologic damage. This latter condition, referred to as benign hyperphenylalaninemia, or mild PKU, is important to recognize because the individuals may well test positive in screening 63] tests but do not develop the stigmata of classic PKU.[ Measurement of serum phenylalanine levels differentiates benign hyperphenylalaninemia and classic PKU. Although dietary restriction of phenylalanine is relatively successful in reducing or preventing the mental retardation associated with PKU, there are problems with long-term compliance (resulting in a decline in mental or behavioral status) Figure 10-17 The phenylalanine hydroxylase system. Figure 10-18 Pathways of galactose metabolism. Figure 10-19 Galactosemia. The liver shows extensive fatty change and a delicate fibrosis. (Courtesy of Dr. Wesley Tyson, The Children's Hospital, Denver, CO.) Figure 10-20 Top, Normal cystic fibrosis transmembrane conductance regulator (CFTR) structure and activation. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD), and a regulatory R domain. Agonists (e.g., acetylcholine) bind to epithelial cells and increase cAMP, which activates protein kinase A, the latter phosphorylating the CFTR at the R domain, resulting in opening of the chloride channel. Bottom, CFTR from gene to protein. The most common mutation in the CFTR gene results in defective protein folding in the Golgi/ER and degradation of CFTR before it reaches the cell surface. Other mutations affect synthesis of CFTR, nucleotide-binding and R domains, and membrane-spanning domains. Figure 10-21 Chloride channel defect in the sweat duct (top) causes increased chloride and sodium concentration in sweat. In the airway (bottom), cystic fibrosis patients have decreased chloride secretion and increased sodium and water reabsorption leading to dehydration of the mucus layer coating epithelial cells, defective mucociliary action, and mucus plugging of airways. CFTR, Cystic fibrosis transmembrane conductance regulator; EnaC, Epithelial sodium channel. Figure 10-22 The many clinical manifestations of mutations in the cystic fibrosis gene, from most severe to asymptomatic. (Redrawn from Wallis C: Diagnosing cystic fibrosis: blood, sweat, and tears. Arch Dis Child 76:85, 1997.) Figure 10-23 Lungs of a patient dying of cystic fibrosis. There is extensive mucus plugging and dilation of the tracheobronchial tree. The pulmonary parenchyma is consolidated by a combination of both secretions and pneumonia—the green color associated with Pseudomonas infections. (Courtesy of Dr. Eduardo Yunis, Children's Hospital of Pittsburgh, Pittsburgh, PA.) Figure 10-24 Mild to moderate cystic fibrosis changes in the pancreas. The ducts are dilated and plugged with eosinophilic mucin, and the parenchymal glands are atrophic and replaced by fibrous tissue. TABLE 10-7 -- Clinical Features and Diagnostic Criteria for Cystic Fibrosis 1. Chronic sinopulmonary disease manifested by •••a. Persistent colonization/infection with typical cystic fibrosis pathogens, including Staphylococcus aureus, nontypeable Hemophilus influenzae, mucoid and nonmucoid Pseudomonas aeruginosa, Burkholderia cepacia •••b. Chronic cough and sputum production •••c. Persistent chest radiograph abnormalities (e.g., bronchiectasis, atelectasis, infiltrates, hyperinflation) •••d. Airway obstruction manifested by wheezing and air trapping •••e. Nasal polyps; radiographic or computed tomographic abnormalities of paranasal sinuses •••f. Digital clubbing 2. Gastrointestinal and nutritional abnormalities, including •••a. Intestinal: meconium ileus, distal intestinal obstruction syndrome, rectal prolapse •••b. Pancreatic: pancreatic insufficiency, recurrent pancreatitis •••c. Hepatic: chronic hepatic disease manifested by clinical or histologic evidence of focal biliary cirrhosis, or multilobular cirrhosis •••d. Nutritional: failure to thrive (protein-calorie malnutrition), hypoproteinemia, edema, complications secondary to fat-soluble vitamin deficiency 3. Salt-loss syndromes: acute salt depletion, chronic metabolic acidosis 4. Male urogenital abnormalities resulting in obstructive azoospermia (congenital bilateral absence of vas deferens) Criteria for Diagnosis of Cystic Fibrosis One or more characteristic phenotypic features, ••OR a history of cystic fibrosis in a sibling, ••OR a positive newborn screening test result AND An increased sweat chloride concentration on two or more occasions ••OR identification of two cystic fibrosis mutations, ••OR demonstration of abnormal epithelial nasal ion transport Adapted with permission from Rosenstein BJ, Cutting GR: The diagnosis of cystic fibrosis: a consensus statement. J Pediatrics 132:589;1998. from onset at birth to onset years later, and from involvement of one organ system to involvement of many. Approximately 5% to 10% of the cases come to clinical attention at birth or soon after because of an attack of meconium ileus. Distal intestinal obstruction can also occur in older individuals, manifesting as recurrent episodes of right lower quadrant pain sometimes associated with a palpable mass in the right iliac fossa. Exocrine pancreatic insufficiency occurs in the majority (85–90%) of patients with cystic fibrosis and is associated with "severe" CFTR mutations on both alleles (e.g., ∆F508/∆F508), whereas 10% to 15% of patients with one "severe" and one "mild" CFTR mutation (∆F508/R117H) or two "mild" CFTR mutations retain enough pancreatic exocrine function so as not to 80 require enzyme supplementation (pancreas sufficient phenotype). [ ] Pancreatic insufficiency is associated with protein and fat malabsorption and increased fecal loss. Manifestations of malabsorption (e.g., large, foul stools, abdominal distention, and poor weight gain) appear during the first year of life. The faulty fat absorption may induce deficiency of the fat-soluble vitamins, resulting in manifestations of avitaminosis A, D, or K. Hypoproteinemia may be severe enough to cause generalized edema. Persistent diarrhea may result in rectal prolapse in up to 10% of children with cystic fibrosis. The pancreas sufficient phenotype is usually not associated with other gastrointestinal complications, and in general, these individuals demonstrate excellent growth and development. The diagnosis of an underlying CFTR mutation in individuals with pancreas sufficient cystic fibrosis is suspected because of abnormal or borderline sweat chloride levels, a positive family history, or because of concomitant infertility in a male patient. "Idiopathic" chronic pancreatitis occurs in a subset of patients with pancreas sufficient cystic fibrosis and is associated with recurrent abdominal pain with 495 85 life-threatening complications.[ ] These patients have other features of cystic fibrosis, such as pulmonary disease. By contrast, "idiopathic" chronic pancreatitis can also occur as an isolated late-onset finding in the absence of other stigmata of cystic fibrosis ( Chapter 19 ); bi-allelic CFTR mutations (usually one "mild", one "severe") are demonstrable in the majority of these individuals, qualifying their inclusion as nonclassic or atypical cystic fibrosis. Endocrine pancreatic insufficiency (i.e., diabetes) is uncommon in cystic fibrosis, and usually accompanied by substantial destruction of pancreatic parenchyma. Cardiorespiratory complications, such as persistent lung infections, obstructive pulmonary disease, and cor pulmonale, are the single most common cause of death (∼80%) in patients in the 86 United States. By age 18, 80% of patients with classic cystic fibrosis harbor P. aeruginosa, and 3.5% harbor Burkholderia cepacia. [ ] With the indiscriminate use of antibiotic prophylaxis against Staphylococcus, there has been an unfortunate resurgence of resistant strains of Pseudomonas in many patients. Individuals who carry a "severe" CFTR mutation on 81 one allele and a "mild" CFTR mutation on the other allele may exhibit late-onset mild pulmonary disease, another example of nonclassic or atypical cystic fibrosis.[ ] Patients with mild pulmonary disease usually have mild or no pancreatic disease. Idiopathic bronchiectasis, a poorly defined entity in adults where no discernible cause for the bronchiectasis can be found, has been linked to CFTR mutations in a subset of cases. Recurrent sinonasal polyps can occur in up to 25% of patients with cystic fibrosis; hence, children who present with this finding should be tested for abnormalities of sweat chloride. Significant liver disease occurs late in the natural history of cystic fibrosis and used to be foreshadowed by pulmonary and pancreatic involvement; however, with increasing life expectancies, liver disease has also received increasing attention. In fact, after cardiopulmonary and transplantation-related complications, liver disease is the most common cause of death in cystic fibrosis. Most studies suggest that symptomatic or biochemical liver disease in cystic fibrosis has its onset at or around puberty, with a prevalence of approximately 13% to 17%. [87] However, asymptomatic hepatomegaly may be present in up to a third of the individuals. Obstruction of the common bile duct may occur due to stones or sludge; it presents with abdominal pain and the acute onset of jaundice. As previously noted, diffuse biliary cirrhosis may develop in up to 5% of individuals with cystic fibrosis. Approximately 95% of males with cystic fibrosis are infertile, as a result of obstructive azoospermia. As mentioned earlier, this is most commonly due to bilateral absence of vas deferens (also called CBAVD). CBAVD can occur as a consequence of several conditions, but bi-allelic CFTR mutations are the most common cause (present in 50% to 75% of cases).[ 88] [89] In most cases, the diagnosis of cystic fibrosis is based on persistently elevated sweat electrolyte concentrations (often the mother makes the diagnosis because her infant tastes salty), characteristic clinical findings (sinopulmonary disease and gastrointestinal manifestations), or a family history. A minority of patients with cystic fibrosis, especially those with at least one "mild" CFTR mutation, may have a normal or near-normal sweat test (<60 mM/L). Measurement of nasal transepithelial potential difference in vivo can be a useful adjunct under these circumstances; individuals with cystic fibrosis demonstrate a significantly more negative baseline nasal potential difference than controls. Sequencing the CFTR gene is, of course, the "gold standard" for diagnosis of cystic fibrosis. Therefore, in patients with suggestive clinical findings or family history (or both), genetic analysis may be warranted. It is important to inform the molecular laboratory whether the individual has classic cystic fibrosis or conforms to one of the nonclassic or atypical variants (CBAVD, late-onset pulmonary disease, or idiopathic chronic pancreatitis), so the appropriate "mild" CFTR mutations are also analyzed. A recent study has demonstrated that a subset of patients with nonclassic or atypical cystic fibrosis may not reveal CFTR mutations in one or both alleles.[ partial phenotype resembling cystic fibrosis. 90] This indicates that other genetic loci (perhaps those that encode proteins interacting with CFTR) may also produce a Advances in management of cystic fibrosis include both improved control of infections and bilateral lung (or lobar), heart-lung, liver, pancreas, or liver-pancreas transplantation. Children and adolescents undergoing bilateral lung transplantation have overall survival rates around 70%. These improvements in management mean that more patients are now surviving to adulthood; the median life expectancy is close to 30 years and continues to increase. In principle, cystic fibrosis, like other single gene disorders, should be amenable to gene therapy. In vitro, it has been possible to correct the chloride defect in epithelial cells of cystic fibrosis patients by both viral and nonviral vector-based transfer of the CFTR gene; even a single copy of the wild-type CFTR gene is able to revert the cystic fibrosis phenotype. Clinical trials with gene therapy in humans are still in their early stages but provide a source of hope for millions of cystic fibrosis patients worldwide. Sudden Infant Death Syndrome (SIDS) SIDS is a disease of unknown cause. The National Institute of Child Health and Human Development defines SIDS as "the sudden death of an infant under 1 year of age which remains unexplained after a thorough case investigation, including performance of a complete autopsy, examination of the death scene, and review of the clinical history."[ that is not stressed in the definition is that the infant usually dies while asleep, hence the pseudonyms of crib death or cot death. 91] An aspect of SIDS Epidemiology. As infantile deaths owing to nutritional problems and microbiologic infections have come under control in countries with higher standards of living, SIDS has assumed greater importance in many countries, including the United States. SIDS is the leading cause of death between age 1 month and 1 year in this country and the third leading cause of death overall in infancy, after congenital anomalies and diseases of prematurity and low birth weight. Due largely to nationwide SIDS awareness campaigns by organizations such as the American Academy of Pediatrics, there has been a significant drop in SIDS-related mortality in the past decade, from over 5000 annual deaths in 1990 to approximately 2600 deaths in 1999. Worldwide, in countries where unexpected infant deaths are diagnosed as SIDS only after postmortem examination, the death rates from SIDS (20 to 100/100,000 live births) are comparable to death rates in the United States (77/100,000 live births). 496 Approximately 90% of all SIDS deaths occur during the first 6 months of life, most between ages 2 and 4 months. This narrow window of peak susceptibility is a unique characteristic that is independent of other risk factors (to be described) and the geographic locale. Most infants who die of SIDS, die at home, usually during the night after a period of sleep. Only rarely is the catastrophic event observed, but even when seen, it is reported that the apparently healthy infant suddenly turns blue, stops breathing, and becomes limp without emitting a cry or struggling. Most infants have had minor manifestations of an upper respiratory infection preceding the fatal event. The term apparent life-threatening event (ALTE) has been applied to those infants who could be resuscitated after such an episode. [ 92] Infants with ALTE are often siblings of SIDS victims and harbor a range of physiologic abnormalities such as frequent or prolonged apnea, diminished chemoreceptor sensitivity to hypercarbia and hypoxia, and impaired control of heart, respiratory rate, and vagal tone.[ to SIDS. 92] Some of these infants later succumb Morphology. At autopsy, a variety of findings have been reported. They are usually subtle and of uncertain significance and are not present in all cases. Multiple petechiae are the most common finding in the typical SIDS autopsy (∼80% of cases); these are usually present on the thymus, visceral and parietal pleura, and epicardium. Grossly, the lungs are usually congested, and vascular engorgement with or without pulmonary edema is demonstrable microscopically in the majority of cases. These changes possibly represent agonal events, since they are found with comparable frequencies in explained sudden deaths in infancy. Within the upper respiratory system (larynx and trachea), there may be some histologic evidence of recent infection (correlating with the clinical symptoms), although the changes are not sufficiently severe to account for death and should not detract from the diagnosis of SIDS. The central nervous system demonstrates astrogliosis of the brain stem and cerebellum. Sophisticated morphometric studies have revealed quantitative brainstem abnormalities such as hypoplasia of the arcuate nucleus or a subtle decrease in brain stem neuronal populations in several cases;[ 93] [94] these observations are not uniform, however, and not amenable to most "routine" autopsy procedures. Nonspecific findings include frequent persistence of hepatic extramedullary hematopoiesis and periadrenal brown fat; it is tempting to speculate that these latter findings relate to chronic hypoxemia, retardation of normal development, and chronic stress. Thus, autopsy usually fails to provide a clear cause of death, and this may well be related to the etiologic heterogeneity of SIDS. The importance of a postmortem examination rests in identifying other causes of sudden unexpected death in infancy, such as unsuspected infection, congenital anomaly, or a genetic disorder ( Table 10-8 ), the presence of any of which would exclude a diagnosis of SIDS, and in ruling out the unfortunate possibility of traumatic child abuse. Pathogenesis. The circumstances surrounding SIDS have been explored in great detail, and it is generally accepted that TABLE 10-8 -- Risk Factors and Postmortem Findings Associated with Sudden Infant Death Syndrome Parental Young maternal age (age <20 years) Maternal smoking during pregnancy Drug abuse in either parent, specifically paternal marijuana and maternal opiate, cocaine use Short intergestational intervals Late or no prenatal care Low socioeconomic group African American and American Indian ethnicity (? socioeconomic factors) Infant Brain stem abnormalities, associated defective arousal, and cardiorespiratory control Prematurity and/or low birth weight Male sex Product of a multiple birth SIDS in a prior sibling Antecedent respiratory infections ? Gastroesophageal reflux Environment Prone sleep position Sleeping on a soft surface Hyperthermia Postnatal passive smoking Postmortem Abnormalities Detected in Cases of Sudden Unexpected Infant Death * Infections • Viral myocarditis • Bronchopneumonia Unsuspected congenital anomaly • Congenital aortic stenosis • Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) Traumatic child abuse • Intentional suffocation (filicide) Genetic and metabolic defects • Long QT syndrome (SCN5A and KCNQ1 mutations) • Fatty acid oxidation disorders (MCAD, LCHAD, SCHAD mutations) • Histiocytoid cardiomyopathy (MTCYB mutations) • Abnormal inflammatory responsiveness (partial deletions in C4a and C4b) *SIDS is not the only cause of sudden unexpected death in infancy but rather is a diagnosis of exclusion. Therefore, performance of an autopsy may often reveal findings that would explain the cause of sudden unexpected death. These cases should not, strictly speaking, be labeled as "SIDS." SCN5A, sodium channel, voltage-gated, type V, alpha polypeptide; KCNQ1, potassium voltage-gated channel, KQT-like subfamily, member 1; MCAD, medium-chain acyl coenzyme A dehydrogenase; LCHAD, long-chain 3-hydroxyacyl coenzyme A dehydrogenase; SCHAD, short-chain 3-hydroxyacyl coenzyme A dehydrogenase; MTCYB, mitochondrial cytochrome b; C4, complement component 4. 497 it is a multifactorial condition, with a variable mixture of contributing factors. A "triple risk" model of SIDS has been proposed, which postulates the intersection of three overlapping 95 factors: (1) a vulnerable infant, (2) a critical developmental period in homeostatic control, and (3) an exogenous stressor(s). [ ] According to this model, several factors make the infant vulnerable to sudden death during the critical developmental period (i.e., age 1 month to 1 year). These vulnerability factors may be attributable to the parents or the infant, while the exogenous stressor(s) is attributable to the environment ( Table 10-8 ). While numerous factors have been proposed to account for a vulnerable infant, the most compelling hypothesis is that SIDS reflects a delayed development of arousal and 96 cardiorespiratory control.[ ] Regions of the brain stem, particularly the arcuate nucleus, located in the ventral medullary surface, play a critical role in the body's "arousal" response to noxious stimuli such as hypercarbia, hypoxia, and thermal stress encountered during sleep. In addition, these areas regulate breathing, heart rate, and body temperature. In certain infants, for yet unexplained reasons, there may be a maldevelopment or delay in maturation of this region, compromising the arousal response to noxious stimuli. This physiologic impairment is compounded by other factors, such as sleeping position or infection (see below). Support of this hypothesis comes from postmortem studies in SIDS victims demonstrating both quantitative abnormalities (e.g., arcuate hypoplasia and decrease in neuronal density) as well as qualitative abnormalities (e.g., reduced serotonergic and muscarinic receptor binding) in the 93 97 98 brain stem.[ ] [ ] [ ] Whether these changes are primary or merely the manifestation of a more "upstream" deficit remains to be elucidated. Recently, some candidate genes have been identified from experimental animal models, which may provide a genetic basis to abnormal neural regulation in the brainstem. For example, Krox20, a homeobox gene, appears to be 99 required for hindbrain segmentation and myelination. Mouse models lacking Krox20 function exhibit abnormally slow respiratory rhythm and prolonged apnea.[ ] Similarly, brain-derived neurotrophic factor (BDNF) is required for normal development of the central respiratory rhythm, including the stabilization of central respiratory output that occurs after birth. Loss of one or both BDNF alleles results in an approximately 50% depression of central respiratory frequency compared with wild-type controls, while hypoxic ventilatory drive is deficient or absent. [100] Whether knowledge gleamed from these animal models of central respiratory dysfunction will be applicable to humans remains to be seen. Epidemiologic studies of infant deaths have found additional risk factors for SIDS ( Table 10-8 ). Infants who are born before term or who are low birth weight are at increased risk, and risk increases with decreasing gestational age or birth weight. Male sex is associated with a slightly greater incidence of SIDS. SIDS in a prior sibling is associated with a fivefold relative risk of recurrence, underscoring the importance of a genetic and/or shared environmental predisposition; traumatic child abuse needs to be carefully excluded under these circumstances. Most SIDS babies have an immediate prior history of a mild respiratory tract infection, but no single causative organism has been isolated. These infections may predispose an already vulnerable infant to even greater impairment of cardiorespiratory control and delayed arousal. In this context, laryngeal chemoreceptors have emerged as a putative "missing link" between 101 upper respiratory tract infections, the prone position (see below), and SIDS. When stimulated, these laryngeal chemoreceptors elicit an apneic and bradycardic reflex.[ ] Stimulation of the chemoreceptors is augmented by respiratory tract infections, which increase the volume of secretions, and by the prone position, which impairs swallowing and clearing of the airways even in healthy infants. In a previously vulnerable infant with impaired arousal, the apneic and bradycardic reflex may prove fatal. Maternal smoking during pregnancy has consistently emerged as a risk factor in epidemiologic studies of SIDS, with children exposed to in utero nicotine having more than double the risk 102 of SIDS compared to children born to nonsmokers. [ ] Young maternal age, frequent childbirths, and inadequate prenatal care are all risk factors associated with increased incidence of SIDS in the offspring. African Americans and American Indians have significantly higher rates of SIDS deaths than Caucasians. It is not obvious whether these ethnic trends represent the effects of genetic make up or the effects of lower socioeconomic status, which by itself is a risk factor for SIDS. 103 Among the potential environmental factors, prone sleeping position, sleeping on soft surfaces, and thermal stress are possibly the most important modifiable risk factors for SIDS.[ ] The prone position predisposes an infant to one or more recognized noxious stimuli (hypoxia, hypercarbia, and thermal stress) during sleep. In addition, the prone position is also associated with decreased arousal responsiveness compared to the supine position. Results of studies from Europe, Australia, New Zealand, and the United States showed clearly increased risk for SIDS in infants who sleep in a prone position, prompting the American Academy of Pediatrics to recommend placing healthy infants on their back when laying them down to sleep. This "Back To Sleep" campaign has resulted in substantial decreases in SIDS-related deaths since its inception in 1994.[ 104] It should be noted that SIDS is not the only cause of sudden unexpected deaths in infancy. In fact, SIDS is a diagnosis of exclusion, requiring careful examination of the death scene and a complete postmortem examination. The latter can reveal an unsuspected cause of sudden death in up to 20% or more of "SIDS" babies ( Table 10-8 ). Infections (e.g., viral myocarditis or bronchopneumonia) are the most common causes of sudden "unexpected" death, followed by an unsuspected congenital anomaly. In part due to advancements in molecular diagnostics and knowledge of the human genome, several genetic causes of sudden "unexpected" infant death have emerged. For example, fatty acid oxidation disorders, characterized by defects in mitochondrial fatty acid oxidative enzymes, may be responsible for up to 5% of sudden death in infancy; of these, a deficiency in medium-chain acyl-coenzyme A dehydrogenase is the most common.[ 105] Retrospective analyses of SIDS cases have also revealed mutations of cardiac sodium and potassium channels, which result in a form of cardiac arrhythmia characterized by prolonged QT intervals; these account for no more than 1% of SIDS deaths.[ 106] 498 Tumors and Tumor-Like Lesions of Infancy and Childhood Other newly emerging genetic causes of explained sudden death are listed in Table 10-8 . Only 2% of all malignant tumors occur in infancy and childhood; nonetheless, cancer (including leukemia) is a leading cause of death from disease in the United States in children over age 4 and up to age 14. Neoplastic disease accounts for approximately 9% of all deaths in this cohort; only accidents cause significantly more deaths. Benign tumors are even more common than cancers. Most benign tumors are of little concern, but on occasion they cause serious disease by virtue of their location or rapid increase in size. It is sometimes difficult to segregate, on morphologic grounds, true tumors or neoplasms from tumor-like lesions in the infant and child. In this context, two special categories of tumor-like lesions should be distinguished from true tumors. The term heterotopia (or choristoma) is applied to microscopically normal cells or tissues that are present in abnormal locations. Examples of heterotopias include a rest of pancreatic tissue found in the wall of the stomach or small intestine or a small mass of adrenal cells found in the kidney, lungs, ovaries, or elsewhere. The heterotopic rests are usually of little significance, but they can be confused clinically with neoplasms. Rarely, they are sites of origin of true neoplasms, producing the paradox of an adrenal carcinoma arising in the ovary. The term hamartoma refers to an excessive but focal overgrowth of cells and tissues native to the organ in which it occurs. Although the cellular elements are mature and identical to those found in the remainder of the organ, they do not reproduce the normal architecture of the surrounding tissue. Hamartomas can be thought of as the linkage between malformations and neoplasms—the line of demarcation between a hamartoma and a benign neoplasm is frequently tenuous and is variously interpreted. Hemangiomas, lymphangiomas, rhabdomyomas of the heart, adenomas of the liver, and developmental cysts within the kidneys, lungs, or pancreas are interpreted by some as hamartomas and by others as true neoplasms. The frequency of these lesions in infancy and childhood and their clinical behavior give credence to the Figure 10-25 Congenital capillary hemangioma at birth (A) and at age 2 years (B) after spontaneous regression. (Courtesy of Dr. Eduardo Yunis, Children's Hospital of Pittsburgh, Pittsburgh, PA.) Figure 10-26 Sacrococcygeal teratoma. Note the size of the lesion compared with that of the infant. TABLE 10-9 -- Common Malignant Neoplasms of Infancy and Childhood 0 to 4 Years 5 to 9 Years Leukemia Leukemia Retinoblastoma Retinoblastoma Neuroblastoma Neuroblastoma 10 to 14 Years Wilms tumor Hepatoblastoma Hepatocarcinoma Hepatocarcinoma Soft tissue sarcoma (especially rhabdomyosarcoma) Soft tissue sarcoma Soft tissue sarcoma Teratomas Central nervous system tumors Central nervous system tumors Ewing sarcoma Lymphoma Osteogenic sarcoma Thyroid carcinoma Hodgkin disease Histologically, many of the malignant pediatric neoplasms are unique. In general, they tend to have a more primitive (embryonal) rather than pleomorphic-anaplastic microscopic appearance, are often characterized by sheets of cells with small, round nuclei, and frequently exhibit features of organogenesis specific to the site of tumor origin. Because of this TABLE 10-10 -- Genetic and Other Useful Markers of Small Round Cell Tumors of Childhood Tumor Type Neuroblastoma Genetic Markers * 17q gain, 1p deletion N-myc amplification Other Diagnostically Useful Features * Clinical elevation in level of urinary catecholamines * DNA hyperdiploidy, near triploidy Neurosecretory granules by electron microscopy † Neuron-specific enolase expression ‡ MIC2 (CD99) gene expression t(11;22), t(21;22), t(7;22) EWS-FLI1 or EWS-ERG fusion transcript Rhabdomyosarcoma t(2;13), ‡* Myogenin and Myo D1 expression (all subtypes) t(1;13)—alveolar rhabdomyosarcoma (ARMS) 11p15.5 deletion—embryonal rhabdomyosarcoma (ERMS) Alternating thick and thin filaments by electron microscopy PAX3-FKHR and PAX7-FKHR fusion transcript (ARMS) Burkitt lymphoma ‡ B-cell phenotype expressing CD19, CD20, CD10, IgM t(8;14), t(2;8), t (8;22) Epstein-Barr virus latent infection (endemic cases) Lymphoblastic lymphoma/ acute lymphoblastic leukemia † Hyperdiploidy (>50), Hypodiploidy(<46) * Terminal deoxynucleotidyl transferase (TdT)+ ‡ † B-lineage: various translocations, including t(12;21) (TEL-AML1), , t * Various B- and T-lineage antigens * (9;22) (BCR-ABL, Philadelphia chromosome), t(4;11) (AF4-MLL) , t (1;19) (PBX-E2A) T-lineage: 1p32 abnormalities (TAL1 gene) Wilms tumor 11p13 (WT1) deletion/mutation 11p15.5 abnormalities of imprinting (e.g., IGF2, H19, p57KIP2 ) * * 16q, 1p, 7p deletion Retinoblastoma 13q14 (RB) deletion/mutation Retinal S antigen expression Medulloblastoma 17p deletion Evidence of neuronal differentiation (synaptophysin expression) or glial differentiation (glial fibrillary acid protein [GFAP] expression) Isochromosome 17q PNET, peripheral neuroectodermal tumor. *Generally associated with a poorer prognosis. †Generally associated with a better prognosis. ‡Most common translocation. latter characteristic, these tumors are frequently designated by the suffix -blastoma, for example, nephroblastoma (Wilms tumor), hepatoblastoma, and neuroblastoma. Owing to their primitive histologic appearance, many childhood tumors have been collectively referred to as small round blue cell tumors. The differential diagnosis of such tumors includes neuroblastoma, Wilms tumor, lymphoma, rhabdomyosarcoma, and Ewing sarcoma/primitive neuroectodermal tumor. Rendering a definitive diagnosis is usually possible on histologic examination alone, or in combination with chromosome analysis, immunoperoxidase stains, and electron microscopy. The diagnostic features associated with the more common childhood neoplasms are summarized in Table 10-10 . Two of these tumors are particularly illustrative and are discussed here: the neuroblastic tumors, specifically neuroblastoma, and Wilms tumor. The remaining tumors are discussed in their respective organ-specific chapters. The Neuroblastic Tumors The term "neuroblastic tumor" includes tumors of the sympathetic ganglia and adrenal medulla that are derived from primordial neural crest cells populating these sites. As a family, neuroblastic tumors demonstrate certain characteristic features such as spontaneous or therapy-induced differentiation of primitive neuroblasts into mature elements, spontaneous tumor regression, and a wide range of clinical behavior and prognosis, which often mirror the extent of histologic differentiation. Neuroblastoma is the most important member of this family. It is the second most common solid malignancy of childhood 501 108 after brain tumors, accounting for 7% to 10% of all pediatric neoplasms, and as many as 50% of malignancies diagnosed in infancy.[ ] Approximately 650 new cases are diagnosed in the United States each year, accounting for an incidence of approximately 9.5 cases per million children. The median age at diagnosis is 22 months; a little more than a third of the cases are diagnosed in infancy. There is a higher incidence of neuroblastoma in Caucasian as compared to African American populations, and males are at a marginally greater risk than females. Alone, it accounts for at least 15% of all childhood cancer deaths, although the 5-year survival rate has improved from 25% in the early 1960s to almost 55% in the mid-1990s. As will be evident later, age and stage have a remarkable effect on prognosis, and, in general, infants tend to have a significantly better prognosis than older individuals. Most occur sporadically, but a few are familial with autosomal dominant transmission, and in such cases the neoplasms may involve both of the adrenals or multiple primary autonomic sites. Morophology. In childhood, about 40% of neuroblastomas arise in the adrenal medulla. The remainder occur anywhere along the sympathetic chain, with the most common locations being the paravertebral region of the abdomen (25%) and posterior mediastinum (15%). Tumors may arise in numerous other sites, including the pelvis and neck and within the brain (cerebral neuroblastomas). Macroscopically, neuroblastomas range in size from minute nodules (the in situ lesions) to large masses more than 1 kg in weight ( Fig. 10-27 ). In situ neuroblastomas are reported to be 40 times more frequent than overt tumors. The great majority of these silent lesions spontaneously regress, leaving only a focus of fibrosis or calcification in the adult. Some neuroblastomas Figure 10-27 Adrenal neuroblastoma in a 6-month-old child. The hemorrhagic, partially encapsulated tumor has displaced the opened left kidney and is impinging on the aorta and left renal artery. (Courtesy of Dr. Arthur Weinberg, University of Texas Southwestern Medical School, Dallas, TX.) Figure 10-28 Adrenal neuroblastoma. This tumor is composed of small cells embedded in a finely fibrillar matrix. Figure 10-29 Ganglioneuromas, arising from spontaneous or therapy-induced maturation of neuroblastomas, are characterized by clusters of large cells with vesicular nuclei and abundant eosinophilic cytoplasm, representing neoplastic ganglion cells (arrow). Spindle-shaped Schwann cells are present in the background stroma. Stage 2A: Localized tumor with incomplete gross resection. Representative ipsilateral nonadherent lymph nodes negative for tumor microscopically. Stage 2B: Localized tumor with or without complete gross excision, ipsilateral nonadherent lymph nodes positive for tumor. Enlarged contralateral lymph nodes, which are negative for tumor microscopically. Stage 3: Unresectable unilateral tumor infiltrating across the midline with or without regional lymph node involvement; or localized unilateral tumor with contralateral regional lymph node involvement. Stage 4: Any primary tumor with dissemination to distant lymph nodes, bone, bone marrow, liver, skin, and/or other organs (except as defined for stage 4S). Stage 4S ("S" = special): Localized primary tumor (as defined for Stages 1, 2A, or 2B) with dissemination limited to skin, liver, and/or bone marrow; Stage 4S is limited to infants <1 yr. Unfortunately, most (60% to 80%) children present with Stage 3 or 4 tumors, and only 20% to 40% present with Stage 1, 2A, 2B, or 4S neuroblastomas. The staging system is of paramount importance in determining prognosis. Clinical Course and Prognostic Features. In young children, under age 2 years, neuroblastomas generally present with large abdominal masses, fever, and possibly weight loss. In older children, they may not come to attention until metastases produce manifestations, such as bone pain, respiratory symptoms, or gastrointestinal complaints. Neuroblastomas may metastasize widely through the hematogenous and lymphatic systems, particularly to liver, lungs, and bones, in addition to the bone marrow. Proptosis and ecchymosis may also be present because the periorbital region is a common metastatic site. Bladder and bowel dysfunction may be caused by paraspinal neuroblastomas that impinge on nerves. In neonates, disseminated neuroblastomas may present with multiple cutaneous metastases with deep blue discoloration to the skin (earning the rather unfortunate designation of "blueberry muffin baby"). About 90% of neuroblastomas, regardless of location, produce catecholamines (similar to the catecholamines associated with pheochromocytomas), which are an important diagnostic feature (i.e., elevated blood levels of catecholamines and elevated urine levels of metabolites, vanillylmandelic acid [VMA], and homovanillic acid [HVA]). Despite the elaboration of catecholamines, hypertension is much less frequent with these neoplasms than with pheochromocytomas ( Chapter 24 ). Ganglioneuromas, unlike their malignant counterparts, tend to produce either asymptomatic mass lesions or symptoms related to compression. The course of neuroblastomas is extremely variable. Several clinical, histopathologic, molecular, and biochemical factors have been identified in neuroblastomas that have a bearing on prognosis (see Table 10-11 ): Age and stage are the most important determinants of outcome. Infants younger than age 1 year have an excellent prognosis regardless of the stage of the neoplasm. Most often in this age 114 group, the neoplasms are Stage 1, 2A, or 2B, and therapy yields a greater than 90% 5-year survival.[ ] At this early age, even when metastases are present, in about half the spread is limited to the liver, bone marrow, and skin (stage 4S), and such infants have at least an 80% 5-year survival with only minimal therapy. In fact, with Stage 4S disease, it is not uncommon 115 for the primary or metastatic tumors to undergo spontaneous regression. [ ] The biologic basis of this welcome behavior is not clear. Even when the dissemination is more widespread in the first year of life or the tumor is accompanied by unfavorable biologic characteristics such as N-myc amplification (see below), the survival is greater than 50%. Children between ages 1 and 5 years have an intermediate prognosis for low-stage tumors that have otherwise favorable 503 TABLE 10-11 -- Prognostic Factors in Neuroblastomas Variable Favorable * Unfavorable Stage 1, 2A, 2B, 4S Stage 3, 4 ≤ 1 year >1 year Present Absent Low High ≤200/5000 cells >200/5000 cells Present Absent Hyperdiploid or near-triploid Diploid, near-diploid, or neartetraploid Not amplified Amplified Chromosome 17q Gain Absent Present Chromosome 1p Loss Absent Present Trk-A Expression Present Absent Telomerase Expression Low or absent Highly expressed MRP Expression Absent Present CD44 Expression Present Absent Stage Age * Histology * ••Evidence of schwannian stroma and gangliocytic differentiation ••Mitotic rate b ••Mitosis-karyorrhexis index c ••Intratumoral calcification DNA ploidy N-myc * * Serum Biochemical Markers a ••Ferritin Normal Elevated ••Lactate Dehydrogenase ≤1500 U/mL > 1500 U/mL Trk-A, tyrosine kinase receptor A; MRP, multidrug resistance-associated protein. *Corresponds to the most commonly used parameters in clinical practice for assessment of prognosis and risk stratification. It is not only the presence but also the amount of schwannian stroma that confers the designation of a favorable histology. At least 50% or more schwannian stroma is required before a neoplasm can be classified as ganglioneuroblastoma or ganglioneuroma. b Mitotic rate is classified as low (≤10 mitoses/10 high power fields) or high (>10 mitoses/10 high power fields). c Mitotic karyorrhexis index (MKI) is defined as the number of mitotic or karyorrhectic cells per 5000 tumor cells in random foci. a biologic characteristics (see below), while those with advanced stage disease have <20% 5-year survival, irrespective of other prognostic variables. In contrast, children older than age 5 years usually have extremely poor outcomes irrespective of stage. 112 Morphology is an independent prognostic variable in neuroblastic tumors.[ ] An age-linked morphologic classification of neuroblastic tumors has recently been proposed that divides them into favorable and unfavorable histologic subtypes. The specific morphologic features that bear in prognosis are listed in Table 10-11 . Ploidy of the tumor cells correlates with outcome. In general, hyperdiploidy and near-triploidy have a correlation with young age, low stage, and a good prognosis, whereas diploidy, neardiploidy, and near-tetraploidy are associated with an unfavorable outcome irrespective of age. For example, in infants and children younger than age 2 years who have advanced disease, the presence of hyperdiploidy or near-triploidy correlates with response to chemotherapy and long-term disease-free survival, while corresponding diploid tumors have a significantly worse prognosis (the beneficial prognostic effects of ploidy tend to be negated in older children with advanced disease). 116 Amplification of the N-myc oncogene in neuroblastomas is a molecular event that has possibly the most profound impact on prognosis. [ ] N-myc is located on the distal short arm of chromosome 2 (2p23-24). Amplification of N-myc does not karyotypically manifest at the resident 2p23-24 site, but rather as extrachromosomal double minute chromatin bodies or homogeneously staining regions on other chromosomes ( Fig. 10-30 ). N-myc amplification is present in about 25% to 30% of primary tumors, most in advanced-stage disease. Up to 300 copies of N-myc have been observed in some tumors; the greater the number of copies, the worse the prognosis. N-myc amplification is currently the most important genetic abnormality used in risk stratification of neuroblastic tumors (see below). 117 Partial gain of the distal long arm of chromosome 17 is the most common karyotypic abnormality in neuroblastomas, present in up to 50% of tumors.[ ] The mechanism of 17q gain is via an unbalanced translocation, where a portion of 17q is translocated to a partner chromosome (most commonly the distal short arm of chromosome 1, or the distal long arm of chromosome 11). Partial gain of 17q demonstrates significant association with adverse outcome in neuroblastomas, independent of other prognostic variables. 118 Deletion of the distal short arm of chromosome 1 in the region of band p36 has been demonstrated in 25% to 35% of primary tumors.[ ] In addition, constitutional deletions of 1p36 have been demonstrated in a subset of patients with neuroblastomas. The loss of genetic material implies that one or more putative tumor suppressor genes in this region may be important in the pathogenesis of neuroblastomas, but their identity remains elusive. At least two distinct loci of deletions on 1p36 have been identified. The first, more distal region appears to demonstrate preferential loss of the maternal allele in tumors, 504 Figure 10-30 Fluorescence in situ hybridization using a fluorescein-labeled cosmid probe for N-myc on a tissue section. Note the neuroblastoma cells on the upper half of the photo with large areas of staining (yellow-green); this corresponds to amplified N-myc in the form of homogeneously staining regions. Renal tubular epithelial cells in the lower half of the photograph show no nuclear staining and background (green) cytoplasmic staining. (Courtesy of Dr. Timothy Triche, Children's Hospital, Los Angeles, CA.) Figure 10-31 Wilms tumor in the lower pole of the kidney with the characteristic tan-to-gray color and well-circumscribed margins. Figure 10-32 Triphasic histology of Wilms' tumor: the stromal component is comprised of spindle-shaped cells in the less cellular area on the left; the immature tubule in the center is an example of the epithelial component and the tightly packed blue cells, of the blastemal elements. (Courtesy of Dr. Charles Timmons, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) 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Curr Opin Pediatr 14:247, 2002. 103. Changing concepts of sudden infant death syndrome: implications for infant sleeping environment and sleep position. American Academy of Pediatrics. Task Force on Infant Sleep Position and Sudden Infant Death Syndrome. Pediatrics 105 (3 Pt 1):650, 2000. 104. Moon RY, Biliter WM: Infant sleep position policies in licensed child care centers after back to sleep campaign. Pediatrics 106:576, 2000. 105. Treem WR: New developments in the pathophysiology, clinical spectrum, and diagnosis of disorders of fatty acid oxidation. Curr Opin Pediatr 12:463, 2000. 106. Valdes-Dapena M, Gilbert-Barness E: Cardiovascular causes for sudden infant death. Pediatr Pathol Mol Med 21:195, 2002. 107. Bourgeois JM, et al: Molecular detection of the ETV6-NTRK3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors. Am J Surg Pathol 24:937, 2000. 108. Kelly DR, Joshi VV: Neuroblastoma and related tumors. In Parham D (ed): Pediatric Neoplasia Morphology and Biology. Philadelphia, Lippincott-Raven, 1996, pp. 105–152. 109. Shimada H, et al: Terminology and morphologic criteria of neuroblastic tumors: recommendations by the International Neuroblastoma Pathology Committee. Cancer 86:349, 1999. 110. Ambros IM, et al: Role of ploidy, chromosome 1p, and Schwann cells in the maturation of neuroblastoma. N Engl J Med 334:1505, 1996. 111. Mora J, et al: Neuroblastic and Schwannian stromal cells of are derived from a tumoral progenitor cell. Cancer Res 61:6892, 2001. 112. Shimada H, et al: The International Neuroblastoma Pathology Classification (the Shimada system). Cancer 86:364, 1999. 113. Smith EI, et al: A surgical perspective on the current staging in neuroblastoma—the International Neuroblastoma Staging System proposal. J Pediatr Surg 24:386, 1989. 114. Brodeur GM, Castleberry RP: Neuroblastoma. In Pizzo PA, Poplack DG (eds): Principles and Practice of Pediatric Oncology. 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Hiyama E, et al: Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med 1:249, 1995. 122. Schilling FH, et al: Neuroblastoma screening at one year of age. N Engl J Med 346:1047, 2002. 123. Woods WG, et al: Screening of infants and mortality due to neuroblastoma. N Engl J Med 346:1041, 2002. 124. Blute ML, et al: Bilateral Wilms tumor. J Urol 138 (4 Pt 2):968, 1987. 125. Grundy P, Coppes MJ, Haber D: Molecular genetics of Wilms tumor. Hematol Oncol Clin North Am 9:1201, 1995. 126. Mueller RF: The Denys-Drash syndrome. J Med Genet 31:471, 1994. 127. Scharnhorst V, van der Eb AJ, Jochemsen AG: WT1 proteins: functions in growth and differentiation. Gene 273:141, 2001. 128. Englert C, et al: Induction of p21 by the Wilms tumor suppressor gene WT1. Cancer Res 57:1429, 1997. 129. Dome JS, Coppes MJ: Recent advances in Wilms tumor genetics. Curr Opin Pediatr 14:5, 2002. 130. Feinberg AP: Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer: an introduction. Cancer Res 59 (7 Suppl):1743s, 1999. 131. Steenman MJ, et al: Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms tumour. Nat Genet 7:433, 1994. 132. Breslow NE, et al: Familial Wilms tumor: a descriptive study. Med Pediatr Oncol 27:398, 1996. 133. Rahman N, et al: Confirmation of FWT1 as a Wilms tumour susceptibility gene and phenotypic characteristics of Wilms tumour attributable to FWT1. Hum Genet 103:547, 1998. 134. McDonald JM, et al: Linkage of familial Wilms tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors. Cancer Res 58:1387, 1998. 135. Maiti S, et al: Frequent association of β-catenin and WT1 mutations in Wilms tumors. Cancer Res 60:6288, 2000. 136. Hennigar RA, O'Shea PA, Grattan-Smith JD: Clinicopathologic features of nephrogenic rests and nephroblastomatosis. Adv Anat Pathol 8:276, 2001. 137. Faria P, et al: Focal versus diffuse anaplasia in Wilms tumor—new definitions with prognostic significance: a report from the National Wilms Tumor Study Group. Am J Surg Pathol 20:909, 1996. 138. Bardeesy N, Beckwith JB, Pelletier J: Clonal expansion and attenuated apoptosis in Wilms tumors are associated with p53 gene mutations. Cancer Res 55:215, 1995. 139. Shearer P, et al: Secondary acute myelogenous leukemia in patients previously treated for childhood renal tumors: a report from the National Wilms Tumor Study Group. J Pediatr Hematol Oncol 23:109, 2001. 509 Section II - Diseases of Organ Systems 510 511 Chapter 11 - Blood Vessels Frederick J. Schoen MD, PhD 512 Diseases of arteries are responsible for more morbidity and mortality than any other type of human disease. Disorders of veins less commonly cause clinically significant problems. Vascular abnormalities cause clinical disease by two principal mechanisms: • Narrowing or completely obstructing the lumens, either progressively (e.g., by atherosclerosis) or precipitously (e.g., by thrombosis or embolism). • Weakening of the walls, leading to dilation or rupture. To understand the diseases that affect blood vessels, we first consider some of the anatomic and functional characteristics of these highly specialized and dynamic tissues. Normal The general architecture and cellular composition of blood vessels are the same throughout the cardiovascular system. However, certain features of the vasculature vary with and reflect distinct functional requirements at different locations (see below). To withstand the pulsatile flow and higher blood pressures in arteries, arterial walls are generally thicker than the walls of veins. Arterial wall thickness gradually diminishes as the vessels become smaller, but the ratio of wall thickness to lumen diameter becomes greater. The basic constituents of the walls of blood vessels are endothelial cells and smooth muscle cells, and extracellular matrix (ECM), including elastin, collagen, and glycosoaminoglycans. The three concentric layers—intima, media, and adventitia—are most clearly defined in the larger vessels, particularly arteries ( Fig. 11-1 ). In normal arteries, the intima consists of a single layer of endothelial cells with minimal underlying subendothelial connective tissue. It is separated from the media by a dense elastic membrane called the internal elastic lamina. The smooth muscle cell layers of the media near the vessel lumen receive oxygen and nutrients by direct diffusion from the vessel lumen, facilitated by holes in the internal elastic membrane. However, diffusion from the lumen is inadequate for the outer portions of the media in large and medium-sized vessels, therefore these areas are nourished by small arterioles arising from outside the vessel (called vasa vasorum, literally "vessels of the vessels") coursing into the outer one half to two thirds of the media. The outer limit of the media of most arteries is a well-defined external elastic lamina. External to the media is the adventitia, consisting of connective tissue with nerve fibers and the vasa vasorum. Based on their size and structural features, arteries are divided into three types: (1) large or elastic arteries, including the aorta, its large branches (particularly the innominate, subclavian, common carotid, and iliac), and pulmonary arteries; (2) medium-sized or muscular arteries, comprising other Figure 11-1 The vascular wall. A, Graphic representation of the cross section of a small muscular artery (e.g., renal or coronary artery). B, Photomicrograph of histologic section containing a portion of an artery (A) and adjacent vein (V). Elastic membranes are stained black (internal elastic membrane of artery highlighted by arrow). Because it is exposed to higher pressures, the artery has a thicker wall that maintains an open, round lumen, even when blood is absent. Moreover, the elastin of the artery is more organized than in the corresponding vein. In contrast, the vein has a larger, but collapsed, lumen, and the elastin in its wall is diffusely distributed. (B, Courtesy of Mark Flomenbaum, M.D., Ph.D., Office of the Chief Medical Examiner, New York City.) TABLE 11-1 -- Endothelial Cell Properties and Functions Maintenance of Permeability Barrier Elaboration of Anticoagulant, Antithrombotic, Fibrinolytic Regulators Prostacyclin Thrombomodulin Heparin-like molecules Plasminogen activator Elaboration of Prothrombotic Molecules Von Willebrand factor Tissue factor Plasminogen activator inhibitor Extracellular Matrix Production (collagen, proteoglycans) Modulation of Blood Flow and Vascular Reactivity Vasconstrictors: endothelin, ACE Vasodilators: NO, prostacyclin Regulation of Inflammation and Immunity IL-1, IL-6, chemokines Adhesion molecules: VCAM-1, ICAM, E-selectin P-selectin Histocompatibility antigens Regulation of Cell Growth Growth stimulators: PDGF, CSF, FGF Growth inhibitors: heparin, TGF-β Oxidation of LDL ACE, angiotensin converting enzyme; NO, nitric oxide; IL, interleukin; PDGF, platelet-derived growth factor; CSF, colony-stimulating factor; FGF, fibroblast growth factor; TGF-β, transforming growth factor-beta; LDL, low-density lipoprotein. Structurally intact ECs can respond to various pathophysiologic stimuli by adjusting their usual (constitutive) functions and by expressing newly acquired (inducible) properties—a process 4 5 termed endothelial activation ( Fig. 11-2 ). [ ] [ ] Inducers of endothelial activation include cytokines and bacterial products, which cause inflammation and septic shock ( Chapter 2 ); hemodynamic stresses and lipid products, critical to the pathogenesis of atherosclerosis (see later); advanced glycosylation end products (important in diabetes, Chapter 24 ), as well as viruses, complement components, and hypoxia. Activated ECs, in turn, express adhesion molecules ( Chapter 2 ), and produce other cytokines and chemokines, growth factors, vasoactive molecules that result either in vasoconstriction or in vasodilation, major histocompatibility complex molecules, procoagulant and anticoagulant moieties, and a variety of other biologically active products. ECs influence the vasoreactivity of the underlying smooth muscle cells through the production of both relaxing factors (e.g., nitric oxide [NO]) and contracting factors (e. g., endothelin). Normal endothelial function is characterized by a balance of these factors and the ability of the vessel to respond appropriately to various pharmacologic stimuli (e.g., vasorelaxation in response to acetylcholine). Endothelial dysfunction, as defined by an altered phenotype that impairs vasoreactivity or induces a surface that is thrombogenic or abnormally adhesive to inflammatory cells, is responsible, at least in part, for the initiation of thrombus formation, Figure 11-2 Endothelial cell response to environmental stimuli: causes (activators) and consequences (induced genes). Figure 11-3 Schematic diagram of the mechanism of intimal thickening, emphasizing smooth muscle cell migration to, and proliferation and extracellular matrix elaboration in, the intima. (Modified and redrawn from Schoen FJ: Interventional and Surgical Cardiovascular Pathology: Clinical Correlations and Basic Principles. Philadelphia, W.B. Saunders Co., 1989, p. 254.) Figure 11-4 American Heart Association classification of human atherosclerotic lesions from the fatty dot (type I) to the complicated type VI lesion. The diagram also includes growth mechanisms and clinical correlations. (Modified from Stary HC, et al: A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. Circulation 92:1355, 1995.) Figure 11-5 Schematic summary of the natural history, morphologic features, main pathogenetic events, and clinical complications of atherosclerosis in the coronary arteries. Figure 11-6 Fatty streak—a collection of foam cells in the intima. A, Aorta with fatty streaks (arrows), associated largely with the ostia of branch vessels. B, Close-up photograph of fatty streaks from aorta of experimental hypercholesterolemic rabbit shown following staining with Sudan red, a lipid-soluble dye, again illustrating the relationship of lesions to branch vessel ostia. C, Photomicrograph of fatty streak in experimental hypercholesterolemic rabbit, demonstrating intimal, macrophage-derived foam cells (arrow). (B and C, Courtesy of Myron I. Cybulsky, M.D., University of Toronto, Canada). Figure 11-7 Schematic depiction of the major components of well-developed intimal atheromatous plaque overlying an intact media. Figure 11-8 Gross views of atherosclerosis in the aorta. A, Mild atherosclerosis composed of fibrous plaques, one of which is denoted by the arrow. B, Severe disease with diffuse and complicated lesions. Figure 11-9 Histologic features of atheromatous plaque in the coronary artery. A, Overall architecture demonstrating fibrous cap (F) and a central necrotic (largely lipid) core (C). The lumen (L) has been moderately narrowed. Note that a segment of the wall is plaque free (arrow). In this section, collagen has been stained blue (Masson's trichrome stain). B, Higher-power photograph of a section of the plaque shown in A, stained for elastin (black), demonstrating that the internal and external elastic membranes are destroyed and the media of the artery is thinned under the most advanced plaque (arrow). C, Higher-magnification photomicrograph at the junction of the fibrous cap and core, showing scattered inflammatory cells, calcification (broad arrow), and neovascularization (small arrows). TABLE 11-2 -- Risk Factors for Atherosclerosis Major Lesser, Uncertain, or Nonquantitated Nonmodifiable Increasing age Obesity Male gender Physical inactivity Family history Stress ("type A" personality) Genetic abnormalities Postmenopausal estrogen deficiency High carbohydrate intake Potentially Controllable Hyperlipidemia Alcohol Hypertension Lipoprotein Lp(a) Cigarette smoking Hardened (trans)unsaturated fat intake Diabetes Chlamydia pneumoniae high blood lipid levels, such as familial hypercholesterolemia, which was discussed in Chapter 5 . Other, nongenetic risk factors, particularly diet, lifestyle, and personal habits, are to a large extent potentially reversible. The four major risk factors potentially responsive to change are hyperlipidemia, hypertension, cigarette smoking, and diabetes. Hyperlipidemia. Hyperlipidemia is a major risk factor for atherosclerosis. Most of the evidence specifically implicates hypercholesterolemia. Elevated levels of serum cholesterol are sufficient to stimulate lesion development, even if other risk Figure 11-10 Estimated 10-year risk of coronary artery disease according to various combinations of risk factor levels, expressed as the probability of an event in 10 years. HDL-C, high density lipoprotein cholesterol (From Kannel WB, et al: An update on coronary risk factors. Med Clin North Am 79:951, 1995.) Figure 11-11 Evolution of arterial wall changes in the response to injury hypothesis. 1, Normal. 2, Endothelial injury with adhesion of monocytes and platelets (the latter to denuded endothelium). 3, Migration of monocytes (from the lumen) and smooth muscle cells (from the media) into the intima. 4, Smooth muscle cell proliferation in the intima. 5, Well-developed plaque (see Fig. 11-7 for details of mature plaque structure). Figure 11-12 Schematic diagram of hypothetical sequence of cellular interactions in atherosclerosis. Hyperlipidemia and other risk factors are thought to cause endothelial injury, resulting in adhesion of platelets and monocytes and release of growth factors, including platelet-derived growth factor (PDGF), which lead to smooth muscle cell migration and proliferation. Foam cells of atheromatous plaques are derived from both macrophages and smooth muscle cells—from macrophages via the very-low-density lipoprotein (VLDL) receptor and low-density lipoprotein (LDL) modifications recognized by scavenger receptors (e.g., oxidized LDL), and from smooth muscle cells by less certain mechanisms. Extracellular lipid is derived from insudation from the vessel lumen, particularly in the presence of hypercholesterolemia, and also from degenerating foam cells. Cholesterol accumulation in the plaque reflects an imbalance between influx and efflux, and high-density lipoprotein (HDL) likely helps clear cholesterol from these accumulations. Smooth muscle cells migrate to the intima, proliferate, and produce extracellular matrix, including collagen and proteoglycans. TABLE 11-3 -- Types and Causes of Hypertension Essential Hypertension Secondary Hypertension ••Renal ••••Acute glomerulonephritis ••••Chronic renal disease ••••Polycystic disease ••••Renal artery stenosis ••••Renal artery fibromuscular dysplasia ••••Renal vasculitis ••••Renin-producing tumors ••Endocrine ••••Adrenocortical hyperfunction (Cushing syndrome, primary aldosteronism, congenital adrenal hyperplasia, licorice ingestion) ••••Exogenous hormones (glucocorticoids, estrogen [including pregnancy-induced and oral contraceptives], sympathomimetics and tyramine-containing foods, monoamine oxidase inhibitors) ••••Pheochromocytoma ••••Acromegaly ••••Hypothyroidism (myxedema) ••••Hyperthyroidism (thyrotoxicosis) ••••Pregnancy-induced ••Cardiovascular ••••Coarctation of aorta ••••Polyarteritis nodosa (or other vasculitis) ••••Increased intravascular volume ••••Increased cardiac output ••••Rigidity of the aorta ••Neurologic ••••Psychogenic ••••Increased intracranial pressure ••••Sleep apnea ••••Acute stress, including surgery Cushing syndrome, pheochromocytoma), narrowing of the renal artery, usually by an atheromatous plaque (renovascular hypertension) or other identifiable cause (secondary hypertension). However, about 95% of hypertension is idiopathic (called essential hypertension). This form of hypertension generally does not cause short-term problems; especially when controlled, is compatible with long life and is asymptomatic, unless a myocardial infarction, cerebrovascular accident, or other complication supervenes. Thus, this subgroup is often called benign hypertension. A small percentage, perhaps 5%, of hypertensive persons show a rapidly rising blood pressure that if untreated, leads to death within a year or two. Called accelerated or malignant hypertension, the clinical syndrome is characterized by severe hypertension (i.e., systolic pressure over 200 mm Hg, diastolic pressure over 120 mm Hg), renal failure, and retinal hemorrhages and exudates, with or without papilledema. It may develop in previously normotensive persons but more often is superimposed on pre-existing benign hypertension, either essential or secondary. Pathogenesis of Hypertension. 36 The multiple mechanisms of hypertension constitute aberrations of the normal physiologic regulation of blood pressure.[ ] Arterial hypertension occurs when the relationship between cardiac output and total peripheral resistance is altered. For many of the secondary forms of hypertension, these factors are reasonably well understood. For example, in renovascular hypertension, renal artery stenosis causes decreased glomerular flow and pressure in the afferent arteriole of the glomerulus. This (1) induces renin secretion, initiating angiotensin IImediated vasoconstriction and increased peripheral resistance, and (2) increases sodium reabsorption and therefore blood volume through the aldosterone mechanism. In pheochromocytoma, a tumor of the adrenal medulla (see Chapter 24 ), catecholamines produced by tumor cells cause episodic vasoconstriction and thus induce hypertension. Regulation of Normal Blood Pressure. Blood pressure is proportional to cardiac output and peripheral vascular resistance ( Fig. 11-13 ). Indeed, the blood pressure level is a complex trait that is determined by the interaction of multiple genetic, environmental, and demographic factors that influence cardiac output and vascular resistance. The major factors that determine blood pressure variation within and between populations include age, gender, body mass index, and diet, principally sodium intake. Cardiac output is highly dependent on blood volume, itself greatly influenced by the whole body sodium homeostasis. Peripheral vascular resistance is determined mainly at the level of the arterioles and is affected by neural and hormonal factors. Normal vascular tone reflects the balance between humoral vasoconstricting influences (including angiotensin II, catecholamines, and endothelin) and vasodilators (including kinins, prostaglandins, and NO). Resistance vessels also exhibit autoregulation, whereby increased blood flow induces vasoconstriction to protect against tissue hyperperfusion. Other local factors such as pH and hypoxia, and the α- and β-adrenergic systems, which influence heart rate, cardiac contraction, and vascular tone, may be important. The integrated function of these systems ensures adequate perfusion of all tissues, despite regional differences in demand. The kidneys play an important role in blood pressure regulation as follows: 527 Figure 11-13 The critical roles of cardiac output and peripheral resistance in blood pressure regulation. NO, nitric oxide. Figure 11-14 Blood pressure regulation by the renin-angiotensin system and the central roles of sodium metabolism in specific causes of inherited and acquired forms of hypertension. Components of the systemic renin-angiotensin system are shown in black. Genetic disorders that affect blood pressure by altering activity of this pathway are indicated in red; arrows indicate sites in the pathway altered by mutation. Genes that are mutated in these disorders are indicated in parentheses. Acquired disorders that alter blood pressure through effects on this pathway are indicated in blue. (From Lifton RP, et al: Molecular genetics of human blood pressure variation. Science 272:676, 1996.) Figure 11-15 Hypothetical scheme for the pathogenesis of essential hypertension, implicating genetic defects in renal excretion of sodium, functional regulation of vascular tone, and structural regulation of vascular caliber. Environmental factors, especially increased salt intake, may potentiate the effects of genetic factors. The resultant increases in cardiac output and peripheral resistance contribute to hypertension. ECF, extracellular fluid. Figure 11-16 Mutations altering blood pressure in humans. A diagram of a nephron, the filtering unit of the kidney, is shown. The molecular pathways mediating NaCl reabsorption in individual renal cells in the thick ascending limb of the loop of Henle (TAL), distal convoluted tubule (DCT), and the cortical collecting tubule (CCT) are indicated, along with the pathway of the renin-angiotensin system, the major regulator of renal salt reabsorption. Single gene defects that manifest as inherited diseases affecting these pathways are indicated, with hypertensive disorders in red and hypotensive disorders in blue. Abbreviations: Al, angiotensin I; ACE, angiotensin converting enzyme; All, angiotensin II; MR, mineralocorticoid receptor; GRA, glucocorticoid-remediable aldosteronism; PHA1, pseudohypoaldosteronism, type 1; AME, apparent mineralocorticoid excess; 11β-HSD2, 11β-hydroxysteroid dehydrogenase-2; and DOC, deoxycorticosterone. (From Lifton RP, et al: Molecular mechanisms of human hypertension. Cell 104:545, 2001.) Figure 11-17 Vascular pathology in hypertension. A, Hyaline arteriolosclerosis. The arteriolar wall is hyalinized, and the lumen is markedly narrowed. B, Hyperplastic arteriolosclerosis (onionskinning) causing luminal obliteration (arrow), with secondary ischemic changes, manifested by wrinkling of the glomerular capillary vessels at the upper left (periodic acid-Schiff [PAS] stain). (Courtesy of Helmut Rennke, M.D., Brigham and Women's Hospital, Boston, MA.) Figure 11-18 True and false aneurysms. Center, Normal vessel. Left, True aneurysm. The wall bulges outward and may be attenuated but is intact. Right, False aneurysm. The wall is ruptured, and there is a collection of blood (hematoma) that is bounded externally by adherent extravascular tissues. Figure 11-19 Abdominal aortic aneurysm. A, External view, gross photograph of a large aortic aneurysm that ruptured; the rupture site is indicated by the arrow. B, Opened view, with the location of the rupture tract indicated by a probe. The wall of the aneurysm is exceedingly thin, and the lumen is filled by a large quantity of layered but largely unorganized thrombus. Figure 11-20 Aortic dissection. A, Gross photograph of opened aorta with proximal dissection, demonstrating a small, oblique intimal tear (demarcated by probe), allowing blood to enter the media, creating an intramural hematoma (thin arrows). Note that the intimal tear has occurred in a region largely free from atherosclerotic plaque, and that propagation of the intramural hematoma was arrested at a site more distally, where atherosclerosis begins (broad arrow). B, Histologic view of the dissection demonstrating an aortic intramural hematoma (asterisk). Aortic elastic layers are black, and blood is red in this section, stained with the Movat stain. Figure 11-21 Medial degeneration. A, Cross-section of aortic media with marked elastin fragmentation and formation of areas devoid of elastin that resemble cystic spaces, from a patient with Marfan syndrome. B, Normal media for comparison, showing the regular layered pattern of elastic tissue. In both A and B, the tissue section is stained to highlight elastin as black. Figure 11-22 Classification of dissection into types A and B. Type A (proximal) involves the ascending aorta, whereas type B (distal) does not. The serious complications predominantly occur in the region from the aortic valve through the arch. TABLE 11-4 -- Classification of Vasculitis Based on Pathogenesis (Not Available) Data from Jennette JC, Falk RJ: Update on the pathobiology of vasculitis. In Schoen FJ, Gimbrone MA (eds); Cardiovascular Pathology: Clinicopathologic Correlations and Pathogenetic Mechanisms. Baltimore, Williams & Wilkins, 1995, p 156. chemical injury, such as irradiation, mechanical trauma, and toxins can also cause vascular damage. Pathogenesis of Noninfectious Vasculitis. The main immunologic mechanisms that initiate noninfectious vasculitis are: (1) immune complex deposition, (2) antineutrophil cytoplasmic antibodies, and (3) anti-endothelial cell antibodies. Immune Complexes. The evidence for involvement of immune complexes in vasculitides can be summarized as follows: • The vascular lesions resemble those found in experimental immune complex-mediated conditions, such as the local Arthus phenomenon and serum sickness ( Chapter 6 ). Immune reactants and complement can be detected in the serum or vessels of patients with vasculitis (e.g., DNA-anti-DNA complexes are present in the vascular lesions of systemic lupus erythematosus-associated vasculitis and IgG, IgM, and complement in cryoglobulinemic vasculitis). • Hypersensitivity to drugs causes approximately 10% of vasculitic skin lesions, largely through vascular deposits of immune complexes. Some, such as penicillin, conjugate serum proteins; others, like streptokinase, are themselves foreign proteins. The manifestations vary and range from small-vessel hypersensitivity and leukocytoclastic vasculitis to polyarteritis nodosa, Wegener granulomatosis, and Churg-Strauss syndrome (see later for descriptions of these entities), and from mild and self-limiting to severe and even fatal. 50 Identification of the disorder as a drug reaction is particularly important, as discontinuation of the offending agent is often followed by rapid improvement. [ ] • In vasculitis associated with viral infections, immune complexes can be found in the serum and in the vascular lesions of some patients, particularly in cases of polyarteritis nodosa (for example, HBsAg-anti-HbsAg in hepatitis-induced vasculitis). Whether immune complexes deposit in vessel walls from the circulation, or are formed in situ, or both, is not known (see Chapter 6 ). However, many small vessel vasculitides show a paucity of vascular immune deposits and therefore other mechanisms have been sought for these so-called pauci-immune vasculitides. Antineutrophil Cytoplasmic Antibodies. 51 Serum from many patients with vasculitis reacts with cytoplasmic antigens in neutrophils, indicating the presence of antineutrophil cytoplasmic antibodies (ANCAs).[ ] ANCAs are a heterogeneous group of autoantibodies directed against enzymes mainly found within the azurophil or primary granules in neutrophils, in the lysosomes of monocytes, and in ECs. The description of these autoantibodies is based on the immunofluorescent patterns of staining of ethanol-fixed neutrophils. Two main patterns are recognized: one shows cytoplasmic localization of the staining (c-ANCA), and the most common target antigen is proteinase-3 (PR3), a neutrophil granule constituent. The second shows perinuclear staining (p-ANCA) and is usually specific for myeloperoxidase (MPO). Either ANCA specificity may occur in a patient with ANCA-associated small-vessel vasculitis but c-ANCA is typically found in Wegener granulomatosis and p-ANCA is found in most cases of microscopic polyangiitis and Churg-Strauss syndrome. The disorders characterized by circulating ANCAs are called the ANCAassociated vasculitides. 52 ANCAs serve as useful quantitative diagnostic markers for these conditions, and their levels may reflect the degree of inflammatory activity.[ ] ANCAs rise in episodes of recurrence, and thus are useful in management. In addition, the close association between ANCA titers and disease activity, particularly c-ANCA in Wegener granulomatosis, suggests that they may be important in the pathogenesis of this disease. Experimental data are consistent with a pathophysiologic mechanism for ANCA and/or ANCA antigen autoimmune responses in these diseases, but the precise mechanisms are unknown. However, there is yet no definitive proof that ANCAs play a causative role in the development of systemic vasculitis. 53 One plausible hypothesis for a causative role of ANCAs in vasculitis is summarized briefly as follows:[ ] (1) An underlying disorder (e.g., an infection) elicits pro-inflammatory cytokines such as TNF, and granulocyte-macrophage colony-stimulating factor, and microbial products such as endotoxin, which together cause neutrophils and other inflammatory cells to express PR3 and MPO on their surfaces. (2) These stimulate the formation of ANCAs. (3) ANCAs react with circulating cytokine-primed neutrophils and cause them to degranulate (4) PMNs activated by ANCA cause endothelial 536 cell toxicity and other direct tissue injury. Interestingly, ANCAs directed against neutrophil constituents other than PR3 and MPO are also found in some patients with a wide range of inflammatory but nonvasculitic disorders such as inflammatory bowel disease, autoimmune liver disease, primary sclerosing cholangitis, and rheumatoid arthritis, and in some patients with malignancies and infections. Anti-endothelial Cell Antibodies. Antibodies to ECs, perhaps induced by defects in immune regulation, may predispose to certain vasculitides, such as those associated with SLE and Kawasaki disease. Classification. The systemic vasculitides are classified on the basis of the size and anatomic site of the involved blood vessels ( Fig. 11-23 ), histologic characteristics of the lesion, and clinical manifestations. There is considerable clinical and pathologic overlap among these disorders summarized in Table 11-5 (Table Not Available) and discussed below. GIANT CELL (TEMPORAL) ARTERITIS 54 Giant cell (temporal) arteritis, the most common form of systemic vasculitis in adults, is an acute and chronic, often granulomatous, inflammation of arteries of large to small size.[ ] It affects principally the arteries in the head—especially the temporal arteries—but also the vertebral and ophthalmic arteries and the aorta, where it may cause thoracic aortic aneurysm. Ophthalmic arterial involvement may lead to permanent blindness. Therefore, visual loss caused by giant cell arteritis is a medical emergency that requires prompt recognition Figure 11-23 Diagrammatic representation of the sites of the vasculature involved by the major forms of vasculitis. The widths of the trapezoids indicate the frequencies of involvement of various portions. LCA, leukocytoclastic angiitis. (Reproduced from Jennette JC, and Falk RJ: Small-vessel vasculitis. New Engl J Med 337:1512, 1997.) TABLE 11-5 -- Classification and Characteristics of Selected Vasculitis (Not Available) Modified from Jennette JC, et al: Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum 37:187, 1994. Figure 11-24 Temporal (giant cell) arteritis. A, H&E stain of section of temporal artery showing giant cells at the degenerated internal elastic membrane in active arteritis (arrow). B, Elastic tissue stain demonstrating focal destruction of internal elastic membrane (arrow) and intimal thickening (IT) characteristic of long-standing or healed arteritis. C, Examination of the temporal artery of a patient with giant-cell arteritis shows a thickened, nodular, and tender segment of a vessel on the surface of head (arrow). (C Reproduced from Salvarani C, et al. Polymyalgia rheumatica and giant-cell arteritis. N Engl J Med 347:261, 2002.) Figure 11-25 Takayasu arteritis. A, Aortic arch angiogram showing narrowing of brachiocephalic, carotid, and subclavian arteries (arrows). B, Gross photograph of two cross-sections of the right carotid artery taken at autopsy of the patient shown in A, demonstrating marked intimal thickening with minimal residual lumen. C, Histologic view of active Takayasu aortitis, illustrating destruction of the arterial media by mononuclear inflammation with giant cells. Figure 11-26 Representative forms of systemic medium-sized to small vessel vasculitis. A, Polyarteritis nodosa. B, Leukocytoclastic vasculitis. C and D, Wegener granulomatosis. E, Thromboangiitis obliterans (Buerger disease). In polyarteritis nodosa (A), there is segmental fibrinoid necrosis and thrombotic occlusion of the lumen of this small artery. Note that part of the vessel wall at the upper right (arrow) is uninvolved. In leukocytoclastic vasculitis (B), shown here from a skin biopsy, there is fragmentation of neutrophils in and around blood vessel walls. In Wegener granulomatosis (C), there is inflammation (vasculitis) of a small artery along with adjacent granulomatous inflammation, in which epithelioid cells and giant cells (arrows) are seen. D, Gross photo from the lung of a patient with fatal Wegener granulomatosis, demonstrating large nodular lesions. In a typical case of Buerger disease (E), the lumen is occluded by a thrombus containing two abscesses (arrow). The vessel wall is infiltrated with leukocytes. (A, and D, courtesy of Sidney Murphree, MD, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX; B, courtesy of Scott Granter, M.D., Brigham and Women's Hospital, Boston.) Figure 11-27 Vasculitis with fibrinoid necrosis in a patient with active systemic lupus erythematosus. Figure 11-28 Raynaud phenomenon. A, Sharply demarcated pallor of the distal fingers resulting from the closure of digital arteries. B, Cyanosis of the fingertips. (Reproduced from Salvarani C, et al.: Polymyalgia rheumatica and giant-cell arteritis. N Engl J Med 347:261, 2002.) TABLE 11-6 -- Classification of Vascular Tumors and Tumor-Like Conditions Benign Neoplasms, Developmental and Acquired Conditions Hemangioma ••Capillary hemangioma ••Cavernous hemangioma ••Pyogenic granuloma (lobular capillary hemangioma) Lymphangioma ••Simple (capillary) lymphangioma ••Cavernous lymphangioma (cystic lymphangioma) Glomus tumor Vascular ectasias ••Nevus flammeus ••Spider telangiectasia (arterial spider) ••Hereditary hemorrhagic telangiectasis (Osler-Weber-Rendu disease) Reactive vascular proliferations ••Bacillary angiomatosis Intermediate-Grade Neoplasms Kaposi sarcoma Hemangioendothelioma Malignant Neoplasms Angiosarcoma Hemangiopericytoma The endothelial derivation of neoplastic proliferations that do not form distinct vascular lumina can usually be confirmed by immunohistochemical demonstration of endothelium-specific markers such as CD31, CD34, or vWF. Because these lesions constitute abnormalities of unregulated vascular proliferation, the possibility of controlling such growth by agents that inhibit blood vessel formation (anti-angiogenic factors) is particularly exciting. BENIGN TUMORS AND TUMOR-LIKE CONDITIONS Hemangioma Difficult to distinguish with certainty from malformations or hamartomas, hemangiomas (angiomas) are most commonly localized; however, some involve large segments of the body such as an entire extremity (called angiomatosis). The majority are superficial lesions, often of the head or neck, but they may occur internally, with nearly one third in the liver. Malignant transformation occurs rarely if at all ( Fig. 11-30 ). Hemangiomas constitute 7% of all benign tumors in infancy and childhood ( Chapter 10 ). Most are present from birth and expand along with the growth of the child. Nevertheless, many of the capillary lesions regress spontaneously at or before puberty. There are several histologic and clinical variants. Capillary Hemangioma. Capillary hemangiomas, the largest single type of vascular tumor, are most common in the skin, subcutaneous tissues, and mucous membranes of the oral cavities and lips, but they may also occur in the liver, spleen, and kidneys. The "strawberry type" of capillary hemangioma (juvenile hemangioma) of the skin of newborns is extremely common (1 in 200 births), may be multiple, grows rapidly in the first few months, begins to fade when the 546 Figure 11-30 Hemangiomas. A, Hemangioma of the tongue. B, Histology of juvenile capillary hemangioma. C, Histology of cavernous hemangioma. D, Pyogenic granuloma of the lip. (A and D, courtesy of John Sexton, M.D., Beth Israel Hospital, Boston; B, courtesy of Christopher D.M. Fletcher, M.D., Brigham and Women's Hospital, Boston; and C, courtesy of Thomas Rogers, M.D., University of Texas Southwestern Medical School, Dallas, TX.) Figure 11-31 Bacillary angiomatosis. A, Photograph of a moist, erosive cutaneous lesion. B, Histologic appearance with acute neutrophilic inflammation and vascular (capillary) proliferation. Inset, demonstration by modified silver (Warthin-Starry) stain of clusters of tangled bacilli (black). (A, courtesy of Richard Johnson, M.D. Beth Israel Deaconess Medical Center, Boston, MA; B and inset, courtesy of Scott Granter, M.D., Brigham and Women's Hospital, Boston, MA.) Figure 11-32 Kaposi sarcoma. A, Gross photograph, illustrating coalescent red-purple macules and plaques of the skin. B, Histology of nodular form, demonstrating sheets of plump, proliferating spindle cells. (B, courtesy of Christopher D.M. Fletcher, M.D., Brigham and Women's Hospital, Boston, MA.) Figure 11-33 Angiosarcoma. A, Gross photograph of angiosarcoma of the heart (right ventricle). B, Photomicrograph of moderately well-differentiated angiosarcoma with dense clumps of irregular, moderate anaplastic cells and distinct vascular lumens. C, Positive immunohistochemical staining of angiosarcoma for the endothelial cell marker CD31, proving the endothelial nature of the tumor cells. Figure 11-34 Balloon angioplasty and endovascular stents. A, Coronary artery with recent balloon angioplasty, in a low-power photomicrograph showing the split encompassing the intima and media (arrow) and partial circumferential dissection. B, Gross photograph of restenosis following balloon angioplasty, demonstrating residual atherosclerotic plaque (left arrow) and a new, glistening proliferative lesion (right arrow). C, Coronary arterial stent implanted long term, demonstrating thickened neointima separating the stent wires (black spot shown by arrow) from the lumen (asterisk). (C, Reproduced from Schoen FJ, Edwards WD. Pathology of cardiovascular interventions, including endovascular therapies, revascularization, vascular replacement, cardiac assist/replacement, arrhythmia control, and repaired congenital heart disease. In Silver MD, Gotlieb AI, Schoen FJ (eds): Cardiovascular Pathology, 3rd ed. Philadelphia, Churchill Livingstone, 2001.) Figure 11-35 Anastomotic hyperplasia at the distal anastomosis of synthetic femoropopliteal graft. A, Angiogram demonstrating constriction (arrow). B, Photomicrograph demonstrating Gore-Tex graft (arrow) with prominent intimal proliferation and very small residual lumen (asterisk). (A, courtesy of Anthony D. Whittemore, M.D., Brigham and Women's Hospital, Boston, MA.) References 1. Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 407:249, 2000. 2. Shin D, et al: Expression of ephrin-B2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization. Dev Biol 230:139, 2001. 3. Karkkainen MJ, et al: Lymphatic endothelium: a new frontier of metastasis research. Nat Cell Biol 4:E2, 2002. 4. 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