Essentials of rubins pathology sixth none edition ebook pdf

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CONTRIBUTORS Michael F. Allard, MD Professor of Pathology and Laboratory Medicine University of British Columbia Cardiovascular Pathologist Department of Pathology and Laboratory Medicine The iCAPTURE Centre St. Paul’s Hospital Vancouver, British Columbia, Canada Mary Beth Beasley, MD Associate Professor of Pathology Mount Sinai Medical Center New York, New York Thomas W. Bouldin, MD Professor of Pathology and Laboratory Medicine Chair for Faculty and Trainee Development University of North Carolina at Chapel Hill Director of Neuropathology McLendon Clinical Laboratories University of North Carolina Hospitals Chapel Hill, North Carolina Diane L. Carlson, MD Assistant Attending Department of Pathology Memorial Sloan-Kettering Cancer Center New York, New York Ivan Damjanov, MD, PhD Professor of Pathology The University of Kansas School of Medicine Pathologist Department of Pathology University of Kansas Medical Center Kansas City, Kansas Jeffrey M. Davidson, PhD Professor of Pathology Vanderbilt University School of Medicine Senior Research Career Scientist Medical Research Service Veterans Affairs Tennessee Valley Healthcare System Nashville, Tennessee David E. Elder, MD, ChB, FRCPA Professor of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Director of Anatomic Pathology Hospital of the University of Pennsylvania Philadelphia, Pennsylvania Alina Dulau Florea, MD Assistant Professor of Pathology Thomas Jefferson University Philadelphia, Pennsylvania

Gregory N. Fuller, MD, PhD Professor of Pathology Chief of Neuropathology The University of Texas MD Anderson Cancer Center Houston, Texas Roberto A. Garcia, MD Assistant Professor of Pathology The Mount Sinai School of Medicine Chief of Orthopaedic and Soft Tissue Pathology The Mount Sinai Hospital New York, New York J. Clay Goodman, MD Professor of Pathology and Neurology Walter Henrick Moursund Endowed Chair in Neuropathology Associate Dean of Undergraduate Medical Education Baylor College of Medicine Houston, Texas Avrum I. Gotlieb, MD, CM, FRCP Professor of Laboratory Medicine and Pathology University of Toronto Staff Pathologist Laboratory Medicine Program University Health Network Toronto, Ontario, Canada Philip N. Hawkins, PhD, FRCP, FRCPath, FMedSci Professor of Medicine Centre for Amyloidosis and Acute Phase Proteins University College London Medical School Head, National Amyloidosis Centre Royal Free Hospital London, England, United Kingdom Steven K. Herrine, MD Professor of Medicine Thomas Jefferson University Philadelphia, Pennsylvania J. Charles Jennette, MD Brinkhous Distinguished Professor and Chair of Pathology and Laboratory Medicine University of North Carolina, School of Medicine Chief of Service Department of Pathology and Laboratory Medicine University of North Carolina Hospitals Chapel Hill, North Carolina Lawrence C. Kenyon, MD, PhD Associate Professor of Pathology, Anatomy & Cell Biology Thomas Jefferson University Pathologist and Neuropathologist Department of Pathology, Anatomy & Cell Biology Thomas Jefferson University Hospital Philadelphia, Pennsylvania

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CONTRIBUTORS Robert Kisilevsky, MD, PhD, FRCPC, FRSC Professor Emeritus Department of Pathology and Molecular Medicine Queen’s University Kingston, Ontario, Canada Michael J. Klein, MD Professor of Pathology and Laboratory Medicine Weill Medical College of Cornell University Pathologist-in-Chief and Director of Pathology and Laboratory Medicine Hospital for Special Surgery New York, New York David S. Klimstra, MD Chief of Surgical Pathology Department of Pathology Memorial Sloan-Kettering Cancer Center New York, New York Gordon K. Klintworth, MD, PhD Professor of Pathology Joseph A.C. Wadsworth Research Professor of Ophthalmology Duke University Durham, North Carolina Amber Chang Liu, MSc University of Toronto Toronto, Ontario, Canada Peter A. McCue, MD Professor of Pathology Thomas Jefferson University Director of Anatomic Pathology Thomas Jefferson University Hospital Philadelphia, Pennsylvania Bruce M. McManus, MD, PhD, FRSC Professor of Pathology and Laboratory Medicine University of British Columbia Director, Providence Heart and Lung Institute St. Paul’s Hospital Vancouver, British Columbia, Canada Maria J. Merino, MD Chief of Translational Pathology Department of Pathology National Cancer Institute Bethesda, Maryland Anna Marie Mulligan, MB, MSc, FRCPath Assistant Professor of Laboratory Medicine and Pathobiology University of Toronto Anatomic Pathologist Department of Laboratory Medicine St. Michael’s Hospital Toronto, Ontario, Canada

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Hedwig S. Murphy, MD, PhD Associate Professor of Pathology University of Michigan Staff Pathologist Department of Pathology and Laboratory Medicine Veterans Affairs Ann Arbor Health System Ann Arbor, Michigan George L. Mutter, MD Associate Professor of Pathology Harvard Medical School Pathologist Department of Pathology Brigham and Women’s Hospital Boston, Massachusetts Victor J. Navarro, MD Professor of Medicine, Pharmacology, and Experimental Therapeutics Medical Director, Liver Transplantation Director, Transplant Hepatology Fellowship Departments of Gastroenterology and Hepatology Thomas Jefferson University Philadelphia, Pennsylvania Frances P. O’Malley, MB, FRCPC Professor of Laboratory Medicine and Pathobiology University of Toronto Staff Pathologist Department of Pathology and Laboratory Medicine Mount Sinai Hospital Toronto, Ontario, Canada Stephen Peiper, MD Peter A. Herbut Professor and Chairman of Pathology, Anatomy & Cell Biology Thomas Jefferson University Philadelphia, Pennsylvania Jaime Prat, MD, PhD, FRCPath Professor of Pathology Director of Pathology Autonomous University of Barcelona Director of Pathology Hospital de la Santa Creu i Sant Pau Barcelona, Spain Martha Quezado, MD Staff Pathologist Laboratory of Pathology National Cancer Institute National Institutes of Health Bethesda, Maryland Jeffrey E. Saffitz, MD, PhD Mallinckrodt Professor of Medicine Harvard Medical School Chairman, Department of Pathology Beth Israel Deaconess Medical Center Boston, Massachusetts

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CONTRIBUTORS

Alan L. Schiller, MD Professor of Pathology Mount Sinai School of Medicine Chairman, Department of Pathology Mount Sinai Hospital New York, New York

Riccardo Valdez, MD Assistant Professor of Pathology Section Head, Hematopathology Department of Laboratory Medicine and Pathology Mayo Clinic Scottsdale, Arizona

David A. Schwartz, MD, MSHyg, FCAP Associate Clinical Professor of Pathology Vanderbilt University School of Medicine Nashville, Tennessee

Jeffrey S. Warren, MD Aldred S. Warthin Endowed Professor of Pathology Director, Division of Clinical Pathology University of Michigan Medical School University of Michigan Hospitals Ann Arbor, Michigan

Gregory C. Sephel, PhD Associate Professor of Pathology Vanderbilt University School of Medicine Nashville, Tennessee Elias S. Siraj, MD Associate Professor of Medicine Section of Endocrinology Temple University School of Medicine Program Director, Endocrinology Fellowship Temple University Hospital Philadelphia, Pennsylvania Edward B. Stelow, MD Associate Professor of Pathology University of Virginia Charlottesville, Virginia Craig A. Storm, MD Assistant Professor of Pathology Dartmouth Medical School Hanover, New Hampshire Staff Dermatopathologist Department of Pathology Dartmouth-Hitchcock Medical Center Lebanon, New Hampshire William D. Travis, MD Professor of Pathology Weill Medical College of Cornell University Attending Thoracic Pathologist Memorial Sloan-Kettering Cancer Center New York, New York

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Bruce M. Wenig, MD Professor of Pathology Albert Einstein College of Medicine Bronx, New York Chairman Department of Pathology and Laboratory Medicine Beth Israel Medical Center St. Luke’s and Roosevelt Hospitals New York, New York Kevin Jon Williams, MD Professor of Medicine Chief, Section of Endocrinology, Diabetes, and Metabolism Temple University School of Medicine Philadelphia, Pennsylvania Robert Yanagawa, MD, PhD Resident in Surgery Division of Cardiac Surgery University of Toronto, Faculty of Medicine Toronto, Ontario, Canada Mary M. Zutter, MD Professor of Pathology and Cancer Biology Vanderbilt University Director of Hematopathology Vanderbilt University Medical Center Nashville, Tennessee

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PREFACE An honest tale speeds best being plainly told.

(Shakespeare, Richard III)

A

s in previous editions of this text, we continue to view pathology as the medical science that deals with all aspects of disease, but with special reference to the essential nature, the causes and the development of abnormal conditions. In this sense, literacy in pathology is the bedrock of practice and research for the student of medical science.

Thus, wherever possible we have related pathologic changes to clinical manifestations of disease.

General pathology emphasizes the towering achievements in the study of cell and molecular biology, biochemistry and immunology, all of which are related to the contemporary understanding of the pathogenesis of disease.

Although systemic pathology is concerned principally with the description of specific maladies, the concepts detailed in general pathology are utilized to explain their underlying causes. In this approach, we are mindful of the admonition of Edmond Halley (1687): “Truth being uniform, and always the same, it is admirable to observe how easily we are enabled to make out very abstruse and difficult matters, when once true and genuine Principles are obtained.”

Particular attention continues to be paid throughout to the impact of molecular genetics on our insights into the causes and manifestations of disease, including the correlations between genotype and phenotypic expression. For reference purposes, we have identified many of the relevant gene mutations and their chromosomal locations. In his treatise On the Natural Faculties, Galen wrote, “the chief merit of language is clearness, and we know that nothing detracts from this as do unfamiliar terms.” In this context, we emphasize important concepts by incorporating short declarative sentences into secondary headings. We continue with the format of designating for individual diseases separate sections of epidemiology, pathogenesis, pathology and clinical features—each identified by a unique icon. Attention is often directed to the key points by the use of bulleted lists and boldface type. We

have also retained the emphasis on graphic representations of the pathogenesis of disease, the complications of various disorders and the sequences of pathological alterations. Because graphic images utilize pattern recognition, one of the most fundamental characteristics of the human brain, they powerfully communicate abstract and complex material. At the same time, we have been guided by Einstein’s admonition that “everything should be made as simple as possible, but not simpler.” We have added numerous new drawings and have revised many of the previous ones. The number of color photographs has also been significantly increased.

Sadly, attempting to edit a comprehensive textbook of pathology without missing any errors is like trying to live without sin—worth the effort, but probably impossible. As Isaac Newton wrote (1703), “To explain all nature is too difficult a task for any one man or even for any one age. Tis much better to do a little with certainty & leave the rest for others that come after you.” However, the inevitability of human mistakes has not deterred us from including new and still controversial concepts. Some of these will stand the test of time; the others will be corrected in the next edition.

Emanuel Rubin

Howard Reisner

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ACKNOWLEDGMENTS This sixth edition of Essentials of Rubin’s Pathology is based on the hard work and insights of all those who made the sixth edition of Rubin’s Pathology possible. In addition, the editors would like to to thank the managing and editorial staff at the Lippincott Williams & Wilkins division of Wolters Kluwer Health and, in particular, Michael Tully, Sirkka Howes and Stacey Sebring for their continuing support. Without their help, this volume would not have been possible.

The editors also acknowledge the contributions made by our colleagues who participated in writing previous editions and those who offered suggestions and ideas for the current edition.

Stuart A. Aaronson Mohammad Alomari Adam Bagg Karoly Balogh Sue Bartow Hugh Bonner Patrick J. Buckley Stephen W. Chensue Daniel H. Connor Jeffrey Cossman John E. Craighead Mary Cunnane Joseph C. Fantone

John L. Farber Gregory N. Fuller Stanley R. Hamiliton Terrence J. Harrist Arthur P. Hays Robert B. Jennings Kent J. Johnson Michael J. Klein William D. Kocher Robert J. Kurman Ernest A. Lack Antonio Martinez-Hernandez Wolfgang J. Mergner

Juan Palazzo Robert O. Peterson Timothy R. Quinn Brian Schapiro Stephen M. Schwartz Benjamin H. Spargo Charles Steenbergen, Jr. Steven L. Teitelbaum Benjamin F. Trump Beverly Y. Wang Jianzhou Wang

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TABLE OF CONTENTS Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

CHAPTER 10: Blood Vessels . . . . . . . . . . . . . . . . . . . . . 255 Avrum I. Gotlieb and Amber Chang Liu

CHAPTER 1: Cell Adaptation, Cell Injury and Cell Death . . . . . . . . . . . . . . . . . . . . . . 1

CHAPTER 11: The Heart. . . . . . . . . . . . . . . . . . . . . . . . . 281

David S. Strayer and Emanuel Rubin

Jeffrey E. Saffitz

CHAPTER 2: Inflammation . . . . . . . . . . . . . . . . . . . . . . 25

CHAPTER 12: The Respiratory System . . . . . . . . . . . . 317

Hedwig S. Murphy

Mary Beth Beasley, William D. Travis, and Emanuel Rubin

CHAPTER 3: Repair, Regeneration and Fibrosis. . . . 49

CHAPTER 13: The Gastrointestinal Tract . . . . . . . . . . 351

Gregory C. Sephel and Jeffrey M. Davidson

CHAPTER 4: Immunopathology. . . . . . . . . . . . . . . . . . 71 Jeffrey S. Warren and David S. Strayer

CHAPTER 5: Neoplasia. . . . . . . . . . . . . . . . . . . . . . . . . . 93

Raphael Rubin

CHAPTER 14: The Liver and Biliary System . . . . . . . 391 Steven K. Herrine, Victor J. Navarro, and Raphael Rubin

David S. Strayer and Emanuel Rubin

CHAPTER 15: The Pancreas . . . . . . . . . . . . . . . . . . . . . . 429

CHAPTER 6: Developmental and Genetic Diseases . . . . . . . . . . . . . . . . . . 123

CHAPTER 16: The Kidney . . . . . . . . . . . . . . . . . . . . . . . 439

David S. Klimstra and Edward B. Stelow

Stephen Peiper and David S. Strayer

J. Charles Jennette

CHAPTER 7: Hemodynamic Disorders . . . . . . . . . . . 155

CHAPTER 17: The Lower Urinary Tract and Male Reproductive System . . . . . . . . . 473

Bruce M. McManus, Michael F. Allard, and Robert Yanagawa

Ivan Damjanov and Peter A. McCue

CHAPTER 8: Environmental and Nutritional Pathology. . . . . . . . . . . . . . . . . . . . . . . . . 173

CHAPTER 18: The Female Reproductive System . . . 497

David S. Strayer and Emanuel Rubin

George L. Mutter, Jaime Prat, and David A. Schwartz

CHAPTER 9: Infectious and Parasitic Diseases . . . . 195

CHAPTER 19: The Breast . . . . . . . . . . . . . . . . . . . . . . . . 531

David A. Schwartz

Anna Marie Mulligan and Frances P. O’Malley

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CONTENTS

CHAPTER 20: Hematopathology . . . . . . . . . . . . . . . . . 543

CHAPTER 25: The Head and Neck. . . . . . . . . . . . . . . . 665

Riccardo Valdez, Mary M. Zutter, Alina Dulau Florea, and Raphael Rubin

Bruce M. Wenig and Diane L. Carlson

CHAPTER 21: The Endocrine System . . . . . . . . . . . . . 591

CHAPTER 26: Bones and Joints . . . . . . . . . . . . . . . . . . 681 Roberto A. Garcia, Michael J. Klein, and Alan L. Schiller

Maria J. Merino and Martha Quezado

CHAPTER 22: Obesity, Diabetes Mellitus and Metabolic Syndrome. . . . . . . . . . . 619 Kevin Jon Williams and Elias S. Siraj

CHAPTER 23: The Amyloidoses . . . . . . . . . . . . . . . . . . 629 Philip N. Hawkins and Robert Kisilevsky

CHAPTER 27: Skeletal Muscle . . . . . . . . . . . . . . . . . . . 725 Lawrence C. Kenyon

CHAPTER 28: The Nervous System . . . . . . . . . . . . . . . 739 Gregory N. Fuller, J. Clay Goodman, and Thomas W. Bouldin

CHAPTER 29: The Eye . . . . . . . . . . . . . . . . . . . . . . . . . . 793 Gordon K. Klintworth

CHAPTER 24: The Skin . . . . . . . . . . . . . . . . . . . . . . . . . 637

Figure Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . 807

Craig A. Storm and David E. Elder

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .809

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1 | Cell Adaptation, Cell Injury and Cell Death

ESSENTIALS

Cell Adaptation, Cell Injury and Cell Death David S. Strayer • Emanuel Rubin

Reactions to Persistent Stress and Cell Injury ATROPHY AS AN ACTIVE RESPONSE HYPERTROPHY MECHANISMS OF HYPERTROPHY POSTMITOTIC CELLS MAY TURN OVER AUTOPHAGY HYPERPLASIA METAPLASIA DYSPLASIA CALCIFICATION Mechanisms and Morphology of Cell Injury HYDROPIC SWELLING SUBCELLULAR CHANGES ISCHEMIC CELL INJURY OXIDATIVE STRESS ISCHEMIA/REPERFUSION INJURY INTRACELLULAR STORAGE

Pathology is the study of structural and functional abnormalities that are expressed as diseases of organs and systems.

Classic theories attributed diseases to imbalances or noxious effects of humors on specific organs. In the 19th century, Rudolf Virchow, often referred to as the father of modern pathology, proposed that injury to the smallest living unit of the body, the cell, is the basis of all disease. To this day, clinical and experimental pathology remain rooted in this concept, which is now extended by an increased understanding of the molecular nature of many disease processes.

A living cell must maintain the ability to produce energy, much of which is spent in establishing a barrier between the internal milieu of the cell and a hostile environment. The plasma membrane, associated ion pumps and receptor molecules serve this purpose. A cell must also be able to adapt to adverse environmental conditions, such as changes in temperature, solute concentrations, oxygen supply, the presence of noxious agents and so on. If an injury exceeds the adaptive capacity of the cell, the cell dies.

How Exogenous Agents Injure Cells IONIZING RADIATION VIRAL CYTOTOXICITY CHEMICALS Cell Death NECROTIC CELL DEATH FROM EXOGENOUS INJURY NECROSIS FROM INTRACELLULAR INSULTS APOPTOSIS (PROGRAMMED CELL DEATH) RECEPTOR–LIGAND INTERACTIONS RELEASE OF CA2 Biologic Aging MAXIMAL LIFE SPAN THE CELLULAR BASIS OF AGING SUMMARY HYPOTHESIS OF AGING

From this perspective, pathology is the study of cell injury and the expression of a cell’s pre-existing capacity to adapt to such injury.

Reactions to Persistent Stress and Cell Injury Cells encounter many stresses as a result of changes in their internal and external environments. Patterns of response to such stresses comprise the cellular basis of disease.

If an injury exceeds the adaptive capacity of the cell, the cell dies. A cell exposed to persistent sublethal injury has limited available responses, the expression of which we interpret as evidence of cell injury. In general, mammalian cells adapt to injury by conserving resources: decreasing or ceasing differentiated functions and focusing exclusively on their own survival.

Persistent stress often leads to chronic cell injury. Although permanent organ injury is associated with the death of individual cells, the cellular response to persistent sublethal injury (whether chemical or physical) reflects adaptation of

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ESSENTIALS OF RUBIN’S PATHOLOGY

the cell to a hostile environment. Again, these changes are, for the most part, reversible on discontinuation of the stress.

The major adaptive responses are atrophy, hypertrophy, hyperplasia, metaplasia, dysplasia and intracellular storage of certain endogenous or exogenous materials. In addition, certain forms of neoplasia may reflect adaptive responses.

ATROPHY IS AN ACTIVE RESPONSE TO AN ALTERED ENVIRONMENT THAT RESULTS IN REDUCED FUNCTION OR SIZE OF CELLS OR ORGANS Clinically, atrophy is often noted as a decrease in size or function of an organ that occurs under pathologic or physiologic circumstances. Therefore, atrophy may result from disuse of skeletal muscle or from loss of trophic signals as part of normal aging. At the level of an individual cell, atrophy may be thought of as an adaptive response, whereby a cell accommodates to changes in its environment while remaining viable. However, most commonly, atrophy is a consequence of harmful processes such as those involved in some chronic diseases and biologic aging (see later). Reduction in an organ’s size may reflect reversible cell atrophy or irreversible loss of cells. For example, atrophy of the brain in Alzheimer disease is secondary to extensive cell death; in this case, the size of the organ cannot be restored (Fig. 1-1).

Atrophy occurs under a variety of conditions: ■

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Reduced functional demand: For example, after immobilization of a limb in a cast, muscle cells atrophy and muscular strength is reduced. When normal activity resumes, the muscle’s size and function return.

Inadequate supply of oxygen: Interference with blood supply to tissues is called ischemia. Although total cessation of oxygen perfusion results in cell death, partial ischemia is often compatible with cell viability. Under such circumstances, cell atrophy may be seen around the inadequately perfused margins of ischemic necrosis (infarcts)

FIGURE 1-1. Atrophy of the brain. Marked atrophy of the frontal lobe is noted in this photograph of the brain. The gyri are thinned and the sulci conspicuously widened.

■

■

■

in the heart, brain and kidney following vascular occlusion in these organs.

Insufficient nutrients: Starvation or inadequate nutrition associated with chronic disease leads to cell atrophy, particularly in skeletal muscle and adipose tissue.

Interruption of trophic signals: The functions of many cells depend on signals transmitted by chemical mediators, of which the endocrine system and neuromuscular transmission are the best examples. Loss of such signals via ablation of an endocrine gland or denervation results in atrophy of the target organ. Atrophy secondary to endocrine insufficiency is not restricted to pathologic conditions. For example, the endometrium atrophies when estrogen levels decrease after menopause (Fig. 1-2).

Persistent cell injury: Persistent cell injury is most commonly caused by chronic inflammation associated with prolonged viral or bacterial infections or by autoimmune

A

B FIGURE 1-2. Proliferative endometrium. A. A section of the uterus from a woman of reproductive age reveals a thick endometrium composed of proliferative glands in an abundant stroma. B. The endometrium of a 75-year-old woman (shown at the same magnification) is thin and contains only a few atrophic and cystic glands.

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■

■

disease. A good example is the atrophy of the gastric mucosa that occurs in association with chronic gastritis (see Chapter 13).

Increased pressure: Physical injury, such as prolonged pressure in inappropriate locations, produces atrophy.

Prolonged bed rest may lead to sustained pressure on the skin, causing atrophy of the skin and consequent decubitus ulcers (bed sores).

Chronic disease: Often, people afflicted with wasting chronic diseases such as cancer, congestive heart failure or acquired immunodeficiency syndrome (AIDS) demonstrate generalized atrophy of many tissues. Importantly, tissue loss exceeds what can be attributed to decreased caloric intake and reflects alterations in cytokines and other mediators.

HYPERTROPHY IS AN INCREASE IN CELL SIZE AND FUNCTIONAL CAPACITY Hypertrophy is an adaptive change that results in an increase in cellular size to satisfy increased functional demand or trophic signals. In some cases, increased cellular number (hyperplasia, see below) may also result. In organs made of terminally differentiated cells (e.g., heart, skeletal muscle), such adaptive responses are accomplished solely by increased cell size (Fig. 1-3). In other organs (e.g., kidney, thyroid), cell numbers and cell size may both increase.

Hypertrophy is associated with an initial increase in the degradation of proteins that do not contribute to the specific need for hypertrophy even as production of proteins that promote hypertrophy tends to increase. Programmed cell death (apoptosis, see below) may be inhibited, thereby resulting in an increase in cell survival.

MECHANISMS OF HYPERTROPHY VARY ACCORDING TO THE STIMULUS Hypertrophy of tissues may follow different pathways, depending on the requirements imposed by the increased functional demand. For instance, increased endurance of

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skeletal muscle entails increased aerobic activity (increased oxygen consumption). By contrast, the response to increased resistance (e.g., weight lifting) results in stimulation of signaling that does not necessitate increased oxygen supply (Fig. 1-4). Similarly, cardiac hypertrophy may occur in response to exercise (physiologic hypertrophy) or increased vascular resistance (pathologic hypertrophy). Each stimulus involves different soluble mediators that in turn activate distinct cellular signaling pathways, with much different consequences (see Chapter 11).

POSTMITOTIC CELLS MAY TURN OVER Historically, neurons, cardiac myocytes and skeletal muscle cells were considered to be incapable of mitosis. This view has generally been interpreted to imply that neurons and myocytes cannot be replaced and, therefore, that the brain, heart and skeletal muscle cannot respond to cell loss or increased demand by adding cells. Today, this is considered only partially correct. Whereas neurons and cardiac myocytes may not undergo mitosis, recent studies have demonstrated the presence of committed progenitor cells, which proliferate and differentiate in response to cell death and injury or, in the case of striated muscle, increased functional demand.

These newer concepts also suggest a natural equilibrium between a low rate of cell loss and replacement in organs that had previously been considered “postmitotic” or “terminally differentiated.” If this equilibrium is tilted to favor cell loss, organ atrophy may result, as seen in the heart, muscle and brain of the very aged. The equilibrium may also be shifted toward predominance of progenitor cell activity, as occurs in muscle hypertrophy.

1 | Cell Adaptation, Cell Injury and Cell Death

CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

Proteasomes and Cellular Homeostasis Cellular homeostasis requires mechanisms that allow the cell to destroy certain proteins selectively. Although there is evidence that more than one such pathway may exist, the bestunderstood mechanism by which cells target specific proteins for elimination is the ubiquitin (Ub)-proteasomal apparatus.

The importance of the proteasome is underscored by the fact that it may comprise up to 1% to 2% of the total protein of the cell. Proteasomes are evolutionarily highly conserved and are present in all eukaryotic cells. Mutations leading to interference with normal proteasomal function are lethal.

Proteasomes exist in two forms. The 20S proteasomes are important in the degradation of oxidized proteins. In 26S proteasomes, proteins to which Ub is bound are degraded.

A variant type of proteasome, the immunoproteasome, is formed in response to cellular production of interferon- (IFN- ). Immunoproteasomes are important in processing proteins to 8- or 9–amino acid peptides to be attached to major histocompatibility complex type I for presentation to the immune system as antigens (see Chapter 4).

Ub and Ubiquitination

FIGURE 1-3. Myocardial hypertrophy. Cross-section of the heart of a patient with long-standing hypertension shows pronounced, concentric left ventricular hypertrophy.

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Proteins to be degraded are identified as such by attaching small chains of Ub molecules to them, thereby serving to identify proteins to be destroyed. Ub is a 76–amino acid protein that is conjugated to proteins as a flag to identify those proteins to be destroyed. The process of attaching Ub to proteins is called ubiquitination (or ubiquitylation). The importance of ubiquitination and specific protein elimination

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ESSENTIALS OF RUBIN’S PATHOLOGY

AMP kinase

AMP:ATP 2+

[Ca ]

Endurance training

Peroxisome activation

Signaling molecules

i

TFAM

Mitochondrion

mt DNA replication mt DNA transcription Mitochondrial energy production

No muscle hypertrophy Slow myosin H chain Endurance

A Protein production by translation Resistance training

IGF-I and other growth factors Signal transduction eIF2 AKT

Transcription of genes that inhibit protein degradation

Fast myosin Muscle hypertrophy

FOXO

mTOR

FOXOPO4

Protein synthesis Ribosome biogenesis

Transcription of genes that promote protein degradations (e.g., Atrogin-1) Nucleus

B FIGURE 1-4. Mechanisms involved in muscle hypertrophy. A. Endurance training. Muscle strengthening for endurance entails repeated or prolonged exercise with small loads and leads to increases in the adenosine monophosphate–to–adenosine triphosphate (AMP:ATP) ratio, leading to increased AMP kinase activity. Such training also increases cytosol calcium concentration ([Ca2 ]i), which increases a number of cellular signaling intermediates. Consequent peroxisome activation increases the transcription factor– activating mitochondrial energy production (TFAM). Increased TFAM in turn activates both replication and transcription of mitochondrial DNA. The consequence is increase in muscle slow myosin H chain and improved endurance without muscle cell hypertrophy. B. Resistance training. Muscle strengthening by lifting weights, in which load, but not repetition, is increased, leads to muscle cell hypertrophy. This is mediated by increased insulin-like growth factor-I (IGF-I) and other growth factors (such as growth hormone), which act via a series of cellular intermediates such as transcription elongation factor eIF2, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). Each of these mediates changes in gene expression and metabolism. AKT phosphorylates and inactivates the forkhead transcription factor FOXO, causing it to leave the nucleus and causing decreased transcription of genes involved in protein degradation (e.g., Atrogin-1).

Simultaneously, increased eIF2 activity activates transcription of genes that inhibit protein degradation. The consequence is increased fast myosin H chain and increased myocyte size (hypertrophy).

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is fundamental to cellular adaptation to stress and injury.

Defective ubiquitination may play a role in several important neurodegenerative diseases. For example, mutations in parkin, a Ub ligase, are implicated in the pathogenesis of Parkinson disease in which undegraded parkin accumulates as Lewy bodies (see Chapter 28). Manipulation of ubiquitination may be important in tumor development.

Thus, papillomavirus strains that are associated with human cervical cancer (see Chapters 5 and 18) produce increased p53 ubiquitination, thereby accelerating p53 degradation.

Impaired ubiquitination may also be involved in some cellular degenerative changes that occur in aging and in some storage diseases.

AUTOPHAGY IS A MULTIFACETED CELLULAR AUTODIGESTIVE SYSTEM THAT IS IMPORTANT IN BOTH CELLULAR ADAPTATION AND PATHOGENESIS OF DISEASE Autophagy (from the Greek, “auto,” self; “phagy,” eat) comprises degradative pathways that operate through the transfer of cytoplasmic constituents to lysosomes for degradation.

This process, which rids cells of materials such as misfolded proteins, microorganisms and damaged organelles, is one

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of the most evolutionarily conserved cellular operations.

Material destined for self-cannibalization is sequestered in vesicles (autophagosomes) that fuse with a lysosome.

There are three recognized types of autophagy, which are illustrated in Figure 1-5. Autophagy normally acts as a cellular housekeeper. It is upregulated when the cell is stressed and represents an alternative source of nutrients for energy production and structural reconstitution for cell survival.

However, the autophagic pathway can also be called upon to kill cells (autophagic cell death).

Autophagy is important for the elimination of mutant or altered proteins that form aggregates. Such proteins are particularly involved in neurodegenerative diseases such as Huntington disease, Parkinson disease and Alzheimer disease. Thus, defects in autophagy are thought to play a key role in these disorders and also in various other conditions, including degenerative and inflammatory diseases, cancer and aging.

1 | Cell Adaptation, Cell Injury and Cell Death

CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

HYPERPLASIA IS AN INCREASE IN THE NUMBER OF CELLS IN AN ORGAN OR TISSUE Hypertrophy and hyperplasia often occur concurrently. The specific stimuli that induce hyperplasia and the mechanisms by which they act vary greatly from one tissue and cell type

A Macroautophagy B Microautophagy Mitochondrion

Bulk cytosol and constituents

Cellular organelles Lysosomal hydrolases

Endoplasmic reticulum

Golgi

C

Secretory organelles Autophagosome

Lysosomal receptor

C

C

Chaperone Unfolded protein

C Chaperone-mediated autophagy

Lysosomal hydrolases

Autolysosome

FIGURE 1-5. Types of autophagy. A. Macroautophagy, in which an autophagosome surrounds bulk cytosol and cytoplasmic organelles. B. Microautophagy, in which selected organelles or foreign materials (e.g., microbial pathogens) are engulfed by lysosomes. C. Chaperone-mediated autophagy, in which selected cellular macromolecules are conducted to lysosomes by chaperones (e.g., hsc70), which are recognized by lysosomal receptor proteins (e.g., lysosomal-associated membrane protein [LAMP]-2A). The targeted material is destroyed by lysosomal degradation.

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ESSENTIALS OF RUBIN’S PATHOLOGY

B

A FIGURE 1-6. Hyperplasia. A. Normal epidermis. B. Epidermal hyperplasia in psoriasis, shown at the same magnification as in A. The epidermis is thickened, owing to an increase in the number of squamous cells.

to the next. Whatever the stimulus, hyperplasia involves stimulating resting cells (G0) to enter the cell cycle (G1) and then to multiply. This may be a response to an altered endocrine milieu, increased functional demand or chronic injury.

These topics are discussed in Chapters 3 and 5.

■

■

■

Hormonal stimulation: Changes in hormone concentrations, whether physiologic or pathologic, can elicit proliferation of responsive cells. The normal increase in estrogens at puberty or early in the menstrual cycle leads to increased numbers of endometrial and uterine stromal cells. Exogenous estrogen administration to postmenopausal women has the same effect. Ectopic hormone production may also result in hyperplasia. For example, erythropoietin production by renal tumors may lead to hyperplasia of erythrocytes in the bone marrow.

Increased functional demand: Hyperplasia, like hypertrophy, may be a response to increased physiologic demand. To illustrate, at high altitudes, low atmospheric oxygen content leads to compensatory hyperplasia of erythrocyte precursors in the bone marrow and increased erythrocytes in the blood (secondary polycythemia).

Chronic blood loss, as in excessive menstrual bleeding, also causes hyperplasia of erythrocytic elements.

Chronic injury: Long-standing inflammation or chronic physical or chemical injury often results in a hyperplastic response. A common cause is pressure from ill-fitting shoes, which causes hyperplasia of the skin of the foot, termed corns or calluses.

Inappropriate hyperplasia can itself be harmful—witness the unpleasant consequences of psoriasis, which is characterized by conspicuous hyperplasia of the skin (Fig. 1-6). Excessive estrogen stimulation, whether from endogenous or exogenous sources, may lead to endometrial hyperplasia.

The cellular and molecular mechanisms responsible for hyperplastic responses clearly relate to control of cell proliferation. These topics are discussed in Chapters 3 and 5 and under the heading “Liver Regeneration” in Chapter 14.

Rubin_6e_CH01.indd 6

METAPLASIA IS CONVERSION OF ONE DIFFERENTIATED CELL TYPE TO ANOTHER Metaplasia is usually an adaptive response to chronic persistent injury, in which a tissue assumes the phenotype that provides it with the best protection from the insult. Most commonly, glandular epithelium is replaced by squamous epithelium. Columnar or cuboidal lining cells may be committed to mucus production but may not be adequately resistant to the effects of chronic irritation or a pernicious chemical. For example, prolonged exposure of the bronchial epithelium to tobacco smoke leads to squamous metaplasia. A similar response occurs in the endocervix afflicted by chronic infection (Fig. 1-7).

The process is not restricted to squamous differentiation. When highly acidic gastric contents reflux chronically into the lower esophagus, the squamous epithelium of the esophagus may be replaced by stomach-like glandular mucosa (Barrett epithelium). This can be thought of as an

FIGURE 1-7. Squamous metaplasia. A section of endocervix shows the normal columnar epithelium at both margins and a focus of squamous metaplasia in the center.

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adaptation to protect the esophagus from injury by gastric acid and pepsin, to which the normal gastric mucosa is resistant. Metaplasia may also consist of replacement of one glandular epithelium by another. Metaplasia of transitional epithelium to glandular epithelium occurs when the bladder is chronically inflamed (cystitis glandularis).

Although metaplasia is often adaptive, it is not necessarily innocuous. For example, squamous metaplasia not only protects a bronchus from tobacco smoke but also impairs mucus production and ciliary clearance. Neoplastic transformation may occur in metaplastic epithelium; cancers of the lung, cervix, esophagus, stomach and bladder often arise in such areas. Metaplasia is usually fully reversible. If the noxious stimulus is removed (e.g., when one stops smoking), the metaplastic epithelium eventually returns to normal.

DYSPLASIA IS DISORDERED GROWTH AND MATURATION OF THE CELLULAR COMPONENTS OF A TISSUE The cells that comprise an epithelium normally exhibit uniformity of size, shape and nuclear structure. Moreover, they are arranged in a regular fashion, as, for example, a squamous epithelium progresses from plump basal cells to flat superficial cells. In dysplasia, this monotonous appearance is disturbed by (1) variation in cell size and shape; (2) nuclear enlargement, irregularity and hyperchromatism and (3) disarray in the arrangement of cells within the epithelium (Fig. 1-8). Dysplasia occurs most often in hyperplastic squamous epithelium, as seen in epidermal actinic keratosis (caused by sunlight) and in areas of squamous metaplasia, such as in the bronchus or the cervix. It is not, however,

exclusive to squamous epithelium. Ulcerative colitis, an inflammatory disease of the large intestine, is often complicated by dysplastic changes in the columnar mucosal cells.

Like metaplasia, dysplasia is a response to a persistent injurious influence and will usually regress, for example, on cessation of smoking or the disappearance of human papillomavirus from the cervix. However, it shares many cytologic features with cancer, and the line between the two may be very fine. For example, it may be difficult to distinguish severe dysplasia from early cancer of the cervix. Dysplasia is preneoplastic in the sense that it is a necessary stage in the multistep cellular evolution to cancer. In fact, dysplasia is included in the morphologic classifications of the stages of intraepithelial neoplasia in various organs (e.g., cervix, prostate, bladder). Severe dysplasia is considered an indication for aggressive preventive therapy to cure the underlying cause, eliminate the noxious agent or surgically remove the offending tissue.

As in the development of cancer (see Chapter 5), dysplasia results from sequential mutations in a proliferating cell population. In other words, dysplasia is the morphologic expression of a molecular disturbance in growth regulation. However, unlike cancer cells, dysplastic cells are not entirely autonomous, and with intervention, tissue appearance may still revert to normal.

1 | Cell Adaptation, Cell Injury and Cell Death

7

CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

CALCIFICATION MAY OCCUR AS PART OF NORMAL DEVELOPMENT OR AS A REFLECTION OF AN ABNORMAL PROCESS Calcium entry into dead or dying cells occurs because of the inability of such cells to maintain a steep calcium gradient.

This cellular calcification is not ordinarily visible except as inclusions within mitochondria.

“Dystrophic” calcification refers to the macroscopic deposition of calcium salts in injured tissues. This type of calcification does not simply reflect an accumulation of calcium derived from the bodies of dead cells but rather represents an extracellular deposition of calcium from the circulation or interstitial fluid. In many locations, calcification has no functional consequences. However, dystrophic calcification may also occur in crucial locations, such as in the mitral or aortic valves, thereby leading to impeded blood flow. “Metastatic” calcification reflects deranged calcium metabolism that has its origin in cell injury. Metastatic calcification is associated with an increased serum calcium concentration (hypercalcemia). In general, almost any disorder that increases the serum calcium level can lead to calcification in such inappropriate locations as the alveolar septa of the lung, renal tubules and blood vessels.

Mechanisms and Morphology of Cell Injury FIGURE 1-8. Dysplasia. Dysplastic epithelium of the uterine cervix lacks normal polarity, and individual cells show hyperchromatic nuclei and a greater than normal nucleus-to-cytoplasm ratio. Normal cervical epithelium is at left. Compare, for example, the size and hyperchromaticity of nuclei in the dysplastic cells (straight arrows) with the characteristics of normal counterparts (curved arrows, left) at comparable height in the adjacent normal cervix. In dysplasia, cellular arrangement is disorderly, largely lacking appropriate histologic maturation, from the basal layers to the surface.

Rubin_6e_CH01.indd 7

All cells have efficient mechanisms to deal with shifts in environmental conditions. Thus, ion channels open or close, harmful chemicals are detoxified, metabolic stores such as fat or glycogen may be mobilized and catabolic processes may lead to the segregation of internal particulate materials. It is when environmental changes exceed the cell’s capacity to maintain normal homeostasis that cell injury occurs. If the stress is removed in time or if the cell can withstand the assault, cell injury is reversible, and complete structural and functional integrity is restored.

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ESSENTIALS OF RUBIN’S PATHOLOGY

FIGURE 1-9. Hydropic swelling. A needle biopsy of the liver of a patient with toxic hepatic injury shows severe hydropic swelling in the centrilobular zone. The affected hepatocytes exhibit central nuclei and cytoplasm distended (ballooned) by excess fluid.

The cell can also be exposed to persistent sublethal stress, as in mechanical irritation of the skin or exposure of the bronchial mucosa to tobacco smoke. In such instances, the cell has time to adapt to reversible injury in several ways, each of which has its morphologic counterpart. On the other hand, if the stress is severe, irreversible injury leads to death of the cell. The precise moment at which reversible injury gives way to irreversible injury, the “point of no return,” cannot be identified at present.

HYDROPIC SWELLING IS A REVERSIBLE INCREASE IN CELL VOLUME Hydropic swelling is characterized by a large, pale cytoplasm and a normally located nucleus (Fig. 1-9). The greater volume refl ects an increased water content. Hydropic

swelling reflects acute, reversible cell injury and may result from such varied causes as chemical and biologic toxins, viral or bacterial infections, ischemia, excessive heat or cold, etc.

By electron microscopy, the number of organelles is unchanged, although they appear dispersed in a larger volume.

The excess fluid accumulates preferentially in the cisternae of the endoplasmic reticulum (ER), which are conspicuously dilated, presumably because of ionic shifts into this compartment (Fig. 1-10). Hydropic swelling is entirely reversible when the cause is removed.

Hydropic swelling results from impairment of cellular volume regulation, a process that controls ionic concentrations in the cytoplasm. This regulation, particularly for sodium (Na ), involves three components: (1) the plasma membrane, (2) the plasma membrane Na pump and (3) the supply of adenosine triphosphate (ATP). The plasma membrane imposes a barrier to the flow of Na down a concentration gradient into the cell and prevents a similar efflux of potassium (K ) from the cell. However, the barrier to Na is imperfect, and the relative leakiness to that ion permits its passive entry into the cell. To compensate for this intrusion, the energy-dependent plasma membrane Na pump (Na /K -ATPase), which is fueled by ATP, extrudes Na from the cell. Injurious agents may interfere with this membrane-regulated process by (1) increasing the permeability of the plasma membrane to Na , thereby exceeding the capacity of the pump to extrude Na ; (2) damaging the pump directly or (3) interfering with the synthesis of ATP, thereby depriving the pump of its fuel. In any event, the accumulation of Na in the cell leads to an increase in water content to maintain isosmotic conditions; the cell then swells.

SUBCELLULAR CHANGES OCCUR IN REVERSIBLY INJURED CELLS ■

Endoplasmic reticulum: The cisternae of the ER are distended by fluid in hydropic swelling. In other forms of acute, reversible cell injury, membrane-bound polysomes may undergo disaggregation and detach from the surface of the rough ER.

A

B FIGURE 1-10. Ultrastructure of hydropic swelling of a liver cell. A. Two apposed normal hepatocytes with tightly organized, parallel arrays of rough endoplasmic reticulum. B. Swollen hepatocyte in which the cisternae of the endoplasmic reticulum are dilated by excess fluid.

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CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

9

1 | Cell Adaptation, Cell Injury and Cell Death

Plama membrane bleb

Disaggregated ribosomes

Increased intracellular volume

Dilated, vesicular endoplasmic reticulum

Aggregated cytoskeletal elements

Mitochondrial swelling and calcification

FIGURE 1-11. Ultrastructural features of reversible cell injury.

■

■

■

Mitochondria: In some forms of acute injury, particularly ischemia, mitochondria swell. This enlargement reflects the dissipation of the energy gradient and consequent impairment of mitochondrial volume control. Amorphous densities rich in phospholipid may appear, but these effects are fully reversible on recovery.

Plasma membrane: Blebs of the cellular plasma membrane— that is, focal extrusions of the cytoplasm—are occasionally noted. These can detach from the membrane into the external environment without the loss of cell viability.

Nucleus: In the nucleus, reversible injury is reflected principally in nucleolar change. The fibrillar and granular components of the nucleolus may segregate.

Alternatively, the granular component may be diminished, leaving only a fibrillar core.

These changes in cell organelles (Fig. 1-11) are reflected in functional derangements (e.g., reduced protein synthesis and impaired energy production). After withdrawal of an acute stress that has led to reversible cell injury, by definition, the cell returns to its normal state.

ISCHEMIC CELL INJURY USUALLY RESULTS FROM OBSTRUCTION TO THE FLOW OF BLOOD When tissues are deprived of oxygen, ATP cannot be produced by aerobic metabolism and is instead generated inefficiently by anaerobic metabolism. Ischemia initiates a series of chemical and pH imbalances, which are accompanied by enhanced generation of injurious free-radical species. The damage produced by short periods of ischemia tends to be reversible if the circulation is restored. However, cells subjected to long episodes of ischemia become irreversibly injured and die. The mechanisms of cell damage are discussed later.

Rubin_6e_CH01.indd 9

OXIDATIVE STRESS LEADS TO CELL INJURY IN MANY ORGANS For human life, oxygen is both a blessing and a curse. Without it, life is impossible, but oxygen metabolism can also produce partially reduced oxygen species that react with virtually any molecule they reach.

Reactive Oxygen Species (ROS) ROS have been identified as the likely cause of cell injury in many diseases and other damaging events. These include: ■ ■ ■

■ ■

The inflammatory process (see Chapter 2) Chemical toxicity Ionizing radiation in which injury is the result of the direct formation of hydroxyl radicals (•OH) from the radiolysis of water (H2O) Chemical carcinogenesis Aging (see below)

ROS have also been implicated in normal cell signaling, including modulation of gene regulation, activation of mitogen-activated protein (MAP) kinases, reversible protein modifications (e.g., phosphorylation and dephosphorylation) and so forth. However, when present in excess under pathologic conditions, ROS may exert profound deleterious effects on signaling pathways. Among these pathways are those leading to cell death via apoptosis (see below).

Cells may also be injured when oxygen is present in concentrations greater than normal. The lungs of adults and the eyes of premature newborns (retrolental fibroplasia) were at one time the major targets of such oxygen toxicity, until the danger was recognized.

Complete reduction of O 2 to H 2O by mitochondrial electron transport involves the transfer of four electrons.

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10

ESSENTIALS OF RUBIN’S PATHOLOGY O2 Cytosolic enzymes

Electron transport chain

Superoxide O2 is produced principally by leaks in mitochondrial electron transport or as part of the inflammatory response (see Chapter 2). Superoxide and other ROS are the principal effectors of cellular oxidative defenses that destroy pathogens, fragments of necrotic cells or other phagocytosed material.

They may also serve as signaling intermediates that elicit the release of proteolytic and other degradative enzymes (see Chapter 2).

P450

O2– CoQ 4H+

2H+

2H2O

O –

SOD

2

Peroxynitrite

SOD OD GPX

H2O2

2H2O2

H2O2

Catalase GPX

O2 + 2H2O

H2O

Peroxisome

Mitochondrion

FIGURE 1-12. Mechanisms by which reactive oxygen radicals are generated from molecular oxygen and then detoxified by cellular enzymes. CoQ, coenzyme Q; GPX, glutathione peroxidase; H , hydrogen ion; H2O, water; H2O2, hydrogen peroxide; O2, oxygen; O2 , superoxide; SOD, superoxide dismutase.

There are three partially reduced species that are intermediate between O2 and H2O, representing transfers of varying numbers of electrons (Fig. 1-12). They are O2 , superoxide anion (one electron); H2O2, hydrogen peroxide (two electrons) and •OH, the hydroxyl radical (three electrons). For the most part, these ROS are produced principally by leaks in mitochondrial electron transport, with an additional contribution from the mixed-function oxygenase (P450) system. The major forms of ROS are listed in Table 1-1.

Peroxynitrite (ONOO ) is formed by the interaction of two free radicals, namely, superoxide (O2 ) and nitric oxide (NO): NO O2 → ONOO . ONOO attacks a wide range of biologically important molecules, including lipids, proteins and DNA. Nitric oxide, a molecule generated in many tissues, is a potent vasodilator and mediator of several important biologic processes.

Hydrogen Peroxide O2 anions are catabolized by superoxide dismutase (SOD) to produce H2O2. H2O2 is also produced directly by several oxidases in cytoplasmic peroxisomes (see Fig. 1-12). By itself, H2O2 is not particularly injurious, and it is largely metabolized to H2O by catalase or glutathione peroxidase in both the cytosol and the mitochondria (see Fig. 1-12).

However, when produced in excess, it is converted to highly reactive •OH. In neutrophils, myeloperoxidase transforms H2O2 to the potent radical hypochlorite (OCl ), which is lethal for microorganisms and cells.

Hydroxyl Radical Hydroxyl radicals (•OH) are formed by (1) the radiolysis of H2O, (2) the reaction of H2O2 with ferrous iron (Fe2 ) (Fenton reaction) and (3) the reaction of O2 with H2O2 (Haber-Weiss reaction). The •OH radical is the most reactive

Table 1-1

Reactive Oxygen Species Molecule

Attributes

Hydrogen peroxide (H2O2)

Forms free radicals via Fe2 -catalyzed Fenton reaction Diffuses widely within the cell

Superoxide anion (O2 )

Generated by leaks in the electron transport chain and some cytosolic reactions Produces other ROS Does not readily diffuse far from its origin

Hydroxyl radical (•OH)

Generated from H2O2 by Fe2 -catalyzed Fenton reaction The intracellular radical most responsible for attack on macromolecules

Peroxynitrite (ONOO •)

Formed from the reaction of nitric oxide (NO) with O2 and damages macromolecules

Lipid peroxide radicals (RCOO•)

Organic radicals produced during lipid peroxidation

Hypochlorous acid (HOCl)

Produced by macrophages and neutrophils during respiratory burst that accompanies phagocytosis Dissociates to yield radical hypochlorite (OCl )

Fe2 , ferrous iron; ROS, reactive oxygen species.

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11

CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

GSSG

GSH

Glutathione peroxidase catalyzes the reduction of H2O2 and lipid peroxides in mitochondria and the cytosol (H2O2 2GSH → 2H2O GSSG).

Scavengers of ROS H 2O

H2O2

■

Fe2+

O2

Fe3+

O2–

■

•OH ■

Lipid peroxidation

Proteins

DNA

■

(inner mitochondrial (oxidative membrane damage) damage)

Vitamin E (␣-tocopherol) is a terminal electron acceptor and, therefore, blocks free-radical chain reactions. Given that it is fat soluble, it exerts its activity in lipid membranes, protecting them against lipid peroxidation.

Vitamin C (ascorbate) is water soluble and reacts directly with O2, OH• and some products of lipid peroxidation.

It also serves to regenerate the reduced form of vitamin E. Retinoids, the precursors of vitamin A, are lipid soluble and function as chain-breaking antioxidants.

NO is the product of constitutive or inducible nitric oxide synthase (NOS). Although it may bind to superoxide to form highly reactive peroxynitrite, NO is also a major mechanism by which ROS are contained. The ability of NO to increase proteasomal activity, and thus decrease cellular iron uptake by the transferrin receptor, may be involved.

1 | Cell Adaptation, Cell Injury and Cell Death

■

CELL DEATH FIGURE 1-13. Mechanisms of cell injury by activated oxygen species.

Fe2 , ferrous iron; Fe3 , ferric iron; GSH, glutathione; GSSG, glutathione disulfide; H2O2, hydrogen peroxide; O2, oxygen; O2 , superoxide anion; •OH, hydroxyl radical.

molecule of ROS, and there are several mechanisms by which it can damage macromolecules.

■

■

■

Lipid peroxidation: This process ultimately results in the destruction of the unsaturated fatty acids of phospholipids and a loss of membrane integrity.

Protein interactions: As a result of oxidative damage caused by •OH, proteins undergo fragmentation, crosslinking, aggregation and eventually degradation.

DNA damage: DNA is an important target of the •OH. A variety of structural alterations include strand breaks, modifi ed bases and cross-links between strands. In most cases, the integrity of the genome can be reconstituted by the various DNA repair pathways. However, if oxidative damage to DNA is sufficiently extensive, the cell dies.

ISCHEMIA/REPERFUSION INJURY REFLECTS OXIDATIVE STRESS Ischemia/reperfusion (I/R) injury is a common clinical problem that arises in occlusive cardiovascular disease, infection, shock and many other settings. I/R injury reflects the interplay of transient ischemia, consequent tissue damage and exposure of damaged tissue to the oxygen that arrives when blood flow is reestablished (reperfusion). Initially, ischemic cellular damage leads to the generation of free-radical species. Reperfusion then provides abundant molecular O2 to combine with free radicals to form ROS. The evolution of I/R injury also involves several other factors, including inflammatory mediators (tumor necrosis factor- [TNF- ], interleukin-1 [IL-1]), platelet-activating factor, NOS, NO, intercellular adhesion molecules, dysregulation of Ca2 homeostasis and many more.

Reperfusion injury can be put into perspective by emphasizing that there are three different degrees of cell injury, depending on the duration of the ischemia: ■

Figure 1-13 summarizes the mechanisms of cell injury by activated oxygen species.

■

Cellular Defenses against Reactive Oxygen Species

■

Cells manifest potent antioxidant defenses against ROS, including detoxifying enzymes such as SOD, catalase and glutathione peroxidase and exogenous free-radical scavengers.

Detoxifying Enzymes ■ ■

SOD is the first line of defense against O2 , converting it to H2O2 and O2 (2O2– 2H → O2 H2O2).

Catalase, principally located in peroxisomes, is one of two enzymes that complete the dissolution of O2 by eliminating H2O2 and, therefore, its potential conversion to OH• (2H2O2 → 2H2O O2).

Rubin_6e_CH01.indd 11

With short periods of ischemia, reperfusion (and, therefore, the resupply of oxygen) completely restores the structural and functional integrity of the cell. Cell injury in this case is completely reversible.

With longer periods of ischemia, reperfusion is not associated with restoration of cell structure and function but rather with deterioration and death of the cells Lethal cell injury may develop during the period of ischemia itself, in which case reperfusion is not a factor.

A longer period of ischemia is needed to produce this third type of cell injury.

INTRACELLULAR STORAGE IS RETENTION OF MATERIALS WITHIN THE CELL The substances that accumulate may be normal (nutrients such as fat and glycogen) or abnormal, endogenous or exogenous, harmful or innocuous (Fig. 1-14).

■

Degraded phospholipids, which result from the turnover of endogenous membranes, are stored in lysosomes

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ESSENTIALS OF RUBIN’S PATHOLOGY

A

B

C

D

E

F FIGURE 1-14. Abnormal intracellular storage. A. Lipid accumulation in macrophages in a cutaneous xanthoma. B. Abnormal cholesterol accumulation in an atherosclerotic plaque. C. Storage of abnormal, mutant, 1-antitrypsin in the liver (red granules). Periodic acid-Schiff (PAS) stain after diastase treatment to remove glycogen. D. Lipofuscin. Photomicrograph of the liver from an 80-year-old man shows golden cytoplasmic granules, which represent lysosomal storage of lipofuscin. E. Melanin storage (arrows) in an intradermal nevus. F. Carbon pigment storage. A mediastinal lymph node, which drains the lungs, exhibits numerous macrophages that contain black anthracotic (carbon) pigment. This material was inhaled and originally deposited in the lungs. G. Iron storage in hereditary hemochromatosis.

Prussian blue stain of the liver reveals large deposits of iron within hepatocellular lysosomes.

G

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CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

■ ■

Fat Abnormal accumulation of fat is most conspicuous in the liver, a subject treated in detail in Chapter 14. When delivery of free fatty acids to the liver is increased, as in diabetes, or when intrahepatic lipid metabolism is disturbed, as in alcoholism, triglycerides accumulate in liver cells. Fatty liver is identified morphologically as lipid globules in the cytoplasm. Other organs, including the heart, kidney and skeletal muscle, also store fat, as do atherosclerotic plaque macrophages (see Fig. 1-14A,B). Fat storage is generally reversible, and there is no evidence that the excess fat by itself interferes with cell function (although such storage may well be associated with disease).

Cholesterol The human body has a love–hate relationship with cholesterol. On the one hand, it is a critical component of all plasma membranes. On the other hand, when stored in excess, it is closely associated with atherosclerosis and cardiovascular disease, the leading cause of death in the Western world (see Chapter 10). Advanced lesions of atherosclerosis are characterized by extracellular deposition of cholesterol (see Fig. 1-14B). In several disorders characterized by elevated blood levels of cholesterol (e.g., familial hypercholesterolemia or primary biliary cirrhosis), macrophages store cholesterol. When clusters of these cells in subcutaneous tissues become grossly visible, they are termed xanthomas (see Fig. 1-14A).

Lipofuscin Lipofuscin is a mixture of lipids and proteins containing a golden brown pigment called ceroid. Lipofuscin tends to accumulate by accretion of oxidized, cross-linked proteins and is not digestible. It occurs mainly in terminally differentiated cells (neurons and cardiac myocytes) or in cells that cycle infrequently (hepatocytes) (see Fig. 1-14D).

It is often more conspicuous in conditions associated with atrophy of an organ. Although lipofuscin was previously thought to be benign, there is increasing evidence that it may be both a result and a cause of increasing oxidant stress in cells. It may impair both proteasomal function and lysosomal degradation of senescent or poorly functioning organelles.

Rubin_6e_CH01.indd 13

Melanin Melanin is an insoluble, brown-black pigment found principally in the epidermal cells of the skin but also in the eye and other organs (see Fig. 1-14E). It is located in intracellular organelles known as melanosomes and results from the polymerization of certain oxidation products of tyrosine. The amount of melanin is responsible for the differences in skin color among the various races, as well as the color of the eyes. It serves a protective function, owing to its ability to absorb ultraviolet light. In white persons, exposure to sunlight increases melanin formation (tanning). Melanin is discussed in detail in Chapter 24.

Exogenous Substances Anthracosis refers to the storage of carbon particles in the lung and regional lymph nodes (see Fig. 1-14F). Virtually all urban dwellers inhale particulates of organic carbon generated by the burning of fossil fuels. These particles accumulate in alveolar macrophages and are also transported to hilar and mediastinal lymph nodes, where the indigestible material is stored indefinitely within macrophages. Although the gross appearance of the lungs of persons with anthracosis may be alarming, the condition is innocuous.

Tattoos are the result of the introduction of insoluble metallic and vegetable pigments into the skin, where they are engulfed by dermal macrophages and persist for a lifetime.

1 | Cell Adaptation, Cell Injury and Cell Death

■

and may be recycled or remain as insoluble pigments (lipofuscin) (see Fig. 1-14D).

Substances that cannot be metabolized accumulate in cells. These include (1) endogenous substrates that are not further processed because a key enzyme is missing (hereditary storage diseases) (see Chapter 6); (2) insoluble endogenous pigments, such as lipofuscin (see previous bulleted item) and melanin (see Fig. 1-14E); (3) aggregates of normal or abnormal proteins and (4) exogenous particulates (e.g., inhaled silica and carbon or injected tattoo pigments).

Overload of normal body constituents, including iron, copper and cholesterol, injures a variety of cells.

Abnormal proteins may be toxic when they are retained within a cell. Examples are Lewy bodies in Parkinson disease and mutant 1-antitrypsin in liver disease (see Fig. 1-14C).

13

Iron and Other Metals Total body iron may be increased by enhanced intestinal iron absorption, as in some anemias, or by administration of ironcontaining erythrocytes in a transfusion. In either case, the excess iron is stored intracellularly as ferritin and hemosiderin (see Fig. 1-14G). Increasing the body’s total iron content leads to progressive accumulation of hemosiderin (a partially denatured form of ferritin that aggregates easily and is recognized microscopically as yellow-brown granules in the cytoplasm), a condition termed hemosiderosis. Intracellular accumulation of iron in hemosiderosis does not usually injure cells. However, if the increase in total body iron is extreme, we speak of iron overload syndromes (see Chapter 14), in which iron deposition is so severe that it damages vital organs— particularly the heart, liver and pancreas. Excessive iron storage in some organs is also associated with an increased risk of cancer. Pulmonary siderosis encountered among certain metal polishers is accompanied by an increased risk of lung cancer. Hereditary hemochromatosis (a genetic abnormality of iron absorption) leads to a higher incidence of liver cancer as well as cirrhosis and cardiac disease.

Excess accumulation of lead, particularly in children, causes mental retardation and anemia. In Wilson disease, a hereditary disorder of copper metabolism, storage of excess copper in the liver and brain may produce severe chronic disease of those organs.

How Exogenous Agents Injure Cells Ionizing radiation, chemicals and viral pathogens injure cells by diverse mechanisms, often by direct interactions with and damage to critical cell components. Other agents

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14

ESSENTIALS OF RUBIN’S PATHOLOGY

may require metabolic activation that produces highly reactive free radicals (ROS), or as is the case with ionizing radiation, directly produce reactive •OH radicals. Viruses may subvert intrinsic cell death pathways (apoptosis, see below) to their advantage or provoke immune-mediated injury.

IONIZING RADIATION DAMAGES CELLS

BY PRODUCTION OF HYDROXYL RADICALS AND DIRECT MUTAGENIC EFFECTS The term ionizing radiation connotes energy that is high enough to cause radiolysis of water, thereby directly forming •OH. As noted previously, •OH interacts with DNA and inhibits DNA replication. For a nonproliferating cell, such as a hepatocyte or a neuron, the inability to divide is of little consequence. For a proliferating cell, however, the prevention of mitosis is a catastrophic loss of function.

Once a proliferating cell can no longer divide, it dies (see below), a process that rids the body of those cells that have lost their prime function. Direct mutagenic effects of ionizing radiation on DNA are also important. The cytotoxic effects of ionizing radiation are dose dependent. Whereas exposure to significant amounts of radiation impairs the replicating capacity of cycling cells, massive doses of radiation may kill both proliferating and quiescent cells directly (see Chapter 8).

CHEMICALS INJURE CELLS DIRECTLY AND INDIRECTLY Innumerable chemicals can damage almost any cell in the body. The science of toxicology attempts to define the mechanisms that determine both target cell specificity and the mechanism of action of such chemicals. Toxic chemicals either (1) are themselves not toxic but are metabolized to yield an ultimate toxin that interacts with the target cell or (2) interact directly with cellular constituents without requiring metabolic activation. Whatever the mechanism, the result is usually necrotic cell death (see below).

Liver Necrosis Caused by the Metabolic Products of Chemicals Acetaminophen, an important constituent of many analgesics, is a well-studied hepatotoxin, which is metabolized by the mixed-function oxidase system of the hepatocyte and causes liver cell necrosis. The drug is innocuous in recommended doses, but when consumed to excess, it is highly toxic to the liver. Most acetaminophen is enzymatically converted in the liver to nontoxic glucuronide or sulfate metabolites. Less than 5% of acetaminophen is ordinarily metabolized by isoforms of cytochrome P450 to N-acetylp-benzoquinone imine (NAPQI), a highly reactive quinone (Fig. 1-15). However, when large doses of acetaminophen overwhelm the glucuronidation pathway, toxic amounts of

VIRAL CYTOTOXICITY IS DIRECT OR IMMUNOLOGICALLY MEDIATED The means by which viruses cause cell injury and death are as diverse as viruses themselves. Unlike bacteria, a virus requires a cellular host to (1) house it; (2) provide enzymes, substrates and other resources for viral replication and (3) serve as a source for dissemination when mature virions are ready to be spread to other cells.

■

■

■

Direct toxicity: Viruses may injure cells directly by subverting cellular enzymes and depleting the cell’s nutrients, thereby disrupting the normal homeostatic mechanisms. The mechanisms underlying virus-induced lysis of cells, however, are complex.

Manipulation of apoptosis (see below): There are many viral activities that can elicit apoptosis. For example, apoptosis is activated when the cell detects episomal (extrachromosomal) DNA replication. Because viruses must avoid cell death before they have produced infectious progeny, they have evolved mechanisms to counteract this effect by upregulating antiapoptotic proteins and inhibiting proapoptotic ones. Some viruses also encode proteins that induce apoptosis once daughter virions are released.

Immunologically mediated cytotoxicity: Both humoral and cellular arms of the immune system protect against the harmful effects of viral infections by eliminating infected cells. These arms of the immune system eliminate virus-infected cells by inducing apoptosis or by lysing the cell with complement (see Chapter 4).

Rubin_6e_CH01.indd 14

Acetaminophen O HNCCH3 15%

80%

Sulfates Glucuronates

Other detoxification pathways

OH 5%

P450 O HNCCH3

NAPQI O

O

HNCCH3

GSH transferase S–G

Covalent binding

Proteins DNA Mitochondria

Oxidative stress

•OH Mercapturic acid NECROSIS

FIGURE 1-15. Chemical reactions involved in acetaminophen hepatotoxicity. GSH, glutathione; NAPQI, N-acetyl-p-benzoquinone imine.

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NAPQI are formed. The conjugation of NAPQI with sulfhydryl groups on liver proteins causes extensive cellular dysfunction and subsequent injury. At the same time, NAPQI depletes the antioxidant glutathione (GSH), rendering the cell more susceptible to free-radical–induced injury. Thus, conditions that deplete GSH, such as starvation, enhance the toxicity of acetaminophen. In addition, acetaminophen toxicity is increased by chronic alcohol consumption, an effect mediated by an ethanol-induced increase in the 3A4 isoform of P450, which results in increased production of NAPQI. Other hepatotoxic compounds (such as carbon tetrachloride [CCl4]) produce metabolites that directly peroxidate and damage cell membrane phospholipids.

Chemicals That Are Not Metabolized Directly cytotoxic chemicals interact with cellular constituents without prior metabolic conversion. The critical cellular targets are diverse and include, for example, mitochondria (heavy metals and cyanide), cytoskeleton (phalloidin from toxic mushrooms) and DNA (chemotherapeutic alkylating agents). The interaction of directly cytotoxic chemicals with GSH (alkylating agents) also weakens the cell’s antioxidant defenses.

Cell Death Three main avenues leading to cell death have been delineated: necrosis, apoptosis and most recently, autophagy.

Necrosis is often defined as accidental and passive cell death caused by a hostile environment to which a cell could not adapt effectively. By contrast, apoptosis is a programmed form of cellular suicide in which the cell participates actively in its own demise. Apoptosis has been appreciated as a mechanism by which individual cells use their own signaling systems to sacrifice themselves both for embryonic development and for the maintenance of the mature organism. Autophagic cell death (see above) is also an active signaling process that is elicited when a stressful environment requires autodigestion of a portion of the cell’s macromolecular constituents.

The various forms of cell death are not strictly separate but rather share molecular effectors and signaling pathways.

For the sake of clarity, mechanisms of cell death by necrosis, apoptosis and autophagy are presented separately, but it is important to understand that all of these processes borrow from each other, involve signaling and collaborate with each other.

15

Whatever the nature of the lethal insult, cell necrosis is heralded by disruption of the permeability barrier function of the plasma membrane. Normally, extracellular concentrations of Na and Ca2 are orders of magnitude greater than intracellular concentrations. The opposite holds for potassium. The selective ion permeability requires (1) considerable energy, (2) structural integrity of the lipid bilayer, (3) intact ion channel proteins and (4) a normal association of the membrane with cytoskeletal constituents. When one or more of these elements is severely damaged, the resulting disturbance of the internal ionic balance is thought to represent the “point of no return” for the injured cell.

The role of calcium in the pathogenesis of cell death deserves special mention. The Ca2 concentration in extracellular fluids is in the millimolar range (10 3 M). By contrast, the cytosolic Ca2 concentration is 10,000-fold lower, on the order of 10 7 M. Many crucial cell functions are exquisitely regulated by minute fluctuations in the cytosolic free calcium concentration. Thus, a massive influx of Ca2 through a damaged plasma membrane ensures the loss of cell viability.

1 | Cell Adaptation, Cell Injury and Cell Death

CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

Coagulative Necrosis Coagulative necrosis refers to light microscopic alterations in a dead cell (Fig. 1-16). The appearance of the necrotic cell has traditionally been termed coagulative necrosis because of its similarity to coagulation of proteins that occurs upon heating. However, the usefulness of this historical term today is questionable.

Shortly after a cell’s death, its outline is maintained.

When stained with the common combination of hematoxylin and eosin, the cytoplasm of a necrotic cell is more deeply eosinophilic than usual. In the nucleus, chromatin is initially clumped and then is redistributed along the nuclear membrane. Three morphologic changes are as follows: ■ ■ ■

Pyknosis: The nucleus becomes smaller and stains deeply basophilic as chromatin clumping continues.

Karyorrhexis: The pyknotic nucleus breaks up into many smaller fragments scattered about the cytoplasm.

Karyolysis: The pyknotic nucleus may be extruded from the cell or it may manifest progressive loss of chromatin staining.

NECROTIC CELL DEATH RESULTS FROM EXOGENOUS CELL INJURY AND IS REFLECTED IN GEOGRAPHIC AREAS OF CELL DEATH At the cellular level, necrosis is characterized by cell and organelle swelling, ATP depletion, increased plasma membrane permeability, release of macromolecules and eventually cell death. The response to this process is usually acute inflammation, which itself may generate further cell injury (see Chapter 2).

Rubin_6e_CH01.indd 15

FIGURE 1-16. Coagulative necrosis. Photomicrograph of the heart in a patient with an acute myocardial infarction. In the center, the deeply eosinophilic necrotic cells have lost their nuclei. The necrotic focus is surrounded by paler staining , viable cardiac myocytes.

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ESSENTIALS OF RUBIN’S PATHOLOGY

Early ultrastructural changes in a dying or dead cell reflect an extension of alterations associated with reversible cell injury. In addition to the nuclear changes described earlier, the dead cell features (1) dilated ER, (2) disaggregated ribosomes, (3) swollen and calcified mitochondria, (4) aggregated cytoskeletal elements and (5) plasma membrane blebs.

After a variable time, depending on the tissue and circumstances, a dead cell is subjected to the lytic activity of intracellular and extracellular enzymes. As a result, the cell disintegrates. This is particularly the case when necrotic cells have elicited an acute inflammatory response (see Chapter 2).

Whereas the morphologic features of individual cell death tend to be uniform across different cell types, the tissue responses are more variable. This diversity is described by several terms that reflect specific histologic patterns that depend on the organ and the circumstances.

Fat Necrosis Fat necrosis specifically affects adipose tissue and most commonly results from pancreatitis or trauma (Fig. 1-18).

The unique feature determining this type of necrosis is the presence of triglycerides in adipose tissue. The process begins when digestive enzymes, normally found only in the pancreatic duct and small intestine, are released from injured pancreatic acinar cells and ducts into the extracellular spaces. On extracellular activation, these enzymes digest the pancreas itself as well as surrounding tissues, including adipose cells. Free fatty acids produced by pancreatic lipases bind calcium and are precipitated as calcium soaps.

Grossly, fat necrosis appears as an irregular, chalky white area embedded in otherwise normal adipose tissue. Traumatic fat necrosis is common in the breast where it may be mistaken for cancer.

Caseous Necrosis Liquefactive Necrosis When the rate of dissolution of the necrotic cells is considerably faster than the rate of repair, the resulting morphologic appearance is termed liquefactive necrosis. The polymorphonuclear leukocytes of the acute inflammatory reaction contain potent hydrolases capable of digesting dead cells. A sharply localized collection of these acute infl ammatory cells, generally in response to bacterial infection, produces rapid cell death and tissue dissolution.

The result is often an abscess (Fig. 1-17), which is a cavity formed by liquefactive necrosis in a solid tissue. Eventually, an abscess is walled off by a fibrous capsule that contains its contents.

Coagulative necrosis of the brain may occur after cerebral artery occlusion and is followed by rapid dissolution— liquefactive necrosis—of the dead tissue by a mechanism that cannot be attributed to the action of an acute inflammatory response. Liquefactive necrosis of large areas of the central nervous system can lead to an actual cavity or cyst that persists permanently.

FIGURE 1-17. Liquefactive necrosis in an abscess of the skin. The abscess cavity is filled with polymorphonuclear leukocytes.

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Caseous necrosis is characteristic of tuberculosis. The lesions of tuberculosis are compact aggregates of macrophages and other inflammatory cells termed granulomas or tubercles (see Chapters 9 and 12, Fig. 12-19). In the center of such caseous granulomas, accumulated mononuclear cells that mediate the chronic inflammatory reaction to the offending mycobacteria are killed. The necrotic cells fail to retain their cellular outlines but do not disappear by lysis, as in liquefactive necrosis. Rather, the dead cells persist indefinitely as amorphous, coarsely granular, eosinophilic debris. Grossly, this debris is grayish white, soft and friable. It resembles clumpy cheese, hence the name caseous necrosis. This distinctive type of necrosis is generally attributed to the toxic effects of the mycobacterial cell wall, which contains complex waxes (peptidoglycolipids) that exert potent biologic effects.

FIGURE 1-18. Fat necrosis . Photomicrograph of peripancreatic adipose tissue from a patient with acute pancreatitis shows an island of necrotic adipocytes adjacent to an acutely inflamed area.

Fatty acids are precipitated as calcium soaps, which accumulate as amorphous, basophilic deposits at the periphery of the irregular island of necrotic adipocytes.

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■

NECROSIS USUALLY INVOLVES ACCUMULATION OF INTRACELLULAR INSULTS

■ ■

The processes by which cells undergo death by necrosis vary according to the cause, organ and cell type. The best studied and most clinically important example is ischemic necrosis of cardiac myocytes, the leading cause of death in the Western world. The mechanisms underlying the death of cardiac myocytes are in part unique, but the basic processes that are involved are comparable to those in other organs. Some of the unfolding events may occur simultaneously, whereas others may be sequential. This complex series of events is summarized below (Fig. 1-19).

Interruption of blood supply decreases delivery of O2 and glucose.

Anaerobic glycolysis leads to overproduction of lactate, decreased intracellular pH and reduced levels of ATP.

■ ■

Blood O2, glucose

ISCHEMIA

Anaerobic glycolysis Aerobic glycolysis

Krebs cycle

Lactate Na+/H+ exchange

ATP

Na+/K+ exchange

H+ Na+

K+

K+

Na+

Na+

H+ Na+ Na+/Ca2+ exchange

Phospholipases Activates

Na+

Na+

Ca2+

Ca2+

Cell membrane damage

NECROSIS

FIGURE 1-19. Mechanisms by which ischemia leads to cell death.

ATP, adenosine triphosphate; Ca2 , calcium ion; H , hydrogen ion; K , potassium ion; Na , sodium ion; O2, oxygen.

Rubin_6e_CH01.indd 17

■

In some instances, necrotic cell death is mediated via signaling cascades. For example, a family of pathogen recognition receptors has been identified that is responsible for recognizing a variety of bacterial and other components. This recognition triggers signaling pathways that can result in damage to mitochondria, lysosomes and cell membranes and are independent of caspase-mediated apoptosis (see below).

Pharmacologic interference with several events involved in the pathogenesis of cell necrosis can preserve cell viability after an ischemic insult. Treatments that increase glucose uptake and redress some of the ionic imbalances may preserve myocyte viability during ischemia.

Apoptosis is a prearranged pathway of cell death triggered by a variety of specific extracellular and intracellular signals. It is part of the balance between the life and death of cells and determines that a cell dies when it is no longer useful or when it may be harmful to the larger organism. As a self-defense mechanism, cells that are infected with pathogens or in which genomic alterations have occurred are destroyed. In this context, many pathogens have evolved mechanisms to inactivate key components of the apoptotic signaling cascades. Apoptosis detects and destroys many cells that harbor dangerous mutations, thereby maintaining genetic consistency and preventing the development of cancer. By contrast, as in the case of acquired antibiotic resistance in infectious agents, successful clones of tumor cells often devise mechanisms to circumvent apoptosis.

Cell membrane

Mitochondria

■

APOPTOSIS, OR PROGRAMMED CELL DEATH, IS A SIGNALING MECHANISM BY WHICH CELLS COMMIT SUICIDE

Artery (e.g., coronary)

Thrombus

■

Distortion of the activities of pumps in the plasma membrane as a result of the lack of ATP and intracellular acidosis skews the ionic balance of the cell.

Ca2ⴙ accumulates in the cell.

Activation of phospholipase A2 (PLA2) and proteases by high intracellular Ca2ⴙ disrupts the plasma membrane and cytoskeleton, thereby causing cell swelling.

The lack of O2 impairs mitochondrial electron transport, thereby decreasing ATP synthesis and facilitating production of ROS. Mitochondrial damage promotes the release of cytochrome c (Cyt c) to the cytosol and initiates the apoptotic cascade.

The cell dies.

1 | Cell Adaptation, Cell Injury and Cell Death

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CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

The Morphology of Apoptosis Apoptotic cells are recognized by pyknosis and nuclear fragmentation, generally against a background of viable cells.

Importantly, individual cells or small groups of cells undergo apoptosis, whereas necrosis characteristically involves larger geographic areas of cell death (Fig. 1-20). Ultrastructural features of apoptotic cells include (1) nuclear condensation and fragmentation, (2) segregation of cytoplasmic organelles into distinct regions, (3) blebs of the plasma membrane and (4) membrane-bound cellular fragments, which often lack nuclei.

Cells that have undergone necrotic cell death tend to elicit strong inflammatory responses. By contrast, inflammation is not generally seen in the vicinity of apoptotic cells. Mononuclear phagocytes may contain cellular debris from apoptotic cells, but recruitment of neutrophils or lymphocytes is

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18

ESSENTIALS OF RUBIN’S PATHOLOGY ■ ■

A

B

FIGURE 1-20. Apoptosis. A viable leukemic cell (A) contrasts with an apoptotic cell (B) in which the nucleus has undergone condensation and fragmentation.

uncommon (see Chapter 2). Once apoptosis has led to DNA fragmentation and cytoskeletal dissolution, only the apoptotic body remains, which is phagocytosed by tissue macrophages.

In view of the numerous developmental, physiologic and protective functions of apoptosis, the lack of inflammation is clearly beneficial to the organism. Apoptosis plays multiple vital roles in normal development and physiology including: ■ ■ ■

■

■

Pruning of nonpersistent structures (such as interdigital tissue) during development Removal of self-reactive clones during the generation of immune diversity Removal of senescent cells in organs continuously repopulated from stem cells (such as the gastrointestinal mucosa, epidermis and hematopoietic system) Regression of hyperplasia in organs responding to changing trophic signals (e.g., postmenopausal atrophy of the endometrium) Deletion of mutant cells after recognition of irreparable DNA damage (in concert with p53)

Apoptosis as a Defense against Dissemination of Infection When a cell “detects” episomal (extrachromosomal) DNA replication, as in a viral infection, it tends to initiate apoptosis.

This effect can be viewed as a means to eliminate infected cells before they can spread the virus. Many viruses have evolved protective mechanisms to manipulate cellular apoptosis. Viral gene products that inhibit apoptosis have been identified for many agents, including human immunodeficiency virus (HIV), human papillomavirus (HPV), adenovirus and many others. In some cases, these viral proteins bind and inactivate certain cellular proteins (e.g., p53) that are important in signaling apoptosis. In other instances, they may act at various points in the signaling pathways that activate apoptosis.

Inflammatory and infectious apoptosis is initiated by intracellular and extracellular infectious agents.

Perforin/granzyme pathway apoptosis is caused by the interaction of cytotoxic T cells with their cellular target and is activated by the transfer of granzyme B from the killer cell to its intended victim.

These pathways are not rigid categories. In fact, the different routes to apoptosis intersect and overlap. However, most intermediate enzymes that transduce proapoptotic signals belong to a family of cysteine proteases called caspases.

RECEPTOR–LIGAND INTERACTIONS AT THE CELL MEMBRANE TRIGGER EXTRINSIC APOPTOSIS Prominent examples of initiation of apoptosis at the cell membrane are the binding of TNF- to its receptor (TNFR) and the recognition of FasL by its receptor, Fas. TNF- is most often a soluble cytokine, whereas FasL is usually situated at the plasma membrane of certain cells, such as cytotoxic effector lymphocytes.

TNFR and Fas, known as death receptors, become activated upon binding their ligands. These transmembrane proteins have specific amino acid sequences, termed death domains, in their cytoplasmic tails that act as docking sites for complementary death domains of other proteins that participate in the apoptotic signaling process. The activated death receptor Fas, a docking protein (Fas-associated death domain [FADD]), and procaspase-8 associate to form a death-inducing signaling complex (DISC) (Fig. 1-21A). The DISC protein complex activates downstream signaling molecules, initially procaspase-8, which is converted to active caspase-8. In turn, caspase-8 stimulates a cascade of other downstream caspases in the apoptosis pathway. These are the effector, or “executioner,” caspases (caspase-3, -6 and -7). Caspase-3 (the most commonly activated effector) in turn activates those enzymes that are responsible for nuclear fragmentation (e.g., caspaseactivated DNAse [CAD], which is responsible for degrading chromosomal DNA). Caspase-3 also destabilizes the cytoskeleton as the cell begins to fragment into apoptotic bodies.

Activation of caspase signaling also occurs when killer lymphocytes, mainly cytotoxic T cells, recognize a cell as foreign. These lymphocytes release perforin and granzyme B. Perforin, as its name suggests, punches a hole in the plasma membrane of a target cell, through which granzyme B enters and directly activates procaspase-10 and procaspase-3 (see Fig. 1-21B). Granzyme A, another constituent of cytotoxic T cells released into target cells, triggers apoptosis by activating a DNA-nicking enzyme, leading to apoptotic degradation of the cell’s genomic DNA.

Apoptosis Comprises Several Signaling Pathways Apoptosis reflects several different pathways that lead to similar end points.

■

■

Extrinsic pathway apoptosis reflects the activation of certain membrane receptors by their ligands (e.g., Fas receptor cluster of differentiation [CD]95 by FAS ligand [FasL] or TNF- receptor by TNF- ).

Intrinsic pathway apoptosis is triggered by diverse intracellular stresses and is characterized by a central role for mitochondria.

Rubin_6e_CH01.indd 18

Intrinsic Apoptosis and Mitochondrial Proteins Mitochondrial proteins of the Bcl-2 family are keys to the balance between cell life and death and are divided into two groups based on the consequences of their overexpression or preponderance. They are either antiapoptotic (e.g., Bcl2, Bcl-XL) or proapoptotic (e.g., Bad, Bak, Bax). The balance between such factors determines the fate of the cell. Heterodimers of proapoptotic and antiapoptotic proteins favor cell survival by sequestering the proapoptotic component.

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CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

19

1 | Cell Adaptation, Cell Injury and Cell Death

TNF

TNFR Cell membrane Death domain Procaspases-3, 6, 7 Docking protein

Death domain Binds and activates Pro-C8 Activates Procaspase-8

Caspase-8

Activates

Endonucleases (PARP)

Caspases-3, 6, 7

Nuclear proteins (lamin)

Effector caspases cleave target proteins

Cytoskeletal proteins (α-fodrin)

APOPTOSIS A B Target cell

Perforin

Activated caspase-10

CTL

Granzyme Procaspase-10

FIGURE 1-21. Mechanisms by which apoptosis may be initiated, signaled and executed.

A. Ligand–receptor interactions that lead to caspase activation. TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor. B. Immunologic reactions in which granzyme released by cytotoxic lymphocytes (CTLs) causes apoptosis. Procaspase-10 is shown as an example of procaspases that are activated by granzyme B.

Rubin_6e_CH01.indd 19

APOPTOSIS

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ESSENTIALS OF RUBIN’S PATHOLOGY

FIGURE 1-21. Continued. C. The equilibrium between proapoptotic and antiapoptotic members of the Bcl-2 family of proteins reflects binding of antiapoptotic members to themselves (homodimers) or to the proapoptotic proteins Bak and Bax. Activation of p53, for example, increases production of proapoptotic members of the Bcl-2 family (e.g., Bim, Puma), which outcompete Bax and Bak for binding to Bcl-2 and Bcl-XL, freeing Bax and Bak to open the mitochondrial permeability transition pore (MPTP).

Activated p53 increases production of Bim, Noxa, etc. Class of proapoptotic activator proteins Bcl- Bax 2

Bcl- Bak XL

p53

Bim Bad

Bcl- Bak XL

BclXL

Bax

UV light

Bim Bik

BclXL

BclXL

Bcl2

p53

Noxa

Bax

Activated p53

Equilibrium between antiapoptotic homodimers and proapoptotic/ antiapoptotic heterodimers

Cyt C MPTP

BclXL

Bik Bcl2

Mitochondria

Bim

Bcl- Noxa 2 Activator proapoptotic proteins outcompete Bax and Bak for antiapoptotic binding and release Bak and Bax

Bax Bax

Bax Bax

Liberated from their antiapoptotic liaisons, Bax and Bak open the mitochondrial transition permeability pane, releasing Cyt C (MPTP)

C However, when, for example, p53 is activated (see below) by stimuli such as ultraviolet (UV) irradiation, production of proapoptotic proteins is increased. As a result, these outcompete Bax and Bak by binding to the antiapoptotic components such as Bcl-2 and Bcl-X (Fig. 1-21C). Free Bax and Bak may then homo-oligomerize and open the mitochondrial permeability transition pore (MPTP), which allows Cyt c to exit the mitochondria and enter the cytosol.

The release of Cyt c into the cytosol is a key step in the initiation of intrinsic apoptosis. In the presence of Cyt c and deoxyadenosine triphosphate (dATP), inactive monomers of an adaptor protein, Apaf-1, homopolymerize into a complex with procaspase-9, which is subsequently activated. The complex of Cyt c, Apaf-1 and the now active caspase-9 is sometimes called the apoptosome. Its formation activates procaspase-3 to caspase-3 (see Fig. 1-21D), which in turn activates enzymes that degrade DNA

RELEASE OF CA2ⴙ FROM THE ENDOPLASMIC RETICULUM MAY TRIGGER APOPTOSIS Ligand-induced and other changes in the intracellular calcium concentration ([Ca2 ]i) are important secondary signals in cellular activation and mitosis. However, changes in [Ca2 ]i may

Rubin_6e_CH01.indd 20

also induce apoptosis. The ER stores considerable calcium, which may be released in response to various stimuli. Under certain circumstances, when ER Ca2 release is activated, and particularly if Ca2 release is prolonged, apoptosis ensues.

Ca2 released by ER may be taken up by mitochondria. The resulting increases in mitochondrial [Ca2 ]i cause the MPTP to open, with release of Cyt c and activation of the intrinsic cascade of apoptosis (see above).

Apoptosis Activated by p53 A pivotal molecule in the cell’s life-and-death dance is the versatile protein p53, which preserves the viability of an injured cell when DNA damage can be repaired, but propels it toward apoptosis after irreparable harm has occurred (p53 is discussed in greater detail in Chapter 5). After it binds to areas of DNA damage, p53 activates proteins, particularly p21WAF/CIP1, that arrest the cell in stage G1 of the cell cycle, allowing time for DNA repair to proceed. It also directs DNA repair enzymes to the site of injury. If DNA damage cannot be repaired, p53 activates mechanisms that lead to apoptosis, principally by altering the balance of Bcl-2 family members at the mitochondria (Fig. 1-22). Stress also leads to the accumulation of p53. Activation of certain oncogenes such as c-myc, hypoxia, depletion of ribonucleotides and loss of cell– cell adhesion during oncogenesis all promote p53-dependent apoptotic pathways.

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CHAPTER 1: CELL ADAPTATION, CELL INJURY AND CELL DEATH

Mitochondrion

Cyt C

DNA damage p53 Activated p 53 PUMA NOXO

WAF/CIP1

Bax

p21 ROS

PTP open

Bcl-2

PTP closed

Bax STOP

G1

1 | Cell Adaptation, Cell Injury and Cell Death

Radiation

Apoptosis

S

STOP

Procaspase-9

M

G2 Mitochondrion

Apaf-1

Apaf-1

FIGURE 1-22. p53-Mediated apoptosis. p53 is recruited to areas of DNA damage following, for example, external irradiation. Activated p53 activates p21WAF/CIP1, which halts cell cycle progression at the G1/S and G2/M transition points. If DNA damage is irreparable, p53 activates transcription of NOXA and PUMA, which alter the balance of proapoptotic and antiapoptotic Bcl-2 family proteins at the mitochondrial membrane in favor of apoptosis. The mitochondrial permeability transition pore (MPTP) is opened (see Fig. 1-21C), leading to apoptosis.

Caspase-9

Procaspase-3

Caspase-3

APOPTOSIS

D FIGURE 1-21. Continued. D. Opening of the MPTP, leading to Apaf-1 activation, thereby triggering the apoptotic cascade. Cyt C, cytochrome c; PTP, permeability transition pores; ROS, reactive oxygen species.

The Equilibrium between Proapoptotic and Antiapoptotic Signals Apoptosis can be viewed as a default pathway, and the survival of many cells is contingent upon continued predominance of antiapoptotic (prosurvival) signals. Such signals are transduced through receptors linked to phosphatidylinositol-3-kinase (PI3K), the enzyme that phosphorylates the signaling molecules phosphatidylinositol bisphosphate and triphosphate (PIP2 and PIP3). By antagonizing apoptosis, PI3K plays a critical role in cell viability. A prototypical receptor that signals via this mechanism is insulin-like growth factor-I receptor (IGF-IR). PI3K exerts antiapoptotic

Rubin_6e_CH01.indd 21

MPTP opens

effects through intracellular mediators, which favor survival by activating protein kinase B (PKB), also called Akt. The latter then inactivates several important proapoptotic proteins. More importantly, Akt activates nuclear factor- B, an important transcription factor that promotes the expression of proteins that prevent the loss of Cyt c from mitochondria, thereby promoting cell survival.

In summary, cells are continually poised between survival and apoptosis; their fate rests on the balance of powerful intracellular and extracellular forces and have signals that constantly act upon and counteract each other. Often, apoptosis functions as a self-protective programmed mechanism that leads to a cell’s suicide when its survival may be detrimental to the organism. At other times, apoptosis is a pathologic process that contributes to many disorders, especially degenerative diseases. Thus, pharmacologic manipulation of apoptosis is an active frontier of drug development.

Biologic Aging Biologic aging can be defined as a constellation of deleterious functional and structural changes that are inevitable consequences of longevity. Importantly, aging must be distinguished from

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DANCE ON STILTS AT THE GIRLS’ UNYAGO, NIUCHI

Newala, too, suffers from the distance of its water-supply—at least the Newala of to-day does; there was once another Newala in a lovely valley at the foot of the plateau. I visited it and found scarcely a trace of houses, only a Christian cemetery, with the graves of several missionaries and their converts, remaining as a monument of its former glories. But the surroundings are wonderfully beautiful. A thick grove of splendid mango-trees closes in the weather-worn crosses and headstones; behind them, combining the useful and the agreeable, is a whole plantation of lemon-trees covered with ripe fruit; not the small African kind, but a much larger and also juicier imported variety, which drops into the hands of the passing traveller, without calling for any exertion on his part. Old Newala is now under the jurisdiction of the native pastor, Daudi, at Chingulungulu, who, as I am on very friendly terms with him, allows me, as a matter of course, the use of this lemon-grove during my stay at Newala.

FEET MUTILATED BY THE RAVAGES OF THE “JIGGER” (Sarcopsylla penetrans)

The water-supply of New Newala is in the bottom of the valley, some 1,600 feet lower down. The way is not only long and fatiguing, but the water, when we get it, is thoroughly bad. We are suffering not only from this, but from the fact that the arrangements at Newala are nothing short of luxurious. We have a separate kitchen—a hut built against the boma palisade on the right of the baraza, the interior of which is not visible from our usual position. Our two cooks were not long in finding this out, and they consequently do—or rather neglect to do—what they please. In any case they do not seem to be very particular about the boiling of our drinking-water—at least I can attribute to no other cause certain attacks of a dysenteric nature, from which both Knudsen and I have suffered for some time. If a man like Omari has to be left unwatched for a moment, he is capable of anything. Besides this complaint, we are inconvenienced by the state of our nails, which have become as hard as glass, and crack on the slightest provocation, and I have the additional infliction of pimples all over me. As if all this were not enough, we have also, for the last week been waging war against the jigger, who has found his Eldorado in the hot sand of the Makonde plateau. Our men are seen all day long—whenever their chronic colds and the dysentery likewise raging among them permit—occupied in removing this scourge of Africa from their feet and trying to prevent the disastrous consequences of its presence. It is quite common to see natives of this place with one or two toes missing; many have lost all their toes,

or even the whole front part of the foot, so that a well-formed leg ends in a shapeless stump. These ravages are caused by the female of Sarcopsylla penetrans, which bores its way under the skin and there develops an egg-sac the size of a pea. In all books on the subject, it is stated that one’s attention is called to the presence of this parasite by an intolerable itching. This agrees very well with my experience, so far as the softer parts of the sole, the spaces between and under the toes, and the side of the foot are concerned, but if the creature penetrates through the harder parts of the heel or ball of the foot, it may escape even the most careful search till it has reached maturity.

Then there is no time to be lost, if the horrible ulceration, of which we see cases by the dozen every day, is to be prevented. It is much easier, by the way, to discover the insect on the white skin of a European than on that of a native, on which the dark speck scarcely shows. The four or five jiggers which, in spite of the fact that I constantly wore high laced boots, chose my feet to settle in, were taken out for me by the all-accomplished Knudsen, after which I thought it advisable to wash out the cavities with corrosive sublimate. The natives have a different sort of disinfectant—they fill the hole with scraped roots. In a tiny Makua village on the slope of the plateau south of Newala, we saw an old woman who had filled all the spaces under her toe-nails with powdered roots by way of prophylactic treatment. What will be the result, if any, who can say?

The rest of the many trifling ills which trouble our existence are really more comic than serious. In the absence of anything else to smoke, Knudsen and I at last opened a box of cigars procured from the Indian store-keeper at Lindi, and tried them, with the most distressing results. Whether they contain opium or some other narcotic, neither of us can say, but after the tenth puff we were both “off,” three-quarters stupefied and unspeakably wretched. Slowly we recovered—and what happened next? Half-an-hour later we were once more smoking these poisonous concoctions—so insatiable is the craving for tobacco in the tropics.

Even my present attacks of fever scarcely deserve to be taken seriously. I have had no less than three here at Newala, all of which have run their course in an incredibly short time. In the early afternoon, I am busy with my old natives, asking questions and making notes. The strong midday coffee has stimulated my spirits to

an extraordinary degree, the brain is active and vigorous, and work progresses rapidly, while a pleasant warmth pervades the whole body. Suddenly this gives place to a violent chill, forcing me to put on my overcoat, though it is only half-past three and the afternoon sun is at its hottest. Now the brain no longer works with such acuteness and logical precision; more especially does it fail me in trying to establish the syntax of the difficult Makua language on which I have ventured, as if I had not enough to do without it. Under the circumstances it seems advisable to take my temperature, and I do so, to save trouble, without leaving my seat, and while going on with my work. On examination, I find it to be 101·48°. My tutors are abruptly dismissed and my bed set up in the baraza; a few minutes later I am in it and treating myself internally with hot water and lemon-juice.

Three hours later, the thermometer marks nearly 104°, and I make them carry me back into the tent, bed and all, as I am now perspiring heavily, and exposure to the cold wind just beginning to blow might mean a fatal chill. I lie still for a little while, and then find, to my great relief, that the temperature is not rising, but rather falling. This is about 7.30 p.m. At 8 p.m. I find, to my unbounded astonishment, that it has fallen below 98·6°, and I feel perfectly well. I read for an hour or two, and could very well enjoy a smoke, if I had the wherewithal—Indian cigars being out of the question.

Having no medical training, I am at a loss to account for this state of things. It is impossible that these transitory attacks of high fever should be malarial; it seems more probable that they are due to a kind of sunstroke. On consulting my note-book, I become more and more inclined to think this is the case, for these attacks regularly follow extreme fatigue and long exposure to strong sunshine. They at least have the advantage of being only short interruptions to my work, as on the following morning I am always quite fresh and fit.

My treasure of a cook is suffering from an enormous hydrocele which makes it difficult for him to get up, and Moritz is obliged to keep in the dark on account of his inflamed eyes. Knudsen’s cook, a raw boy from somewhere in the bush, knows still less of cooking than Omari; consequently Nils Knudsen himself has been promoted to the vacant post. Finding that we had come to the end of our supplies, he began by sending to Chingulungulu for the four sucking-pigs which we had

bought from Matola and temporarily left in his charge; and when they came up, neatly packed in a large crate, he callously slaughtered the biggest of them. The first joint we were thoughtless enough to entrust for roasting to Knudsen’s mshenzi cook, and it was consequently uneatable; but we made the rest of the animal into a jelly which we ate with great relish after weeks of underfeeding, consuming incredible helpings of it at both midday and evening meals. The only drawback is a certain want of variety in the tinned vegetables. Dr. Jäger, to whom the Geographical Commission entrusted the provisioning of the expeditions—mine as well as his own—because he had more time on his hands than the rest of us, seems to have laid in a huge stock of Teltow turnips,[46] an article of food which is all very well for occasional use, but which quickly palls when set before one every day; and we seem to have no other tins left. There is no help for it—we must put up with the turnips; but I am certain that, once I am home again, I shall not touch them for ten years to come.

Amid all these minor evils, which, after all, go to make up the genuine flavour of Africa, there is at least one cheering touch: Knudsen has, with the dexterity of a skilled mechanic, repaired my 9 × 12 cm. camera, at least so far that I can use it with a little care.

How, in the absence of finger-nails, he was able to accomplish such a ticklish piece of work, having no tool but a clumsy screw-driver for taking to pieces and putting together again the complicated mechanism of the instantaneous shutter, is still a mystery to me; but he did it successfully. The loss of his finger-nails shows him in a light contrasting curiously enough with the intelligence evinced by the above operation; though, after all, it is scarcely surprising after his ten years’ residence in the bush. One day, at Lindi, he had occasion to wash a dog, which must have been in need of very thorough cleansing, for the bottle handed to our friend for the purpose had an extremely strong smell. Having performed his task in the most conscientious manner, he perceived with some surprise that the dog did not appear much the better for it, and was further surprised by finding his own nails ulcerating away in the course of the next few days. “How was I to know that carbolic acid has to be diluted?” he mutters indignantly, from time to time, with a troubled gaze at his mutilated finger-tips.

Since we came to Newala we have been making excursions in all directions through the surrounding country, in accordance with old habit, and also because the akida Sefu did not get together the tribal elders from whom I wanted information so speedily as he had promised. There is, however, no harm done, as, even if seen only from the outside, the country and people are interesting enough.

The Makonde plateau is like a large rectangular table rounded off at the corners. Measured from the Indian Ocean to Newala, it is about seventy-five miles long, and between the Rovuma and the Lukuledi it averages fifty miles in breadth, so that its superficial area is about two-thirds of that of the kingdom of Saxony. The surface, however, is not level, but uniformly inclined from its south-western edge to the ocean. From the upper edge, on which Newala lies, the eye ranges for many miles east and north-east, without encountering any obstacle, over the Makonde bush. It is a green sea, from which here and there thick clouds of smoke rise, to show that it, too, is inhabited by men who carry on their tillage like so many other primitive peoples, by cutting down and burning the bush, and manuring with the ashes. Even in the radiant light of a tropical day such a fire is a grand sight.

Much less effective is the impression produced just now by the great western plain as seen from the edge of the plateau. As often as time permits, I stroll along this edge, sometimes in one direction, sometimes in another, in the hope of finding the air clear enough to let me enjoy the view; but I have always been disappointed.

Wherever one looks, clouds of smoke rise from the burning bush, and the air is full of smoke and vapour. It is a pity, for under more favourable circumstances the panorama of the whole country up to the distant Majeje hills must be truly magnificent. It is of little use taking photographs now, and an outline sketch gives a very poor idea of the scenery. In one of these excursions I went out of my way to make a personal attempt on the Makonde bush. The present edge of the plateau is the result of a far-reaching process of destruction through erosion and denudation. The Makonde strata are everywhere cut into by ravines, which, though short, are hundreds of yards in depth. In consequence of the loose stratification of these beds, not only are the walls of these ravines nearly vertical, but their upper end is closed by an equally steep escarpment, so that the

western edge of the Makonde plateau is hemmed in by a series of deep, basin-like valleys. In order to get from one side of such a ravine to the other, I cut my way through the bush with a dozen of my men.

It was a very open part, with more grass than scrub, but even so the short stretch of less than two hundred yards was very hard work; at the end of it the men’s calicoes were in rags and they themselves bleeding from hundreds of scratches, while even our strong khaki suits had not escaped scatheless.

NATIVE PATH THROUGH THE MAKONDE BUSH, NEAR MAHUTA

I see increasing reason to believe that the view formed some time back as to the origin of the Makonde bush is the correct one. I have no doubt that it is not a natural product, but the result of human occupation. Those parts of the high country where man—as a very slight amount of practice enables the eye to perceive at once—has not yet penetrated with axe and hoe, are still occupied by a splendid timber forest quite able to sustain a comparison with our mixed forests in Germany. But wherever man has once built his hut or tilled his field, this horrible bush springs up. Every phase of this process

may be seen in the course of a couple of hours’ walk along the main road. From the bush to right or left, one hears the sound of the axe— not from one spot only, but from several directions at once. A few steps further on, we can see what is taking place. The brush has been cut down and piled up in heaps to the height of a yard or more, between which the trunks of the large trees stand up like the last pillars of a magnificent ruined building. These, too, present a melancholy spectacle: the destructive Makonde have ringed them— cut a broad strip of bark all round to ensure their dying off—and also piled up pyramids of brush round them. Father and son, mother and son-in-law, are chopping away perseveringly in the background—too busy, almost, to look round at the white stranger, who usually excites so much interest. If you pass by the same place a week later, the piles of brushwood have disappeared and a thick layer of ashes has taken the place of the green forest. The large trees stretch their smouldering trunks and branches in dumb accusation to heaven—if they have not already fallen and been more or less reduced to ashes, perhaps only showing as a white stripe on the dark ground.

This work of destruction is carried out by the Makonde alike on the virgin forest and on the bush which has sprung up on sites already cultivated and deserted. In the second case they are saved the trouble of burning the large trees, these being entirely absent in the secondary bush.

After burning this piece of forest ground and loosening it with the hoe, the native sows his corn and plants his vegetables. All over the country, he goes in for bed-culture, which requires, and, in fact, receives, the most careful attention. Weeds are nowhere tolerated in the south of German East Africa. The crops may fail on the plains, where droughts are frequent, but never on the plateau with its abundant rains and heavy dews. Its fortunate inhabitants even have the satisfaction of seeing the proud Wayao and Wamakua working for them as labourers, driven by hunger to serve where they were accustomed to rule.

But the light, sandy soil is soon exhausted, and would yield no harvest the second year if cultivated twice running. This fact has been familiar to the native for ages; consequently he provides in time, and, while his crop is growing, prepares the next plot with axe and firebrand. Next year he plants this with his various crops and

lets the first piece lie fallow. For a short time it remains waste and desolate; then nature steps in to repair the destruction wrought by man; a thousand new growths spring out of the exhausted soil, and even the old stumps put forth fresh shoots. Next year the new growth is up to one’s knees, and in a few years more it is that terrible, impenetrable bush, which maintains its position till the black occupier of the land has made the round of all the available sites and come back to his starting point.

The Makonde are, body and soul, so to speak, one with this bush.

According to my Yao informants, indeed, their name means nothing else but “bush people.” Their own tradition says that they have been settled up here for a very long time, but to my surprise they laid great stress on an original immigration. Their old homes were in the south-east, near Mikindani and the mouth of the Rovuma, whence their peaceful forefathers were driven by the continual raids of the Sakalavas from Madagascar and the warlike Shirazis[47] of the coast, to take refuge on the almost inaccessible plateau. I have studied African ethnology for twenty years, but the fact that changes of population in this apparently quiet and peaceable corner of the earth could have been occasioned by outside enterprises taking place on the high seas, was completely new to me. It is, no doubt, however, correct.

The charming tribal legend of the Makonde—besides informing us of other interesting matters—explains why they have to live in the thickest of the bush and a long way from the edge of the plateau, instead of making their permanent homes beside the purling brooks and springs of the low country.

“The place where the tribe originated is Mahuta, on the southern side of the plateau towards the Rovuma, where of old time there was nothing but thick bush. Out of this bush came a man who never washed himself or shaved his head, and who ate and drank but little.

He went out and made a human figure from the wood of a tree growing in the open country, which he took home to his abode in the bush and there set it upright. In the night this image came to life and was a woman. The man and woman went down together to the Rovuma to wash themselves. Here the woman gave birth to a stillborn child. They left that place and passed over the high land into the valley of the Mbemkuru, where the woman had another child, which

was also born dead. Then they returned to the high bush country of Mahuta, where the third child was born, which lived and grew up. In course of time, the couple had many more children, and called themselves Wamatanda. These were the ancestral stock of the Makonde, also called Wamakonde,[48] i.e., aborigines. Their forefather, the man from the bush, gave his children the command to bury their dead upright, in memory of the mother of their race who was cut out of wood and awoke to life when standing upright. He also warned them against settling in the valleys and near large streams, for sickness and death dwelt there. They were to make it a rule to have their huts at least an hour’s walk from the nearest wateringplace; then their children would thrive and escape illness.”

The explanation of the name Makonde given by my informants is somewhat different from that contained in the above legend, which I extract from a little book (small, but packed with information), by Pater Adams, entitled Lindi und sein Hinterland. Otherwise, my results agree exactly with the statements of the legend. Washing?

Hapana—there is no such thing. Why should they do so? As it is, the supply of water scarcely suffices for cooking and drinking; other people do not wash, so why should the Makonde distinguish himself by such needless eccentricity? As for shaving the head, the short, woolly crop scarcely needs it,[49] so the second ancestral precept is likewise easy enough to follow. Beyond this, however, there is nothing ridiculous in the ancestor’s advice. I have obtained from various local artists a fairly large number of figures carved in wood, ranging from fifteen to twenty-three inches in height, and representing women belonging to the great group of the Mavia, Makonde, and Matambwe tribes. The carving is remarkably well done and renders the female type with great accuracy, especially the keloid ornamentation, to be described later on. As to the object and meaning of their works the sculptors either could or (more probably) would tell me nothing, and I was forced to content myself with the scanty information vouchsafed by one man, who said that the figures were merely intended to represent the nembo—the artificial deformations of pelele, ear-discs, and keloids. The legend recorded

by Pater Adams places these figures in a new light. They must surely be more than mere dolls; and we may even venture to assume that they are—though the majority of present-day Makonde are probably unaware of the fact—representations of the tribal ancestress.

The references in the legend to the descent from Mahuta to the Rovuma, and to a journey across the highlands into the Mbekuru valley, undoubtedly indicate the previous history of the tribe, the travels of the ancestral pair typifying the migrations of their descendants. The descent to the neighbouring Rovuma valley, with its extraordinary fertility and great abundance of game, is intelligible at a glance—but the crossing of the Lukuledi depression, the ascent to the Rondo Plateau and the descent to the Mbemkuru, also lie within the bounds of probability, for all these districts have exactly the same character as the extreme south. Now, however, comes a point of especial interest for our bacteriological age. The primitive Makonde did not enjoy their lives in the marshy river-valleys.

Disease raged among them, and many died. It was only after they had returned to their original home near Mahuta, that the health conditions of these people improved. We are very apt to think of the African as a stupid person whose ignorance of nature is only equalled by his fear of it, and who looks on all mishaps as caused by evil spirits and malignant natural powers. It is much more correct to assume in this case that the people very early learnt to distinguish districts infested with malaria from those where it is absent.

This knowledge is crystallized in the ancestral warning against settling in the valleys and near the great waters, the dwelling-places of disease and death. At the same time, for security against the hostile Mavia south of the Rovuma, it was enacted that every settlement must be not less than a certain distance from the southern edge of the plateau. Such in fact is their mode of life at the present day. It is not such a bad one, and certainly they are both safer and more comfortable than the Makua, the recent intruders from the south, who have made USUAL METHOD OF good their footing on the western edge of the CLOSING HUT-DOOR plateau, extending over a fairly wide belt of country. Neither Makua nor Makonde show in their dwellings anything of the size and comeliness of the Yao houses in the plain, especially at Masasi, Chingulungulu and Zuza’s. Jumbe Chauro, a Makonde hamlet not far from Newala, on the road to Mahuta, is the

most important settlement of the tribe I have yet seen, and has fairly spacious huts. But how slovenly is their construction compared with the palatial residences of the elephant-hunters living in the plain.

The roofs are still more untidy than in the general run of huts during the dry season, the walls show here and there the scanty beginnings or the lamentable remains of the mud plastering, and the interior is a veritable dog-kennel; dirt, dust and disorder everywhere. A few huts only show any attempt at division into rooms, and this consists merely of very roughly-made bamboo partitions. In one point alone have I noticed any indication of progress—in the method of fastening the door. Houses all over the south are secured in a simple but ingenious manner. The door consists of a set of stout pieces of wood or bamboo, tied with bark-string to two cross-pieces, and moving in two grooves round one of the door-posts, so as to open inwards. If the owner wishes to leave home, he takes two logs as thick as a man’s upper arm and about a yard long. One of these is placed obliquely against the middle of the door from the inside, so as to form an angle of from 60° to 75° with the ground. He then places the second piece horizontally across the first, pressing it downward with all his might.

It is kept in place by two strong posts planted in the ground a few inches inside the door. This fastening is absolutely safe, but of course cannot be applied to both doors at once, otherwise how could the owner leave or enter his house? I have not yet succeeded in finding out how the back door is fastened.

MAKONDE LOCK AND KEY AT JUMBE CHAURO

This is the general way of closing a house. The Makonde at Jumbe Chauro, however, have a much more complicated, solid and original one. Here, too, the door is as already described, except that there is only one post on the inside, standing by itself about six inches from one side of the doorway. Opposite this post is a hole in the wall just large enough to admit a man’s arm. The door is closed inside by a large wooden bolt passing through a hole in this post and pressing with its free end against the door. The other end has three holes into which fit three pegs running in vertical grooves inside the post. The door is opened with a wooden key about a foot long, somewhat curved and sloped off at the butt; the other end has three pegs corresponding to the holes, in the bolt, so that, when it is thrust through the hole in the wall and inserted into the rectangular opening in the post, the pegs can be lifted and the bolt drawn out.[50]

MODE OF INSERTING THE KEY

With no small pride first one householder and then a second showed me on the spot the action of this greatest invention of the Makonde Highlands. To both with an admiring exclamation of “Vizuri sana!” (“Very fine!”). I expressed the wish to take back these marvels with me to Ulaya, to show the Wazungu what clever fellows the Makonde are. Scarcely five minutes after my return to camp at Newala, the two men came up sweating under the weight of two heavy logs which they laid down at my feet, handing over at the same time the keys of the fallen fortress. Arguing, logically enough, that if the key was wanted, the lock would be wanted with it, they had taken their axes and chopped down the posts—as it never occurred to them to dig them out of the ground and so bring them intact. Thus I have

two badly damaged specimens, and the owners, instead of praise, come in for a blowing-up.

The Makua huts in the environs of Newala are especially miserable; their more than slovenly construction reminds one of the temporary erections of the Makua at Hatia’s, though the people here have not been concerned in a war. It must therefore be due to congenital idleness, or else to the absence of a powerful chief. Even the baraza at Mlipa’s, a short hour’s walk south-east of Newala, shares in this general neglect. While public buildings in this country are usually looked after more or less carefully, this is in evident danger of being blown over by the first strong easterly gale. The only attractive object in this whole district is the grave of the late chief Mlipa. I visited it in the morning, while the sun was still trying with partial success to break through the rolling mists, and the circular grove of tall euphorbias, which, with a broken pot, is all that marks the old king’s resting-place, impressed one with a touch of pathos.

Even my very materially-minded carriers seemed to feel something of the sort, for instead of their usual ribald songs, they chanted solemnly, as we marched on through the dense green of the Makonde bush:—